Polymorphisms in Th1/Th2 Cytokine Genes, Hormone Replacement Therapy, and Risk of Non-Hodgkin Lymphoma

We conducted a population-based case–control study in Connecticut women to test the hypothesis that genetic variations in Th1 and Th2 cytokine genes modify the relationship between hormone replacement therapy (HRT) and risk of non-Hodgkin lymphoma (NHL). Compared to women without a history of HRT use, women with a history of HRT use had a significantly decreased risk of NHL if they carried IFNGR2 (rs1059293) CT/TT genotypes (OR = 0.5, 95%CI: 0.3–0.9), IL13 (rs20541) GG genotype (OR = 0.6, 95%CI: 0.4–0.9), and IL13 (rs1295686) CC genotype (OR = 0.6, 95%CI: 0.4–0.8), but not among women who carried IFNGR2 CC, IL13 AG/AA, and IL13CT/TT genotypes. A similar pattern was also observed for B-cell lymphoma but not for T-cell lymphoma. A statistically significant interaction was observed for IFNGR2 (rs1059293 Pfor interaction = 0.024), IL13(rs20541 Pfor interaction = 0.005), IL13 (rs1295686 Pfor interaction = 0.008), and IL15RA (rs2296135 Pfor interaction = 0.049) for NHL overall; IL13 (rs20541 Pfor interaction = 0.0009), IL13(rs1295686 Pfor interaction = 0.0002), and IL15RA (rs2296135 Pfor interaction = 0.041) for B-cell lymphoma. The results suggest that common genetic variation in Th1/Th2 pathway genes may modify the association between HRT and NHL risk.

and interleukin (IL)-2] produced by Th1 cells drive cellular immunity to fight intracellular pathogens including viruses, and remove cancerous cells, while Th2 cytokines (i.e., IL-4, IL-5, IL-9, IL-10, and IL-13) secreted by Th2 cells control humoral immunity by upregulating antibody production to protect against extracellular pathogens (Mosmann et al., 1986;Romagnani, 1991;Bouman et al., 2005;Lehrnbecher et al., 2005;Croxford and Buch, 2011). Immune dysfunction resulting from imbalanced regulation and expression of Th1 and Th2 cytokines play an important role in the development of NHL (Mori et al., 2001;Chiu and Weisenburger, 2003). Single nucleotide polymorphisms (SNPs) in several Th1/ Th2 cytokine genes (i.e., IL4, IL5, IL6, IL10, IFNGR2, IL12A, IL13, IL7R, and TNF) have been reported to be associated with the risk of NHL and its major subtypes (Lan et al., 2006;Chen et al., 2011). It is possible that genetic variation in the Th1/Th2 cytokine genes may modify the relationship between HRT and NHL risk. As such, we analyzed data from a population-based case-control study in Connecticut women to test the hypothesis.

Materials and Methods study population
The study population has been described in detail in other studies by our group (Zhang et al., 2004a;Chen et al., 2011). Briefly, all histologically confirmed incident cases of NHL (ICD-O, introduction Female sex hormones play an important role in modulation of immune system function and autoimmune disease activities (Olsen and Kovacs, 1996;Medina et al., 2000). Non-Hodgkin lymphoma (NHL) is a tumor originating in the immune system (Hoover, 1992) and autoimmune disease is one of the few established risk factors. Epidemiological studies, however, provided inconsistent results linking hormone replacement therapy (HRT) use and risk of NHL with some studies (Cerhan et al., 1997;Nelson et al., 2001;Glaser et al., 2003;Zhang et al., 2004b) suggesting a decreased risk and others (Bernstein and Ross, 1992;Cerhan et al., 2002) suggesting an increased risk. While the mechanisms underlying the association between HRT and NHL remain unclear, it has been suggested that estrogen may act as a systemic anti-inflammatory treatment to lower the production of, or response to, pro-inflammatory cytokines (Saucedo et al., 2002). These cytokines can modulate lymphoid development and immune function (Hofmann et al., 2002;Keen, 2002;Gergely et al., 2004). Therefore, some of the inconsistent findings linking HRT and NHL risk may be explained by genetic variation in cytokine genes.
T-helper cells are vital to human immune responses. The T-helper cell response is defined by two distinct pathways involving two different subtypes of T-helper cells, T-helper 1 (Th1) cells, and T-helper 2 (Th2) cells. Th1 cytokines [i.e., interferon-γ (IFN-γ) sample. If there was suggestive evidence, or if we had a priori knowledge that a given SNP was associated with risk of NHL, genotype analysis would include subjects who provided only buccal cell samples.
Duplicate samples from 100 study subjects to 40 replicate samples from each of two blood donors were interspersed throughout the plates used for genotype analysis. The concordance rates for quality control (QC) samples were between 99 and 100% for all assays. The genotype frequencies for three SNPs (rs231775, rs2243250, and rs2070874) were not consistent with Hardy-Weinberg equilibrium (HWE) among non-Hispanic white controls using a chi-square test (p < 0.05) and were excluded from the final analysis. To increase the statistical power for the gene-environmental interaction analysis, another five SNPs (rs2069822, rs2069818, rs2069807, 3024509, and rs361525) with minor allele frequency less than 10% were also excluded from the final analysis. A total of 31 SNPs in 17 Th1/Th2 genes: IL10 (rs1800871, rs1800872, rs1800896, rs3024496, rs3024491, rs1800890), and TNF (rs1800629, rs1799724) were included in the final analysis.

statistical analysis
Unconditional logistic regression models were used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for associations between HRT, and risk of NHL and its subtypes in different genotype strata adjusting for age, menopausal status, and family history of hematopoietic cancers in first degree relatives. We conducted analyses by separate heterozygous and homozygous variant genotypes and found that the risks were similar between heterozygous and homozygous variant genotypes. Since the numbers for homozygous variant genotypes in several genes were very small, the risk estimates were unstable. As such, heterozygous and homozygous variant genotypes were combined for all genes to increase the statistical power. Adjustments for other variables, such as race, education, tobacco use, or alcohol consumption, did not result in material change of the observed associations, and thus were not included in the final models reported here. Significance of gene-HRT interaction was assessed by adding an interaction term in the logistic regression models. The false discovery rate (FDR) method set at 0.2 was used to control for multiple comparisons (Benjamini and Hochberg, 1995). All p values presented are two-sided and all analyses were performed using SAS Software, version 9.2 (SAS Institute, Cary, NC, USA).

results
The distributions of selected characteristics of study population are presented in Table 1. Compared to controls, cases were more likely to have family history of hematopoietic cancers (p = 0.02). The proportion of postmenopausal women was greater in cases than in controls (p = 0.01). The distributions of age, race and HRT between cases and controls were not significantly different. M-9590-9642, 9690-9701, 9740-9750) diagnosed between 1996 and 2000 in Connecticut were identified through the Yale Cancer Center's Rapid Case Ascertainment Shared Resource (RCA). Enrollment criteria included age between 21 and 84 years, residence in Connecticut, female, alive at the time of interview, and without a previous diagnosis of cancer except for non-melanoma skin cancer. Of 832 eligible cases, 601 (72%) completed in-person interviews, and 231 (28%) refused to participate in the study. Pathology slides (or tissue blocks) from all patients were obtained from the original pathology departments and reviewed by two independent pathologists. All cases were classified according to the 2001 WHO classification (Alsheikh et al., 2001).
Female population-based controls from Connecticut were recruited by: (1) random-digit dialing methods for those younger than 65 years of age; or (2) random selection from the Centers for Medicare and Medicaid Services records for those aged 65 years or older. Controls were frequency matched on age (±5 years) to cases. The participation rate was 69% among persons identified via the random-digit dialing and 47% among persons identified from the Centers for Medicare and Medicaid Services. Approximately 75% of the study subjects (76.7% of the cases and 74.6% of the controls) provided blood samples, and approximately 10% of the subjects (11.0% of the cases and 10.4% of the controls) provided buccal cell samples for genotyping.

data collection
The study was approved by the institutional review boards at Yale University, the Connecticut Department of Public Health, and the National Cancer Institute. Participation was voluntary and written informed consent was obtained from all participants. Those who signed consent forms were interviewed by trained study nurses at the subject's home or at a convenient location using a standardized and structured questionnaire. Information on anthropometrics, demographics, family history of cancer, smoking, and alcohol consumption, occupational exposure, medical conditions and medication use, and diet were collected through in-person interview. An open-ended question was used to ask whether the subject had taken any medicine at least once a day for a period of 6 months or longer previous to 1 year ago, which included HRT. If yes, the age at first and last use, and the total months of use of the medicine were also ascertained (Zhang et al., 2004b).

GenotypinG
Genotyping was performed at the National Cancer Institute Core Genotyping Facility 1 . All TaqMan assays (Applied Biosystems, Foster City, CA, USA) for this study were optimized on the ABI 7900HT detection system with 100% concordance with sequence analysis of 102 individuals as listed on the SNP500 Cancer website 2 . A total of 39 SNPs in 20 Th1/Th2 immune genes were selected for genotyping based on the following criteria: minor allele frequencies more than 5%, laboratory evidence of function, or prior association with human disease studies (Lan et al., 2006). Due to a limited amount of DNA available for subjects who provided only buccal cells, we first genotyped subjects who provided a blood Zhu et al.

discussion
To our knowledge, this is the first comprehensive analysis of interaction between HRT, genetic polymorphisms in Th1/Th2 pathway genes, and the risk of NHL and its subtypes. Significant interactions were observed for IFNGR2 (rs1059293), IL13 (rs20541, rs1295686), and IL15RA (rs2296135) for NHL overall and/or B-cell NHL subtypes.
The study suggested that IL13 polymorphisms modify the association between HRT use and risk of B-cell lymphoma, particularly for follicular lymphoma. The IL13 gene encodes the IL-13 cytokine which exerts anti-apoptotic functions and is linked to leukemogenesis (Waldele et al., 2004). In vitro study also suggested that IL-13 was a weak inducer and an amplifier of IL6 expression in vascular endothelial cells (Sironi et al., 1994). Estrogen has been shown to downregulate IL6 gene expression by endocrinological feedback mechanisms (Dijsselbloem et al., 2004). Studies have shown that higher serum levels of IL-6 were associated with an increased risk of B-cell lymphoma (Preti et al., 1997). It is biologically plausible that IL-6 expression may play an important role in our observed interaction between IL13 polymorphisms and HRT on the risk of B-cell lymphoma. Although it is currently unclear whether the two IL13 polymorphisms (rs20541 and rs1295686) causes over expression or enhanced function of IL-13, rs20541 has been linked to the risk of NHL (Wang et al., 2009). Zhu et al.
HRT, cytokine gene, and NHL  HRT and NHL. As such, the observed interaction between genetic variation of IL15RA and HRT on the risk of NHL could be due to the change of IL-2 cytokine network. Several strengths are included in our study. First, it is a population-based case-control study with histologically confirmed incident NHL cases which minimized potential disease misclassification. Second, this study used a rapid case identification system to identify all eligible NHL cases eliminated survival bias given the aggressive nature of NHL. Eligible cases were identified within 1 month after their diagnosis through the RCA. And finally, this study, for the first time, reported the effect modification of Th1/ Th2 genes on the association between HRT and NHL.
While our study included more than 1,000 study subjects, the statistical power is limited when investigating the relationship by NHL subtypes. Given the number of SNPs investigated in the study, chance cannot be ruled out for some of the significant findings. However, several significant findings remained after adjusted for multiple comparisons using the FDR approach.
In summary, our study provided the first suggestive evidence that common genetic variations in the Th1/Th2 pathway genes may modify the association between HRT and risk of NHL. The observed results could not only advance our understanding of the relationship between HRT use and risk of NHL but also have a potential impact on future clinical practices using HRT. Further larger population-based studies or pooled analyses with greater power are needed to replicate the results.

acknowledGMents
This study was supported by the NIH grant CA62006 and CA105666, the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, and the National Institutes of Health training grants HD70324-01, 1D43TW008323-01, and 1D43TW007864-01.
Our study also suggested IFNGR2 polymorphism (rs1059293) modified the association between HRT and NHL. The gene IFNGR2 encodes the non-ligand-binding beta chain of the IFN-γ located on chromosome 21 (Mogensen et al., 1999). Initiation of the IFN-γ signal transduction cascade, serves to directly inhibit viral replication and serves to stimulate and modulate the immune system. A recent study suggested that HRT could improve postmenopausal women's immune system by inducing a significant decrease in the production of IL-10 and IFN-γ (Deguchi et al., 2001). Effect modification was observed for NHL suggesting the IFN-γ transduction pathway could play a role in the relationship between HRT and risk of NHL. Further knowledge of the functional impact of IFNGR2 polymorphism (rs1059293) on IFNGR2 gene is needed to help elucidate its role between HRT and NHL.
Potential effect modification by IL15RA (rs2296135) polymorphism was observed. The IL15RA gene encodes the alpha chain of the IL-15 receptor which is expressed in a variety of immune and non-immune cell types from different tissues and generates multiple splicing events of functional importance (Bouchaud et al., 2008;Diniz et al., 2010). IL-15 and IL-2 receptors share the beta and gamma(c) subunits with private alpha chains, which presumably ensure the binding of the appropriate cytokine and the specificity of the immune response (Vamosi et al., 2004). IL-15 and IL-2 can activate similar janus kinase/signal transducer and activator of transcription (JAK/STAT)-dependent signaling pathway at the presence of both beta and gamma(c) subunits, suggesting a significant overlap between the functions of IL-2 and IL-5 (Lin et al., 1995). Recent study demonstrated that both IL-2 and IL-5 alpha subunits co-expressed in a supramolecular receptor cluster in lipid rafts of the T cells (Vamosi et al., 2004). HRT has been found to reduce IL-2 production (Stopinska-Gluszak et al., 2006) suggesting that IL-2 cytokine network plays a role in the association between Zhu et al. HRT, cytokine gene, and NHL (Continued) appendix