Abstract
Background/Aim: Nasopharyngeal carcinoma (NPC) is a virally associated epithelial malignancy with a high prevalence in East Asia. While Epstein-Barr virus (EBV) infection is a well-established risk factor, the role of host immune-related genetic variants remains insufficiently understood. Interleukin-12 (IL-12), a key immunoregulatory cytokine, is essential for EBV-specific T-cell responses, however, the impact of IL-12A gene polymorphisms on NPC susceptibility remains unknown.
Materials and Methods: This case-control study investigated the association between IL-12A rs568408 and rs2243115 genotypes and NPC risk in a Taiwanese population comprising 208 patients with NPC and 416 matched cancer-free controls. Genotype and allele frequencies were assessed, and gene-lifestyle interactions were evaluated based on smoking, alcohol consumption, and betel quid chewing.
Results: No significant associations were observed between either IL-12A rs568408 or rs2243115 genotypes and overall NPC risk (p for trend=0.5286 and 0.4910). However, stratified analysis revealed that the rs568408 AA variant genotype conferred significantly elevated NPC risk among smokers [odds ratio (OR)=5.47, 95% confidence interval (95%CI)=1.03-29.05, p=0.0397], and the AG variant genotype conferred significantly elevated NPC risk among betel quid users (OR=2.61, 95%CI=1.32-5.14, p=0.0080). In contrast, no significant associations were observed among non-smokers, non-betel quid chewers, alcohol drinkers, or non-alcohol drinkers (all p>0.05). No any significant interaction was found for rs2243115 genotypes.
Conclusion: IL-12A rs568408 may interact with tobacco and betel quid exposures to influence NPC susceptibility. These findings highlight the importance of integrating genetic and environmental factors in NPC risk assessment and warrant further validation in larger, multi-ethnic NPC cohorts.
- Betel quid chewing
- genotype
- interleukin-12A
- nasopharyngeal carcinoma
- polymorphism
- smoking
- stratified analysis
- Taiwan
Introduction
Nasopharyngeal carcinoma (NPC) is an aggressive malignancy originating from the epithelial lining of the nasopharyngeal mucosa, with recent global estimates reporting approximately 120,416 new cases annually (1). The etiology of NPC is complex and multifactorial, encompassing persistent Epstein-Barr virus (EBV) infection, host genetic susceptibility, and aberrant immune regulation (2). EBV contributes significantly to NPC development through the expression of latent viral proteins such as LMP1, LMP2, and EBNA1, which collectively promote immune evasion, oncogenic transformation, and suppression of host immune surveillance mechanisms (3, 4). Although genome-wide association studies (GWAS) have revealed several genetic loci linked to NPC susceptibility (5, 6), translating these findings into clinically actionable biomarkers remains a formidable hurdle. Notably, population-specific genetic indicators, particularly those pertinent to Taiwanese cohorts hold greater promise for integration into comprehensive public health strategies, such as nationwide genomic screening programs (7-9). The advancement of precision medicine in NPC hinges on translational efforts to identify robust, clinically relevant biomarkers that can enhance diagnostic accuracy, guide therapeutic decisions, and improve patient outcomes.
Interleukin-12 (IL-12) is a critical immunoregulatory cytokine that bridges innate and adaptive immune mechanisms (10). It plays a pivotal role in potentiating cytotoxic T lymphocyte responses against EBV antigens by promoting CD8+ T-cell activation and facilitating the clearance of EBV-infected cells (11). Evidence has shown a positive association between IL-12 signaling and T-cell reactivity to EBV-derived peptides in patients with NPC, highlighting its role in mediating immune surveillance against EBV-driven oncogenesis (12). The tumor microenvironment in NPC is frequently dominated by immunosuppressive populations, including regulatory T cells and myeloid-derived suppressor cells, which secrete anti-inflammatory cytokines such as IL-10 and TGF-β that suppress Th1-mediated immunity (13, 14). Through the induction of interferon-gamma (IFN-γ), IL-12 can antagonize these immunosuppressive pathways, thereby reinvigorating effector T cells and natural killer (NK) cell activity and mitigating tumor-mediated immune evasion (15). Despite its recognized immunostimulatory functions, studies examining IL-12 expression at mRNA or protein level in NPC remain exceedingly limited (12, 15, 16). Even more, there has been no investigation into the genetic variants of IL-12 and their potential contributions to NPC susceptibility.
Structurally, IL-12 is a heterodimeric cytokine composed of a 35-kDa α-chain (p35 subunit) and a 40-kDa β-chain (p40 subunit), the latter of which is also a component of IL-23, another key cytokine within the IL-12 family (17). The p35 and p40 subunits are encoded by the IL-12A and IL-12B genes, respectively. Numerous studies have explored the association between IL-12A genetic polymorphisms and various malignancies, including cancers of the oral cavity (18), esophagus (19, 20), stomach (21, 22), liver (23, 24), colon and rectum (25), lung (26), breast (27, 28), and cervix (29, 30), and non-Hodgkin leukemia (31). These studies predominantly focused on two single nucleotide polymorphisms (SNPs) in IL-12A, namely rs568408 and rs2243115. Surprisingly, despite IL-12’s established immunological relevance in virus-associated malignancies, no prior studies have evaluated the impact of IL-12A variants on NPC susceptibility. In light of this knowledge gap, the present study aimed to investigate the potential association between IL-12A rs568408 and rs2243115 polymorphisms (Figure 1) and the risk of developing NPC in a Taiwanese population comprising 208 patients with NPC and 416 matched healthy controls. Additionally, our study aimed to assess potential gene-environment interactions involving these polymorphisms and common lifestyle factors, including cigarette smoking, alcohol intake, and betel quid chewing.
Location of the IL-12A rs568408 and rs2243115 polymorphic sites together with the neighbouring DNA sequences.
Materials and Methods
Recruitment of patients with NPC and matched controls. A total of 208 patients diagnosed with NPC were enrolled from the Department of General Surgery at China Medical University Hospital in Taichung, Taiwan. Participation was voluntary, and each individual completed a structured, self-administered questionnaire and provided a peripheral blood sample for subsequent analyses. The control group consisted of non-cancer individuals selected at a 2:1 ratio relative to the number of NPC cases, matched meticulously by sex, age (within a ±5-year range), and lifestyle exposures, including tobacco use, alcohol consumption, and betel quid chewing. Individuals with a personal history of any malignancy, metastatic disease of unknown origin or non-NPC etiology, or any hereditary/genetic disorder were excluded from the control group to minimize potential confounders. To ensure uniformity in data collection across cohorts, lifestyle information -including tobacco, alcohol, and betel quid use- was obtained using the same standardized questionnaire administered to both groups. Participants were classified as “ever” users if they had engaged in any of these behaviors more than twice weekly for a duration exceeding one year. The frequency and intensity of such exposures were subsequently categorized as discrete variables for statistical evaluation. All enrolled subjects were of self-reported Taiwanese ethnicity. Ethical approval for the study protocol was obtained from the Institutional Review Board of China Medical University Hospital (DMR101-IRB1-306), and all research procedures were conducted in accordance with the ethical standards outlined in the Declaration of Helsinki. A summary of demographic and clinical characteristics for both cases and controls is provided in Table I.
Demographic characteristics of the 416 control subjects and 208 patients with nasopharyngeal carcinoma.
Genotyping procedures for IL-12A polymorphisms. Genomic DNA was isolated from peripheral blood leukocytes using the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC), in accordance with protocols validated in prior investigations (32, 33). Primer design, restriction enzyme selection, and polymerase chain reaction (PCR) conditions for detecting IL-12A rs568408 and rs2243115 followed the methodological framework established in our previous publications (34). To ensure high genotyping fidelity, analyses of rs568408 and rs2243115 were independently conducted by at least two trained investigators, with all procedures executed under double-blind conditions. Each DNA sample underwent repeated genotyping, and the consistency rate across all replicates was 100%, affirming the robustness and reproducibility of the assay.
Statistical analysis approaches. To determine whether the control group reflected the genetic distribution of the general population, genotype frequencies of IL-12A polymorphisms were assessed for conformity with Hardy-Weinberg equilibrium (HWE) using the goodness-of-fit chi-square test. Differences in mean age between NPC cases and controls were evaluated using the unpaired Student’s t-test. The distribution of IL-12A genotypes and alleles among cases and controls was examined using Pearson’s chi-square test with Yates’ correction for continuity; in instances where expected frequencies in any cell were fewer than five, Fisher’s exact test was applied. A two-sided p-value less than 0.05 was considered statistically significant for all comparisons. To quantify the association between specific IL-12A genotypes and the risk of NPC, odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated using unconditional logistic regression models. Stratified analyses were subsequently conducted to evaluate potential gene-environment interactions between IL-12A variants and lifestyle factors, including smoking, alcohol intake, and betel quid chewing. These models were adjusted for potential confounders such as age, sex, and co-exposures to other lifestyle risk factors.
Results
Comparison of baseline characteristics between patients with NPC and controls. Table I presents the distribution of demographic and lifestyle variables for the 208 patients with NPC and the 416 cancer-free controls. Frequency matching was implemented to achieve comparable age and sex profiles between the two groups, with no statistically significant differences observed (p=0.4639 and 1.0000 for age and sex, respectively). This matching strategy also yielded similar distributions of major lifestyle exposures, including cigarette smoking (40.9% in cases versus 38.0% in controls), alcohol consumption (45.9% versus 40.4%), and betel quid chewing (38.6% versus 37.5%). Histopathological classification of NPC cases revealed that only eight individuals (3.8%) were diagnosed with keratinizing squamous cell carcinoma (WHO type I), while the remaining 200 patients (96.2%) presented with non-keratinizing carcinoma (WHO type II). Within this non-keratinizing group, 32 patients (16.0%) had the differentiated subtype (WHO type IIa), whereas the majority, 168 patients (84.0%), were diagnosed with the undifferentiated subtype (WHO type IIb), as detailed in Table I.
Association between IL-12A rs568408 and rs2243115 genotypes and NPC risk. Table II presents the genotype distributions of IL-12A rs568408 and rs2243115 polymorphisms among 208 patients with NPC and 416 matched cancer-free controls. Genotypic frequencies for both SNPs in the control group conformed to HWE (p=0.1521 and 0.0796), suggesting no deviation from expected population level distribution. Comparative analysis revealed no statistically significant difference in rs568408 genotype frequencies between NPC cases and controls (p for trend=0.5286). Although carriers of the heterozygous AG and homozygous AA genotypes exhibited a trend toward elevated NPC risk, the associations were not statistically significant (OR=1.20 and 1.47, 95%CI=0.80-1.79 and 0.55-3.95, p=0.4311 and 0.6083). Similarly, when analyzed under recessive (AA versus GG+AG) and dominant (AG+AA versus GG) genetic models, no meaningful associations were observed (OR=1.41 and 1.23, 95%CI=0.53-3.77 and 0.84-1.80, p=0.6638 and 0.3390). For the rs2243115 variant, neither the heterozygous GT nor the homozygous GG genotype demonstrated a significant relationship with NPC susceptibility (OR=1.10 and 2.05, 95%CI=0.68-1.79 and 0.59-7.18, p=0.7905 and 0.4190). Likewise, analyses using recessive (GG vs. TT+GT) and dominant (GT+GG vs. TT) inheritance models did not yield statistically significant findings (OR=2.02 and 1.18, 95%CI=0.58-7.07 and 0.75-1.87, p=0.4301 and 0.5511). These results collectively suggest that neither IL-12A rs568408 nor rs2243115 genotypes are significantly associated with altered risk for NPC in the Taiwanese population studied.
Distribution of IL-12A rs568408 and rs2243115 variant genotypes among the controls and patients with nasopharyngeal carcinoma.
Allelic frequency distributions of IL-12A rs568408 and rs2243115 and their associations with NPC susceptibility. To complement the genotypic data presented in Table II, an allelic frequency analysis was performed to further evaluate the potential involvement of IL-12A rs568408 and rs2243115 variants in NPC risk. In alignment with the genotype-based findings, the frequency of the rs568408 variant A allele did not differ significantly between NPC cases and healthy controls (p=0.2792). Individuals harboring the A allele demonstrated a slightly increased, yet statistically non-significant, risk of developing NPC compared to those with the wild-type G allele (OR=1.22, 95%CI=0.87-1.71; Table III). Likewise, carriers of the rs2243115 G allele exhibited a marginally elevated, but non-significant, likelihood of NPC compared to T allele carriers (OR=1.24, 95%CI=0.82-1.88, p=0.3632; Table III). These allelic trends are concordant with the genotype-based analyses and reinforce the notion that neither rs568408 nor rs2243115 exerts a substantial influence on NPC susceptibility in this Taiwanese cohort.
Allelic frequencies for IL-12A rs568408 and rs2243115 genotypes in the control and nasopharyngeal carcinoma patient groups.
Stratified analysis of IL-12A rs568408 and rs2243115 genotypes in relation to lifestyle factors. To explore potential gene–environment interactions, a stratified analysis was conducted to evaluate the joint effects of IL-12A rs568408 genotypes with lifestyle exposures -namely smoking, alcohol consumption, and betel quid chewing- on NPC susceptibility (Table IV, Table V, Table VI). Notably, statistically significant interactions were identified between IL-12A rs568408 and both smoking (p for trend=0.0359, Table IV) and betel quid chewing (p for trend=0.0043, Table VI). Among smokers, individuals with the homozygous AA genotype exhibited a significantly elevated risk of NPC compared to those carrying the wild-type GG genotype (OR=5.47, 95%CI=1.03-29.05, p=0.0397). This association remained significant after adjusting for potential confounders, including age, sex, alcohol use, and betel quid chewing (adjusted OR=6.89, 95%CI=1.06-26.48, Table IV). In contrast, no statistically significant interaction was observed between IL-12A rs568408 genotypes and alcohol consumption, irrespective of drinking status (Table V). However, a strong association was detected between the heterozygous AG genotype and increased NPC risk among betel quid chewers (OR=2.61, 95%CI=1.32-5.14, p=0.0080, Table VI), which persisted after multivariate adjustment (adjusted OR=2.53, 95%CI=1.50-4.82, Table VI). A borderline association was also noted for the AA genotype in this subgroup (OR=4.15, 95%CI=0.96-17.99, p=0.0545), which became statistically significant following adjustment for covariates (adjusted OR=3.98, 95%CI=1.07-14.36, Table VI). In contrast to rs568408, the IL-12A rs2243115 polymorphism did not exhibit significant interactions with smoking (Table VII), alcohol intake (Table VIII), or betel quid use (Table IX), suggesting a limited role of this variant in modifying NPC risk through environmental exposures.
Distribution of IL-12A rs568408 genotypes among 208 nasopharyngeal carcinoma cases and 416 controls after stratification by smoking status.
Distribution of IL-12A rs568408 genotypes among 208 nasopharyngeal carcinoma cases and 416 controls after stratification by alcoholism status.
Distribution of IL-12A rs568408 genotypes among 208 nasopharyngeal carcinoma cases and 416 controls after stratification by betel quid chewing. status.
Distribution of IL-12A rs2243115 genotypes among 208 nasopharyngeal carcinoma cases and 416 controls after stratification by smoking status.
Distribution of IL-12A rs2243115 genotypes among 208 nasopharyngeal carcinoma cases and 416 controls after stratification by alcoholism status.
Distribution of IL-12A rs2243115 genotypes among 208 nasopharyngeal carcinoma cases and 416 controls after stratification by betel quid chewing status.
Discussion
As discussed in the introduction, IL-12 is a multifunctional cytokine that bridges the innate immune response (via NK cells) and the acquired immune response (via cytotoxic T lymphocytes) (10). Given this, it is plausible to speculate that polymorphisms within IL-12A could influence IL-12 gene expression, potentially impairing functional IL-12 protein synthesis. Such disruptions could lead to immune system dysfunction and subsequently increased risk of malignant diseases, including NPC. In 2008, He et, al. conducted an extensive study on the antitumor immune responses triggered by recombinant IL-12 gene transfection into dendritic cells, finding that over-expression of IL-12 enhances anti-cancer immunity against colon tumors (35). Clinical investigations have demonstrated that serum IL-12 levels correlate with the severity of gastric cancer (36) and the progression of colorectal cancer (37). In our study, we initially examined the contribution of IL-12A rs568408 and rs2243115 polymorphisms to NPC susceptibility. Our results showed that neither polymorphism was significantly associated with an altered NPC risk (Table II). However, we observed that the variant genotypes of IL-12A rs568408 may interact with smoking (Table IV) and betel quid chewing behaviors (Table VI), contributing to an increased NPC risk among the Taiwanese population. While the precise mechanisms remain unclear, it is important to focus attention on individuals carrying these risk alleles in conjunction with smoking and/or betel quid chewing behaviors (Table VI), as they may face a higher risk of NPC. It is important to note that the etiological factors contributing to NPC in East Asia (Taiwan) such as the consumption of pickled foods, EBV infection status, smoking, and betel quid chewing may differ from those in other regions. Notably, East Asia has borne the heaviest global burden of NPC from 1990 to 2021 (38). These findings necessitate further studies in diverse populations to validate the observed associations and explore the underlying mechanisms.
In addition to evaluating the role of IL-12 genotypes in NPC risk, our study also explored potential interactions between the IL-12A rs568408 and rs2243115 genotypes and other factors associated with NPC susceptibility. No significant differences were observed in NPC risk between individuals with variant genotypes of IL-12A rs568408 or rs2243115, regardless of alcohol consumption status (Table V, Table VI, Table VII, Table VIII). The World Health Organization classifies NPC into three pathological subtypes: keratinizing squamous, non-keratinizing, and basaloid squamous. Furthermore, non-keratinizing NPC can be further subdivided into differentiated and undifferentiated forms (39-41). In this context, we analyzed the association between IL-12A rs568408 and rs2243115 genotypes and NPC pathological subtypes. However, no significant differences were identified among the three subtypes (data not shown). Age is also a well-established risk factor for NPC, with the highest proportion of cases occurring in individuals over 50 years of age worldwide (42). The relative risk of NPC is reported to peak at approximately 55 years old (43). To investigate this further, we conducted a stratified analysis to assess the association between age and IL-12A genotypes, but no significant differences were observed across age-stratified subgroups (data not shown). Similarly, no significant association was found between sex and any IL-12A genotypes (data not shown).
The antineoplastic activity of interferon-alpha (IFN-α) has been demonstrated in animal models, where administration of recombinant IFN-α not only inhibited NPC tumor progression but also triggered G1-phase cell cycle arrest. This was accompanied by upregulation of tumor suppressor proteins p16 and pRb, downregulation of cyclin D1 (CCND1) and cyclin-dependent kinase 6 (CDK6), and a marked elevation in circulating IL-12 levels (15). In colorectal cancer, reprogramming of tumor cells was achieved via activation of nucleotide-binding oligomerization domain-like receptor family CARD domain-containing 4 (NLRC4), which promoted the maturation of human dendritic cells and enhanced a Th1-polarized immune response, largely mediated by IL-12 secretion (44). Despite these promising preclinical insights, clinical applications of IL-12 have yielded limited success due to its substantial toxicity when administered systemically, and current trials have not specifically addressed its therapeutic potential in NPC management (45).
In summary, our results indicate that the IL-12A polymorphisms rs568408 and rs2243115 may modestly contribute to NPC susceptibility. Notably, the rs568408 variant genotypes appear to exert a synergistic effect in individuals with specific lifestyle exposures, particularly tobacco smoking and betel quid chewing. These findings underscore the critical importance of integrating detailed exposure history with genetic profiling screening in cancer risk assessment. To validate and broaden the clinical relevance of IL-12A genotypes as predictive biomarkers, further investigations with larger cohorts and ethnically diverse populations are in urgent need.
Acknowledgements
The Authors are grateful to Yu-Hsin Yen and Yu-Cheng Lou for their excellent technical assistance. This study was supported by Taichung Veterans General Hospital (TCVGH-1134905B). The funders had not involved in the study design, data collection, analysis, or annotation of the manuscript.
Footnotes
Authors’ Contributions
Research design: Chen KY, Bau DT, and Tsai CW; patient and questionnaire summaries: Hsu SW, Chen KY, Hsu CL, and Liu YF; experimental work: Hsu SW, Wang YC, Shih HY, Chang WS, and Tsai CW; data clearance and identification: Chen KY, Hsu SW, and Hsu CL; statistical analysis: Bau DT, Hsu SW, and Chang WS; literature review and manuscript writing: Chen KY, Hsu SW, Tsai CW and Bau DT; review and revision: Tsai CW and Bau DT.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
Artificial Intelligence (AI) Disclosure
The Authors announce that no AI tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received April 18, 2025.
- Revision received April 25, 2025.
- Accepted April 28, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
