Abstract
Background/Aim: Breast cancer (BC) remains the leading cause of cancer-related mortality among women worldwide. Interleukin-12 (IL-12), a cytokine pivotal in immune regulation, has shown antitumor properties and may contribute to BC pathogenesis.
Materials and Methods: This study investigated the association between IL-12B rs3212227 polymorphism and BC risk in a Taiwanese population comprising 1,232 BC patients and 1,232 age-matched cancer-free controls.
Results: Genotype frequencies among controls conformed to Hardy-Weinberg equilibrium (p=0.0581). Compared to the AA genotype, carriers of the AC (OR=0.88, 95%CI=0.73-1.06, p=0.2105) and CC (OR=0.84, 95%CI=0.68-1.04, p=0.1307) genotypes exhibited a non-significant reduction in BC susceptibility. Similarly, neither dominant (AC+CC versus AA: OR=0.87, 95%CI=0.73-1.03, p=0.1225) nor recessive (CC versus AA+AC: OR=0.90, 95%CI=0.75-1.09, p=0.3198) models reached statistical significance. Allelic analysis showed a marginally reduced risk associated with the C allele (OR=0.91, 95%CI=0.81-1.02, p=0.1038). Notably, age-stratified analysis revealed significant protective effects of the AC (OR=0.79, 95%CI=0.64-0.98, p=0.0331) and CC (OR=0.68, 95%CI=0.52-0.87, p=0.0034) genotypes in individuals aged ≤55 years, but not in older subjects. No significant associations were found between IL-12B rs3212227 and risk of triple-negative BC (TNBC) or non-TNBC subtypes (p for trend=0.2072 and 0.8291, respectively).
Conclusion: IL-12B rs3212227 is not a major genetic determinant of overall BC susceptibility in this population but may exert age-dependent protective effects. Further studies integrating IL-12 expression profiling and additional IL-12 pathway variants are warranted to clarify the immunogenetic basis of BC.
Introduction
Breast cancer (BC) ranks as the most frequently diagnosed malignancy among women in 157 of 185 countries, accounting for approximately 670,000 deaths worldwide in 2022 (1, 2). As the leading cause of cancer-related mortality in women, BC significantly impacts both survival outcomes and quality of life (3). The disease is characterized by substantial pathological and molecular heterogeneity, with genetic variability contributing to its diverse clinical manifestations across different populations (4, 5). Among the various BC subtypes, triple-negative breast cancer (TNBC), first delineated in the early 2000s, remains the most therapeutically challenging. This subtype is associated with an aggressive clinical course marked by early recurrence, high metastatic potential, and reduced overall survival (6, 7). The absence of well-established molecular markers in TNBC has hindered early diagnosis and targeted treatment, contributing to poor outcomes. Notably, early-stage TNBC (stage I-III) presents a recurrence rate of up to 50%, and over one-third of affected individuals succumb to the disease within five years of diagnosis (8, 9). In response to these challenges, recent efforts in translational research have increasingly focused on uncovering novel biomarkers for BC, with particular emphasis on TNBC. These investigations aim to improve prognostic accuracy and facilitate the development of personalized therapeutic strategies (10-12).
The multifaceted involvement of the immune system in BC pathogenesis and progression has become increasingly evident in recent years (13, 14). Consequently, immunologically based approaches for the diagnosis and treatment of BC have garnered growing scientific interest. Interleukin-12 (IL-12), a heterodimeric cytokine composed of p35 and p40 subunits, serves as a pivotal mediator in immune regulation (15). It is particularly influential in orchestrating adaptive immune responses (16). Preclinical studies using murine models have consistently demonstrated the antitumor potential of IL-12. Its administration inhibits tumor proliferation, invasion, and metastatic spread across various cancer types, including breast malignancies (17-21). Moreover, IL-12 has demonstrated efficacy against a wide range of tumors, including sarcoma, melanoma, lung carcinoma, and most notably, BC (22-26). Importantly, the therapeutic potency of IL-12 can be augmented when used in combination with other immunomodulators. Synergistic effects have been observed in co-administration with agents such as oxaliplatin (27, 28), IL-18 (29, 30) and interferon-γ (31). Notably, elevated IL-12 expression has been detected in breast tumor tissues compared to adjacent normal controls, suggesting its relevance in disease biology (32). Taken together, these findings support IL-12 as a compelling biomarker candidate for BC prediction and a potential target for immunotherapeutic intervention.
The human IL-12A and IL-12B genes are located on chromosomes 3 and 5, respectively. A number of molecular epidemiological studies have investigated the association between polymorphisms in IL-12-related genes and the risk of various malignancies, including brain tumors (33), oral squamous cell carcinoma (34), nasopharyngeal carcinoma (35), esophageal cancer (36), lung cancer (37), hepatocellular carcinoma (38-40), gastric cancer (41, 42), colorectal carcinoma (43, 44), osteosarcoma (45), and cervical cancer (46-48). These collective findings imply that IL-12 gene variants may contribute to individual susceptibility to cancer, including BC. Specifically, prior research has examined the relationship between IL-12A polymorphisms, particularly rs568408 and rs2243115, and BC risk (49). However, no statistically significant association was identified between these IL-12A genotypes and increased BC risk in the studied populations (49). Although these results suggest that IL-12A variants at rs568408 and rs2243115 may have limited predictive value in BC risk stratification, the potential role of IL-12B polymorphisms in BC remains less clearly defined. To address this knowledge gap, our study focused on evaluating the impact of IL-12B rs3212227 genotypes on BC susceptibility in a Taiwanese population. The analysis included a case-control cohort comprising 1,232 patients with BC and an equal number of age-matched non-cancerous controls in Taiwan. The chromosomal localization of IL-12B rs3212227 is illustrated in Figure 1. In addition, we aimed to explore whether this genetic variant might serve as a potential predictor of risk specifically for TNBC within the Taiwan population.
Physical map of the genomic region surrounding the IL-12B rs3212227 polymorphic site.
Materials and Methods
Study population. This study included 1,232 female patients diagnosed with BC, all of whom were recruited from the outpatient clinics of the Department of General Surgery at China Medical University Hospital, Taiwan. All participants were of Taiwanese ethnicity. Detailed inclusion and exclusion criteria have been described previously (50, 51). Clinical and pathological data were collected and validated by surgical specialists. Histopathological classifications and biomarker assessments were independently reviewed by at least two board-certified pathologists. Immunohistochemical analyses were used to determine the status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2/neu). Tumors were classified as receptor-positive when ≥10% of tumor nuclei showed specific immunostaining. A Ki67 proliferation index greater than 30% was considered indicative of high proliferative activity. HER-2/neu evaluation followed the diagnostic criteria established by the American Society of Clinical Oncology and the College of American Pathologists (52). All BC participants provided written informed consent, completed a structured self-reported questionnaire, and donated peripheral blood samples for genetic analysis. The control group consisted of 1,232 age-matched healthy female volunteers, randomly selected from the hospital’s Health Examination Cohort. Individuals with a prior history of cancer, metastasis from non-breast origins, or any hereditary disorders were excluded from the control population. Both cases and controls completed a lifestyle and medical history questionnaire. The study protocol was reviewed and approved by the Institutional Review Board of China Medical University Hospital (IRB No. DMR-99-IRB-108). An overview of the demographic and clinical characteristics of the study population is presented in Table I.
Demographics of the 1,232 breast cancer patients and the 1,232 non-cancerous controls.
Genotyping procedure for IL-12B rs3212227. Genomic DNA was isolated from peripheral blood leukocytes of both BC patients and control participants using a commercially available extraction kit (Blossom, Taipei, Taiwan, ROC), in accordance with standard biomedical procedures (53-55). Genotyping of the IL-12B rs3212227 single nucleotide polymorphism (SNP) was carried out using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique, as previously optimized by our laboratory (37). Specifically, the targeted SNP region was amplified using the following primer pair: forward 5′-GATATCTTTGCTGTATTTGTATAGTT-3′ and reverse 5′-AATATTTAAATAGCATGAAGGC-3′, generating a 118-base pair amplicon. The PCR product was then subjected to enzymatic digestion with Taq I (New England BioLabs, Ipswich, MA, USA). The variant allele (C) produced two digested fragments of 92 bp and 26 bp, while the wild-type allele (A) remained intact at 118 bp. To ensure analytical accuracy, all genotyping was independently performed in a double-blinded manner by at least two trained researchers. Each DNA sample underwent repeat genotyping to confirm reproducibility. Remarkably, the genotyping assay achieved a 100% success rate, with complete concordance observed across all samples analyzed.
Statistical analysis. The Hardy-Weinberg equilibrium for the IL-12B rs3212227 polymorphism in the control group was evaluated using a chi-square goodness-of-fit test. Differences in age distribution between BC cases and non-cancerous controls were expressed as mean±standard deviation (SD), and group comparisons were performed using an unpaired Student’s t-test. To assess differences in genotype frequencies, Pearson’s chi-square test with Yates’ continuity correction was applied. The association between IL-12B rs3212227 genotypes and BC risk was estimated using odds ratios (ORs) and 95% confidence intervals (CIs) under various genetic models, including allele, codominant, dominant, and recessive models. A two-tailed p-value of less than 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA).
Results
The distribution of genotypes for the IL-12B rs3212227 polymorphism among the 1,232 cancer-free controls adhered to Hardy-Weinberg equilibrium (p=0.0581), as shown in Table II. When genotype frequencies were compared between BC cases and controls, no statistically meaningful differences were observed (p for trend=0.2498). Specifically, individuals carrying the heterozygous AC or homozygous variant CC genotypes showed a modest, though statistically non-significant, decrease in BC susceptibility relative to those with the reference AA genotype. The calculated odds ratios were 0.88 (95%CI=0.73-1.06, p=0.2105) for the AC genotype and 0.84 (95%CI=0.68-1.04, p=0.1307) for the CC genotype. Under a dominant inheritance model (AC + CC compared to AA), the presence of at least one C allele was associated with a non-significant 10% reduction in BC risk (OR=0.87, 95%CI=0.73-1.03, p=0.1225). Likewise, analysis using a recessive model (CC versus AA + AC) yielded an odds ratio of 0.90 (95%CI=0.75-1.09, p=0.3198), again without statistical significance. Consistent with these genotype-based findings, the allelic analysis revealed that the C allele was marginally associated with a reduced risk of BC, although the association did not reach statistical significance (OR=0.91, 95%CI=0.81-1.02, p=0.1038), as shown in Table III.
IL-12B rs3212227 genotypes among the 1,232 patients with breast cancer and 1,232 non-cancerous controls.
Allelic frequencies for IL-12B rs3212227 among the 1,232 patients with breast cancer and 1,232 non-cancerous controls.
To further explore the potential interaction between IL-12B rs3212227 polymorphisms and age in modulating BC risk, stratified analyses were conducted based on age groups among both cases and controls (Table IV). Among participants older than 55 years, neither the heterozygous AC nor the homozygous CC genotype exhibited a statistically significant association with BC susceptibility. The odds ratios for AC and CC genotypes in this age group were 1.26 (95%CI=0.87-1.84, p=0.2588) and 1.52 (95%CI=0.99-2.33, p=0.0662), respectively (Table IV, right panel). In contrast, for individuals aged 55 years or younger, both variant genotypes demonstrated a statistically significant inverse association with BC risk. Specifically, carriers of the AC genotype had a 21% reduced risk (OR=0.79, 95%CI=0.64-0.98, p=0.0331), while those with the CC genotype experienced a 32% risk reduction (OR=0.68, 95%CI=0.52-0.87, p=0.0034), as presented in the left panel of Table IV. These findings suggest a potential age-dependent protective effect of IL-12B rs3212227 variant alleles against BC development.
IL-12B rs3212227 genotypes in breast cancer risk after stratification by age.
Although IL-12B rs3212227 genotypes did not demonstrate a significant association with overall BC susceptibility (Table II), we further assessed their potential utility as predictive markers for TNBC specifically (Table V). To this end, BC patients were categorized into TNBC and non-TNBC subgroups. The analysis revealed no statistically significant correlation between IL-12B rs3212227 genotypic variants and TNBC risk (p for trend=0.2072), consistent with the findings in the non-TNBC group (p for trend=0.8291). Within both subtypes, neither the heterozygous AC nor the homozygous variant CC genotype exhibited a meaningful association with disease risk, as all corresponding p-values exceeded 0.05 (Table V). These results suggest that IL-12B rs3212227 is unlikely to serve as a reliable genetic biomarker for distinguishing TNBC from other BC subtypes.
Association of IL-12B rs3212227 genotypes with breast cancer risk according to triple negative breast cancer (TNBC), non-TNBC, or non-cancerous controls.
Discussion
The IL-12B gene is situated on the long arm of human chromosome 5 (5q31-33), with the rs3212227 SNP positioned within its 3′-untranslated region. To date, numerous genetic variants have been identified in IL-12B, though rs3212227 has garnered the most attention due to its potential functional significance (56-58). This particular polymorphism has been linked to altered cytokine production and has been implicated in elevating susceptibility to a broad spectrum of malignancies (59, 60). Regarding BC, three studies have examined the relationship between IL-12B rs3212227 and disease risk (61-63). In 2012, Kaarvatn and his colleagues initially observed a statistically significant protective association between the CC genotype and BC risk within a cohort of 382 BC patients and 388 non-cancerous controls from Croatia (61). Conversely, a study from Iran by Jafarzadeh’s team have conducted an investigation among 100 BC patients and 100 non-cancerous individuals in Iran, finding no significant association (62). In addition, they have provided evidence that the serum IL-12 was of the similar level among patients with BC and healthy subjects (62). More recently, Núñez-Marrero and colleagues conducted a haplotype-based investigation in a Puerto Rican population and confirmed the protective role of IL-12B variants. Interestingly, their data also implicated signal transducer and activator of transcription 4 (STAT4) polymorphisms in risk reduction, while variations in interleukin-12 receptor subunit beta-1 (IL-12RB1) were associated with increased susceptibility (63). In contrast, our study in a Taiwanese cohort found no significant association between IL-12B rs3212227 genotypes and BC risk (Table II and Table III). These discrepancies may reflect interethnic genetic diversity. Notably, the minor allele frequency (MAF) of rs3212227 varies considerably across populations, being highest in East Asians (0.4653) and lowest in Europeans (0.2014) (64). Our control group showed a MAF of 0.4821, consistent with East Asian data (Table VI). Although our study benefits from a robust sample size (total n=2,464), further validation in ethnically diverse populations is essential to elucidate the role of rs3212227 in BC susceptibility.
Minor allelic frequencies of IL-12B rs3212227 among different populations.
Understanding the genotype-phenotype relationship is essential for clarifying how genetic variants influence disease biology. However, the impact of the IL-12B rs3212227 polymorphism on circulating IL-12 levels remains largely ambiguous. Prior studies in individuals with type 1 diabetes have reported significantly elevated IL-12 concentrations among those harboring the AA genotype compared to AC or CC carriers, suggesting genotype-dependent regulation of cytokine expression (65, 66). This SNP has also been implicated in modulating IL-12 protein production and linked to susceptibility to T helper 1 (Th1)-mediated conditions, including various cancers and inflammatory disorders (67-71). Interestingly, cross-regulation between IL-12A and IL-12B has been proposed, wherein rs3212227 variant genotypes may enhance IL-12A secretion while exerting minimal influence on IL-12B output (65). Clinically, serum IL-12 levels have been associated with disease severity in gastric cancer (72), and shown to influence the progression of colorectal carcinoma (43). Recent therapeutic investigations further reinforce IL-12’s anti-tumor potential, demonstrating that administration of recombinant IL-12 elevates both IL-12 and interferon-γ levels, reduces primary tumor burden, inhibits metastatic spread, and prolongs survival outcomes (73). In the context of BC, however, findings remain inconclusive. While one study reported no significant differences in serum IL-12 between patients and controls (62), another identified markedly higher IL-12 expression within tumor tissues compared to adjacent non-tumorous regions (32).
Several limitations of the present study warrant consideration. First, the absence of long-term follow-up data restricts our ability to assess the prognostic value of IL-12B rs3212227 genotypes in BC, including their potential influence on survival outcomes, metastatic progression, and recurrence risk. Second, the lack of comprehensive IL-12 expression profiling, at both mRNA and protein levels, limits our capacity to establish robust genotype-phenotype correlations. Third, while our analysis focused on a single polymorphism within the IL-12B gene, the contribution of additional genetic variants in immunologically relevant loci, such as the IL12RB1 and STAT4 genes, in BC risk determination cannot be excluded. In summary, our findings revealed no statistically significant relationship between IL-12B rs3212227 genotypes and overall BC risk, suggesting that this SNP alone may not serve as a reliable genetic biomarker for BC in this population. However, age-stratified analysis indicated a potential protective effect of the AC and CC genotypes in individuals aged 55 years or younger, highlighting the possible influence of age in modulating the genetic effect of IL-12B rs3212227 on BC development. Despite these age-specific findings, no significant associations were observed between IL-12B rs3212227 and risk of TNBC or non-TNBC subtypes, further limiting its utility in subtype-specific risk prediction. Comprehensive studies incorporating IL-12 expression profiling, additional IL-12-related genetic variants, and longitudinal clinical data are warranted to better understand the immunogenetic contributions to BC.
Acknowledgements
The Authors are grateful to the colleagues at Tissue Bank of China Medical University Hospital for their excellent sample collection and technical assistance. The technical assistance from Dr. Liang-Chih Liu, Dr. Hwei-Chung Wang and Ai-Chia Tung were very helpful. This study was supported by China Medical University and Asia University (CMU114-ASIA-02) and Taichung Veterans General Hospital (TCVGH-1141601A).
Footnotes
Authors’ Contributions
Research design: Hung CC, Bau DT and Tsai CW; patient and questionnaire summaries: Liu CH, Hung CC and Su CH; experimental work: Wang YC, Chang WS, CH SU and Shih HY; statistical analysis: Lin ML, Chen SS, and Tsai CW; data clearance and validation: Wang YC, Hsia TC, Tsai CW and Yang YC; article writing: Hung CC, Tsai CW, Bau DT and Wang YC; correction of manuscript: Tsai CW, Wang YC and Liu CH; review and revision: Hung CC, Tsai CW and Bau DT.
Conflicts of Interest
The Authors declare no conflicts of interest with any company or person.
Artificial Intelligence (AI) Disclosure
No artificial intelligence tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received July 30, 2025.
- Revision received August 18, 2025.
- Accepted August 19, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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