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We conducted a population-based case-control study in Connecticut women to test the hypothesis that genetic variations in Th1 and Th2 cytokine genes may modify the association between blood transfusion and risk of non-Hodgkin lymphoma (NHL). Compared with women without blood transfusion, women with a history of transfusion had an increased risk of NHL if they carried IL10RA (rs9610) GG genotype [odds ratio (OR) = 1.9, 95% confidence interval (CI): 1.1–3.2] or TNF (rs1800629) AG/AA genotypes (OR = 1.6, 95% CI: 0.9–2.7). We also found women with a history of transfusion had a decreased risk of NHL if they carried IL10RA (rs9610) AG/AA genotypes (OR = 0.6, 95% CI: 0.4–0.9) or TNF (rs1800629) GG genotype (OR = 0.7, 95% CI: 0.5–1.0). A similar pattern was also observed for B-cell lymphoma but not for T-cell lymphoma. Statistically significant interactions with blood transfusion were observed for IL10RA (rs9610) (Pforinteraction = 0.003) and TNF (rs1800629) (Pforinteraction = 0.012) for NHL overall and IL10RA (rs9610) (Pforinteraction = 0.001) and TNF (rs1800629) (Pforinteraction = 0.019) for B-cell lymphoma. The results suggest that genetic polymorphisms in TNF and IL10RA genes may modify the association between blood transfusion and NHL risk.
Non-Hodgkin lymphoma (NHL) is the fifth most frequently diagnosed cancer in the United States, with 65,540 new cases and 20,210 deaths estimated in 2010 . Various immune dysfunctions resulting from human immunodeficiency virus infection, immunosuppressive therapy, and autoimmune diseases are well-established risk factors for NHL [2,3]. However, the established risk factors can only explain a small portion of the cases, which leaves majority of NHL cases unexplained.
Blood transfusion particularly allogeneic blood transfusion can induce immunosupression and has been suggested as a risk factor for NHL [4–7]. Epidemiological studies linking blood transfusion to the risk of NHL, however, provided inconsistent results [8–11]. Different genetic susceptibility in different study populations may explain some of the conflicting results.
T helper 1 (Th1) and T helper 2 (Th2) lymphocyte cytokines play a crucial role in regulation of key pathways of the immune system. Imbalanced regulation and expression of Th1 and Th2 lymphocyte cytokines have been linked to the development of NHL [12,13]. Single nucleotide polymorphisms (SNPs) in several Th1/Th2 cytokine genes (i.e., TNF and IL10) have been reported to be associated with the risk of NHL and its major subtypes [14,15]. Damaged autologous erythrocytes during blood transfusion has been shown to augment the cytokines TNF-α and IL-10 production of the mononuclear phagocyte system in humans . We speculate that genetic variations in TNF and IL-10 and/or other Th1 and Th2 cytokine genes may modify the relationship between blood transfusion and NHL risk. As such, we analyzed data from a population-based case-control study in Connecticut women.
The study population has been described in detail elsewhere [11,17]. Briefly, all histologically confirmed incident cases of NHL (ICD-O, M-9590-9642, 9690–9701, and 9740–9750) diagnosed between 1996 and 2000 in Connecticut were identified through the Yale Cancer Center’s Rapid Case Ascertainment Shared Resource. Enrollment criteria included age between 21 and 84 years, residents in Connecticut, female, alive at the time of interview, and without a previous diagnosis of cancer except for nonmelanoma skin cancer. Of 832 eligible cases, 601 (72%) completed in-person interviews. Pathology slides (or tissue blocks) from all patients were obtained from the original pathology departments and reviewed by two independent pathologists. All cases in this article were classified according to the World Health Organization classification system.
Female population-based controls from Connecticut were recruited by random-digit dialing methods for those younger than 65 years of age; or random selection from the Centers for Medicare and Medicaid Services records for those aged 65 years or older. Controls (n = 717) 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. As more than 90% of the study subjects were non-Hispanic Caucasians, this project was limited to non-Hispanic Caucasians. Among those, ~75% of the study subjects (76.7% of the cases and 74.6% of the controls) provided blood samples, and ~10% of the subjects (11.0% of the cases and 10.4% of the controls) provided buccal cell samples for genotyping. We further excluded women who had autologous blood transfusion (one case and three controls), and the final study included 482 cases and 541 controls. The selected characteristics of the study population were presented in Table I.
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 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, environmental and lifestyle factors, and medical conditions and medication use were collected through in-person interview.
Regarding blood transfusion history, subjects were asked whether they had ever had a blood transfusion during their lifetime. If so, subjects were asked to provide information regarding the year and reason for each blood transfusion they had received. For this analysis, 31 subjects who had a blood transfusion within 1 year before diagnosis or interview were considered not to have received a blood transfusion as recent blood transfusion might be related to the disease itself. Autologous blood transfusion was defined as a subject who reported having a blood transfusion using her own blood. In contrast, allogeneic blood transfusion was defined as a subject who reported having a blood transfusion without using her own blood. This article was focused on allogeneic blood transfusion.
Genotyping was performed at the National Cancer Institute Core Genotyping Facility (http://cgf.nci.nih.gov). All TaqMan assays (Applied Biosystems, Foster City, CA) for this study were optimized on the ABI 7900HT detection system with 100% concordance with sequence analysis of 102 individuals as listed on the SNP500Cancer website (http://snp500cancer.nci.nih.gov). 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 . Due to a limited amount of DNA available for subjects who provided only buccal cells, we first genotyped subjects who provided a blood sample. If there was suggestive evidence, or if we had a relatively high prior 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 and 40 replicate samples from each of two blood donors were interspersed throughout the plates used for genotype analysis. The concordance rates for quality control samples were between 99% and 100% for all assays. The genotype frequencies for four SNPs (rs1059293, rs231775, rs2243250, and rs2070874) were not consistent with Hardy–Weinberg equilibrium among non-Hispanic white controls using a chi-square test (P < 0.05) and were excluded from the final analysis. Another five SNPs (rs2069822, rs2069818, rs2069807, rs3024509, and rs361525) with minor allele frequency less than 10% were also excluded from the final analysis. A total of 30 SNPs in 17 Th1/Th2 genes: IFNG (rs1861494 and rs2069705), IFNGR1 (rs3799488), IFNGR2 (rs9808753), IL10RA (rs9610), IL12A (rs568408 and rs582054), IL13 (rs20541, rs1800925, and rs1295686), IL15 (rs10833), IL15RA (rs2296135), IL2 (rs2069762), IL4 (rs2243248, rs2243290, and rs2243268), IL4R (rs2107356), IL5 (rs2069812), IL6 (rs1800795 and rs1800797), IL7R (rs1494555), JAK3 (rs3008), IL10 (rs1800871, rs1800872, rs1800896, rs3024496, rs3024491, and rs1800890), and TNF (rs1800629 and rs1799724) were included in the final analysis.
Unconditional logistic regression was used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for associations between blood transfusion, and risk of NHL and its subtypes in different genotype strata. To increase statistical power, heterozygous and homozygous variant genotypes were combined for all genes. The only potential confounding variable included in the final model was age (< 55 years, 55–70 years, ≥ 70 years). Adjustments for other variables, such as cigarette smoking, alcohol consumption, and family history, did not result in material changes in the observed associations and these variables were not included in the final models reported here. Significance of gene-blood transfusion interaction was assessed by adding an interaction term in the logistic models. The false discovery rate (FDR) threshold of 0.2 was used to control for multiple comparisons . All P values presented are two-sided, and all analyses were performed using SAS software, version 9.2 (SAS Institute, Cary, NC).
The association between blood transfusion and risk of NHL overall and NHL subtypes are presented in Table II. Blood transfusion was not associated with the risk of NHL overall (OR = 0.9, 95% CI: 0.7–1.2), B-cell lymphoma (OR = 0.8, 95% CI: 0.6–1.2) and T-cell lymphoma (OR = 1.2, 95% CI: 0.5–2.7). The results were comparable with the results in the overall population that we reported previously .
Compared with women without a history of blood transfusion, women with a history of transfusion had an increased risk of NHL overall and B-cell lymphoma if they carried IL10RA (rs9610) GG genotype (OR = 1.9, 95% CI: 1.1–3.2; OR = 2.1, 95% CI: 1.2–3.9, respectively, Table III), and a reduced risk if they carried IL10RA (rs9610) AG/AA genotypes (OR = 0.6, 95% CI: 0.4–0.9 for NHL overall; OR = 0.5, 95% CI: 0.3–0.8 for B-cell lymphoma). The interaction was statistically significant (Pforinteraction = 0.003 and 0.001 for NHL overall and B-cell lymphoma, respectively) after adjustment for FDR.
Among women who carried TNF (rs1800629) (Table III) AG/AA genotypes, blood transfusion was associated with an increased risk of NHL overall (OR = 1.6, 95% CI: 0.9–2.7) and B-cell lymphoma (OR = 1.5, 95% CI: 0.8–2.7). Among women who carried TNF (rs1800629) GG genotype, blood transfusion was associated with a decreased risk of NHL overall (OR = 0.7, 95% CI: 0.5–1.0) and B-cell lymphoma (OR = 0.7, 95% CI: 0.5–1.0). A statistically significant interaction between TNF (rs1800629) and blood transfusion was observed for NHL overall (Pforinteraction = 0.012), and for B-cell lymphoma (Pforinteraction = 0.019). Only the interaction for NHL overall remained statistically significant after adjustment for FDR.
After stratification by common B-cell lymphoma subtypes (Table IV), a significant association between blood transfusion and NHL was only observed for small lymphocytic lymphoma/ chronic lymphocytic leukemia (SLL/CLL) if women carried IL10RA (rs9610) GG genotypes (OR = 5.3, 95% CI: 1.5–18.9) and for follicular lymphoma if women carried IL10RA (rs9610) AG/AA genotypes (OR = 0.3, 95% CI: 0.1–0.7) with Pforinteraction of 0.008 for SLL/CLL and 0.012 for follicular lymphoma. However, none of these interactions remained statistically significant after adjustment for FDR. Increased or decreased risks were also observed for several other cytokine polymorphisms, but none of them showed a statistically significant interaction with blood transfusion and risk of NHL and its subtypes after adjustment for FDR (Supporting Information Tables I and II).
To our knowledge, this is the first comprehensive analysis of interaction between blood transfusion, genetic polymorphisms in Th1/Th2 pathway genes, and the risk of NHL and its subtypes. Significant interactions were observed for IL10RA (rs9610) and TNF (rs1800629) for NHL overall and B-cell lymphoma. No interactions were observed for T-cell lymphoma; however, they were based on small numbers. Future larger studies with greater statistical power are needed to clarify the association with T-cell lymphoma.
IL10 and TNF are key genes to be linked to lymphomagenesis. Both genes encode immunoregulatory cytokines that are critical mediators of inflammation, apoptosis, and Th1/Th2 balance, and function as autocrine growth factors in lymphoid tumors [19–21]. Studies of IL10 and TNF knockout mice have shown that each cytokine affects B-cell lymphomagenesis directly or indirectly [22–24]. Prolonged red blood cell storage before transfusion has been reported can increase intracellular iron, which can exacerbate the systemic inflammatory response syndrome and lead to deleterious consequences . Blood transfusions also can suppress cellular immunity and transmit infectious agents and allogeneic cells . Therefore, it is possible that genetic variation in TNF and IL10RA genes may modify the association between blood transfusion and risk of NHL.
Our study found that IL10RA polymorphisms modified the association between blood transfusion and risk of NHL, particularly for B-cell lymphoma. IL10RA gene encodes the interleukin 10 receptor-alpha chain of the IL10 receptor complex and plays a dominant role in mediating high affinity ligand binding and signal transduction [27,28]. The IL10R signaling causes transcriptional activation of several hundred genes, which play important roles in immunological diseases . IL10RA expression levels correlate with the strengths of the effects of IL10 on immune cells . Serum IL10 levels have been found to be higher in NHL patients . On the other hand, studies have shown that allogeneic nonleukofiltered red cell transfusion can significantly increase the production of IL10  and damaged autologous erythrocytes during blood transfusion can also elevate IL10 production . Although the underlying mechanism has yet to be established, effect modification observed in this study suggesting the IL10RA pathway could play a role in the relationship between blood transfusion and risk of NHL. IL10RA (rs9610) does not lead to amino acid substitution, but its importance to gene expression (translation and mRNA stability) cannot be ruled out.
The relationship between TNF (rs1800629) polymorphism and NHL risk has been reported in several studies [33–36], including this study . The TNF (rs1800629) polymorphism has been found to be associated with higher constitutive and inducible expression of TNF-α and increased susceptibility to several infectious and inflammatory conditions . High levels of TNF-α promote activation of the transcription factor NF-κB, which has antiapoptotic effects on B cells . Inappropriate activation of NF-κB has also been linked to lymphomagenesis  and to autoimmune diseases, which are known risk factors for NHL . Study also found that TNF (rs1800629) polymorphism can induce immune suppression through an increased production of TNF-α [16,40]. Recent evidence suggests that blood transfusion can increase serum levels of TNF-α . As such, the observed interaction between genetic variation of TNF (rs1800629) and blood transfusion on the risk of NHL could be due to the change of TNF-α production.
Strengths and limitations should be considered in explaining the observed results. This study is a population-based case-control study with histologically confirmed incident NHL cases, which minimized potential disease misclassification. The study has a modest sample size, and therefore, the statistical power is limited particularly for NHL subtype analysis. As such, chance cannot be ruled out for some of the significant findings. Because of a number of SNPs being examined in the study, we adjusted for multiple comparisons using FDR approach. The study only included women, so the results may not be generalizable to men.
In summary, this is the first study to examine the effect modifications of common genetic variations in the Th1/Th2 pathways genes on the association between blood transfusion and risk of NHL. The observed significant interactions between IL10RA and TNF and blood transfusion on the risk of NHL need to be replicated in other study populations with greater statistical power.
Contract grant sponsor: NIH; Contract grant numbers: CA62006, 1D43TW008323-01, 1D43TW007864-01, HD70324-01, CA165923-01; Contract grant sponsor: Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute.
Additional Supporting Information may be found in the online version of this article.
Conflict of interest: Nothing to report.