Abstract
Background/Aim: Oral cancer incidence is highest worldwide in Taiwan, and practical markers for personalized therapeutic strategies such as immunotherapies, is lacking. Interleukin-12 (IL12) is a cytokine that is reported to exhibit potent tumoricidal effects, however, its genotypic contribution to oral cancer is still largely unknown. We aimed to examine whether IL12A rs568408 and rs2243115 genotypes are associated with oral cancer risk in Taiwan. Materials and Methods: Genotypic characteristics of IL12A were determined among 958 oral cancer cases and age- and gender-matched individuals via typical polymerase chain reaction–restriction fragment length polymorphism methodology. Results: The variant genotypes of IL12A rs568408 and rs2243115 were not found to be significantly associated with elevated oral cancer risk (all p>0.05). Moreover, there was no interaction between IL12A genotypes and personal smoking, alcohol drinking and betel quid chewing behaviors (all p>0.05). Conclusion: IL12A rs568408 and rs2243115 genotypes may not serve as good predictors for oral cancer risk.
According to the 2017 Statistics of Causes of Death published by the Ministry of Health and Welfare, oral cancer is the fourth most common cancer in males and the fourth leading cause of cancer death in males in Taiwan, where the incidence of oral cancer is the highest in the world (1-3). Epidemiological studies have shown that the development and susceptibility to oral cancer for Taiwanese is determined by subtle genomic variations [such as single nucleotide polymorphisms (SNPs)] and several lifestyle factors (such as alcohol/tobacco/betel quid consumption, poor oral hygiene habits) and viral infectious status (4-7). Thus, a better target or marker for advanced personalized therapeutic approaches such as immunotherapies are an urgent need. To fulfill this aim, mounting studies have provided evidence for that specific genotypes are associated with increased oral cancer risk in cigarette smokers (8, 9), and betel quid chewers (10, 11) using genotyping. These findings are extremely valuable in elucidating the contribution of genomic, environmental and personal behavioral aspects to oral cancer development and providing a better consulting etiology for therapeutic decision making and genomic pharmacology for each patient with oral cancer.
Interleukin 12 (IL12) is a multi-functional cytokine originally identified as stimulatory factor for natural killer cells and maturation factor for lymphocytes (12, 13). IL12 has been found to stimulate the proliferation and cytolytic capacity of natural killer cells, and activate their capacity for cytokine production, particularly interferon-γ (IFNγ) (14, 15). Furthermore, IL12 has been reported to serve in bridging innate and adaptive immunity by promoting the differentiation of T-helper 1 (Th1) cells (16, 17). In animal model experiments, IL12 has been found to have antitumor capacity since mice lacking IL12 subunit p35 had earlier and more papilloma development compared with wild-type mice (18). Moreover, mice deficient in IL12 receptor chain (IL12Rβ2) had a faster growth of B16 melanomas than did wild-type ones (19). Lastly, exogenous administration of IL12 significantly suppressed the growth of sarcoma, melanoma, lung carcinoma and breast carcinoma in transplanted mouse models (20-22). Most interesting, although IL12 has certain side-effects, its curative effect is impressive and clinically significant. For instance, phase I/II trials of IL12 in patients with B-cell lymphoma or Kaposi sarcoma were both very successful in 2002 and 2007 (23, 24). IL12 treatment can have synergistic effects with IL18 in restoring the intratumoral natural killer cell functions in major histocompatibility complex class I-deficient tumors (23, 24). Combined with natural killer cell-secreted IFNγ, IL12 can inhibit tumor angiogenesis and suppress tumor growth (25).
Human IL12A and IL12B genes are located at chromosomes 3 and 5, respectively. Among the SNPs of IL12A, rs568408 is the polymorphic site most frequently examined. IL12A rs568408 is located in the 3’-untranslated region (UTR) and may influence the level of IL12 production (26, 27). Another polymorphic site, IL12A rs2243115, is situated in the 5’UTR, and its functional significance has not been well examined or conclusively reported. In the current study, we aimed to investigate the contribution of IL12A genotypes to the risk of oral cancer and to examine the joint effect of personal behavioral habits and IL12A genotypes on oral cancer risk in Taiwan.
Materials and Methods
Population sampling methodology. Nine hundred and fifty-eight patients diagnosed with oral cancer were recruited with help from the cancer team at the Outpatient Clinics of General Surgery, collecting the clinical characteristics of patients, including histological details, which were all graded and defined by experts in pathology. Patients with history of any other cancer were excluded from the databank, and all participants were Taiwanese and voluntarily completed a self-administered questionnaire and provided their blood sample for genotyping studies. Some number of healthy volunteers as controls were selected by matching for age, gender, and behavioral factors alcohol, tobacco and areca-nut consumption after initial random sampling from the Health Examination Cohort of the China Medical University Hospital. The exclusion criteria of the controls included previous malignancy, metastasized cancer from other or unknown origin, and any genetic or familial disease. The study was approved by the Institutional Review Board of the China Medical University Hospital (DMR101-IRB1-306) and written informed consent was obtained from all participants. Several selected characteristics of all the investigated participants are summarized in Table I.
IL12A polymerase chain reaction (PCR)-restriction fragment length polymorphism genotyping conditions. Genomic DNA from the peripheral blood leucocytes of each patient with oral cancer and control was prepared using the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC) (28, 29) and further processed in typical PCR processes as previously published (30, 31). For IL12A rs2243115 and rs568408, the forward primers introduce a mismatched A to replace C and a mismatched T to replace C, respectively, at -3 bp from the polymorphic sites to create BstNI- and NdeI- (New England BioLabs, Ipswich, MA, USA) digestible restriction sites, respectively. For IL12A rs568408, the primers were 5’-AGAAAAGACCTGTGAACAAAACGACT-3’ (forward) and 5’-AGATGGCTCACTAGATGCCAGG-3’ (reverse). For IL12A rs2243115, the primers were 5’-GAAGGATGGGACYATTACATCCATAT-3’ (forward) and 5’-CAGGATGGATATTTTCCCTTCT-3’ (reverse). The wild-type allele IL12A rs2243115T generated a full fragment of 122 bp, while the variant allele IL12A rs2243115G produced two fragments of 93 and 29 bp after PCR. For IL12A rs568408, the wild-type allele IL12A rs568408G produced two fragments of 98 and 23 bp and the variant allele IL12A rs568408A resulted in a full fragment of 121 bp.
The PCR cycling was set as: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. Agarose gel used was 3% and the electrophoresis conditions were 100 V for exactly 20 min. The genotype analysis was performed by three researchers independently and blindly. For each IL12A SNP, 5% of the PCR products were randomly selected for direct sequencing and the results from PCR-restriction fragment length polymorphism and direct sequencing were 100% concordant to each other.
Statistical analyses. Student's t-test was used to compare the difference of age between case and control groups. Pearson's chi-square test was used to compare the distribution of the IL12A genotypes between the two groups. The associations between the IL12A genotypes and cancer risk were estimated by computing odds ratios (ORs) and corresponding 95% confidence intervals (CIs) from logistic regression analysis. Any comparison result with p<0.05 was recognized as statistically significant.
Results
The basic and clinical data including age, gender, personal habits for the investigated 958 patients with oral cancer and the 958 matched controls are summarized and compared in Table I. In addition, the tumor sites of all the patients are also presented in Table I. Firstly, there was no difference between the case and control groups in the aspects of age and gender (p=0.3755 and >0.99, respectively) (Table I). Secondly, the proportions of smokers, alcohol drinkers and betel quid chewers were found to be higher in the oral cancer group than those in the age- and gender-matched control group (p=0.0107, 0.0377 and 0.0001, respectively), which indicates that these behavioral habits are associated with oral cancer for Taiwanese (Table I). Lastly, the most prevalent tumor sites of oral cancer were the tongue (41.4%) and buccal mucosa (37.2%) among this Taiwan oral cancer population (Table I).
The distribution of genotypic frequencies of IL12A rs568408 and rs2243115 among the controls and cases are summarized in Table II. Firstly, the genotypic frequencies for the two SNPs investigated fit well with the Hardy–Weinberg equilibrium for the control group (p>0.05). Secondly, there was no significant difference in the distribution of IL12A rs568408 genotypes between oral cancer and control groups (p for trend=0.4570). The frequencies of the heterozygous variant AG and homozygous variant GG of IL12A rs568408 were 24.1% and 4.0% in the oral cancer group and not significantly different from those in the control group (26.1% and 3.2%, respectively). Thus, neither the AA (OR=1.21, 95% CI=0.74-1.96, p=0.4526) nor AG (OR=0.91, 95% CI=0.74-1.12, p=0.3624) genotype at IL12A rs568408 can serve as a potential biomarker for oral cancer in Taiwanese. Moreover, considering the mere presence of the A allele by combining variant AG with AA at IL12A rs568408 also did not alter the risk for oral cancer risk among Taiwanese compared with the wild-type GG genotype (OR=0.94, 95% CI=0.77-1.15, p=0.5445; Table II). Similarly, there was no significant difference in the distribution of IL12A rs2243115 genotypes between oral cancer and control groups (p for trend=0.5756). The frequencies of the heterozygous variant GT and homozygous variant GG of IL12A rs2243115 were 15.7% and 1.5% for the oral cancer group, non-significantly different from those of the control group (14.4% and 1.2%, respectively). Thus, neither the GG (OR=1.30, 95% CI=0.59-2.88, p=0.5190) nor GT (OR=1.12, 95% CI=0.87-1.43, p=0.3897) genotype at IL12A rs2243115 can serve as a potential biomarker for Taiwan oral cancer. The presence of a G allele at IL12A rs2243115 also did not alter the risk for oral cancer risk among Taiwanese compared with the wild-type TT genotype (OR=1.13, 95% CI=0.89-1.44, p=0.3234) (Table II).
Characteristics of the investigated 958 patients with oral cancer and 958 healthy controls.
Distribution of interleukin-12A (IL12A) rs568408 and rs2243115 genotype among 958 patients with oral cancer and 958 controls.
The distribution of allelic frequencies of IL12A rs568408 and rs2243115 are summarized in Table III. Consistent with the findings in Table II, the presence of allele A at IL12A rs568408 was not associated with an altered risk of oral cancer compared with allele G (p=0.8263). Similarly, the variant G allele of IL12A rs2243115 was not associated with any significantly altered risk for oral cancer (p=0.2798; Table III).
Since betel quid chewing behavior and those of cigarette smoking and alcohol drinking may contribute to the etiology of oral cancer, we were also interested in the interaction of the genotype of IL12A with these personal behaviors on the determining oral cancer susceptibility. The joint effects of IL12A rs568408 with betel quid chewing status are presented in Table IV. In the Table, patients with oral cancer and matched controls were stratified according to their betel quid chewing status and ORs were analyzed. The results show that non-betel quid chewers carrying the variant genotypes at IL12A rs568408 did not have altered risk of oral cancer (p=0.8930). Similarly, there was no significantly elevated oral cancer risk for ever chewers with variant genotypes at IL12A rs568408 (p=0.5802). There were also no significant findings for variants of IL12A rs2243115 (Table V). Furthermore, we found no significant interaction between these IL12A genotypes and cigarette smoking status nor alcohol drinking status for oral cancer risk (data not shown).
Discussion
In the current study, the contributions of IL12A rs568408 and rs2243115 genotypes to oral cancer susceptibility were investigated for the first time in a Taiwanese population. From the genotyping results, neither IL12A rs568408 nor rs2243115 were found to be suitable genomic biomarkers for detection and prediction of oral cancer risk among Taiwanese (Table II). The findings were further validated by the allelic frequency analysis revealing neither of the variant alleles at IL12A rs568408 and rs2243115 were associated with altered oral cancer risk (Table III). These findings support the idea that any involvement of IL12 gene and its protein in the carcinogenesis of oral cancer may simply not be critical at IL12A rs568408 and rs2243115.
Distributions of interleukin-12A (IL12A) rs568408 and rs2243115 allelic frequencies among the 958 patients with oral cancer and the 958 controls.
The genotypes of pro-inflammatory cytokine gene IL12A are reported to be associated with many human diseases. For instance, The AA genotype of IL12A rs568408 is associated with higher risk of Graves' disease in Chinese populations (32). The variant AA genotype at IL12A rs568408 is also associated with higher risk of asthma in Taiwan (33). On the other hand, Sima and colleagues found that variant G allele carriers of IL12A rs2243115 were at higher risk of brain tumors compared with those carrying the wild-type T allele (34). Almost at the same time, Wang and colleagues provided evidence that the variant G allele at IL12A rs2243115 also contributes to higher risk of chronic obstructive pulmonary disease (35). To our knowledge, there is no other report examining the contribution of IL12A genotypes to oral cancer. Interestingly, a higher A allele frequency was found among patients with oral lichen planus than controls in an East Chinese cohort study (36). Although it is reported by a meta-analysis that the genotypes of IL12A rs568408 were found to significantly associated with overall cancer risk, especially among Asian ethnicities (37), in the current study, we did not find any association between IL12A rs568408 or rs2243115 genotypes with oral cancer risk in Taiwanese.
We have to take into consideration that the phenotype of IL12 is determined not only by the genotype of IL12A but also by that of IL12B. Frequently, the genotype of IL12B seems to play a more role critical in determining the serum level of IL12. For instance, the serum levels of IL12 were found to be much higher in patients with type 1 diabetes with IL12B rs3212227 AA genotype than those with AC or CC genotypes (38, 39). On the contrary, the levels of IL12 were reported to be higher in peripheral blood mononuclear cells from individuals with CC genotype at IL12B rs3212227 than those with AC or AA genotypes, upon stimulation with lipopolysaccharide (40). Furthermore, IL12A and IL12B may control the secretion of each other. It has been shown that the presence of the variant genotype IL12B rs3212227 correlated with increased secretion of IL12A but not of IL12B itself (39). In addition, the combinatory effects of IL12A and IL12B need to be examined. In 2019, although the we found the genotypes of IL12B were not associated with altered oral cancer risk, the IL12B rs3212227 genotype was significantly associated with oral cancer risk specifically for betel quid chewers but not non-chewers (40). There is no synergistic effect for either IL12A rs568408 or rs2243115 with IL12B rs3212227 on determination of oral cancer risk (data not shown).
Distribution of interleukin-12A (IL12A) rs568408 genotypes among the 958 patients with oral cancer and the 958 controls after stratification by betel quid chewing behavior.
Distribution of interleukin-12A (IL12A) rs2243115 genotypes among the 958 oral cancer patients and the 958 controls after stratification by betel quid chewing behavior.
In this study, the interaction analysis of the genotype of IL12A at rs568408 and rs2243115 and the major personal behavioral risk factors for Taiwan oral cancer were evaluated. The results showed that those people with variant allele at IL12A rs568408 (Table IV) or rs2243115 (Table V) with or without betel quid chewing habit had similar susceptibility to oral cancer. No genotype–habit interaction was found among cigarette smokers or alcohol consumers either (data not shown). In the present research, we did not find any genotype–phenotype correlation among the investigated Taiwanese individuals. Further studies using primarily cultured cells from tumor sites of patients with oral cancer are strongly recommended to explore the effects of betel quid compounds on primarily cultured cells with different IL12A rs568408, IL12A rs2243115 and IL12B rs3212227 genotypes in order to validate the genotype–phenotype correlation of these SNPs.
In conclusion, the study indicated that genotypes of IL12A rs568408 and rs2243115 are not determiners of oral cancer risk among Taiwanese. The findings should be validated in more populations in similar or different ethics.
Acknowledgements
The Authors are grateful to the Tissue Bank of China Medical University Hospital and doctors/nurses for their excellent sample collection and technical assistance. The technical assistance from Yu-Chen Hsiau, Yi-Ru Huang, Yu-Hsin Lin, Tzu-Yu Wang, Tzu-Hsuan Wang and the consultants of statistical analysis from Cheng-Li Lin were very helpful in article preparation. This study was supported mainly by China Medical University Hospital (CMU108-ASIA-01) and partially by Taichung Veterans General Hospital (TCVGH-1094904C).
Footnotes
Authors' Contributions
Research design was performed by LCH, SLC. Patient and questionnaire summaries were provided by SLC and HCL. Experimental work was carried out by WYC and CWS. Statistical analysis was performed by LCY and LHT. LCY, TCW, and BDT wrote the article; BDT, CWS, and TCW reviewed it and are responsible for the revision.
Conflicts of Interest
The Authors declare no conflicts of interest with any company or person.
- Received June 2, 2020.
- Revision received June 18, 2020.
- Accepted June 22, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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