Supplementary Table 1 from Genetic Variation in the Inhibin Pathway and Risk of Testicular Germ Cell Tumors

Beginning in 1985, the United States military has consistently maintained repositories of frozen human serum for force health protection reasons. The separate repositories created by the Army, Navy, and Air Force during the startup of their human immunodeficiency virus (HIV) screening programs were fully combined by 1996, along with the Defense Medical Surveillance System, to form the DoD Serum Repository (DoDSR). Currently comprised of 450,000 square feet of storage space at a constant -30 degrees Celsius, the DoDSR, operated by the Armed Forces Health Surveillance Center (AFHSC), receives approximately 2 million new serum specimens per year as a result of current HIV screening programs and pre- and post-deployment serum collection. Following initial testing for HIV when required, each specimen remains frozen until needed for clinical testing or a public health study, and its physical location is carefully tracked. Certain militarily-relevant research studies occur, though the serum from a specific individual is never allowed to be fully exhausted. AFHSC maintains careful control over the repository, utilizing a scientific review board to determine which requests for serum will be granted. As of 2012, only 0.42% of all of the frozen specimens in the DoDSR had been thawed for any type of use. The addition of new specimen processing capacity and significant changes to policy would be required if more of the specimens were to be used to answer relevant epidemiological, operational, or medical research questions.

Specimens in the United States Department of Defense (DoD) Serum Repository have accumulated in frozen storage since 1985 when the DoD began universal screening for human immunodeficiency virus. Use of the stored serum for health research has been carefully controlled, but the resulting publications have never been systematically identified or described. The Armed Forces Health Surveillance Center (AFHSC) information systems and open (online) sites were used as data sources. Through 2012, the repository contained 54,542,658 serum specimens, of which 228,610 (0.42%) have been accessed for any purpose. Between 2001 (the first year that comprehensive, digital records were available) and 2012, 65.2% of all approved requests for serum were for healthcare or public health investigations, but greater than 99% of all shipped samples were for research. Using two different methods - a structure search of PubMed and an exhaustive online search based on records from AFHSC - we identified 76 articles published between October 1988 and March 2013 that covered a multitude of infectious diseases, injuries, environmental exposures and mental health conditions through analysis of antibodies, biological metabolic, signaling and regulatory substances, Vitamin D, organochlorines, dioxin, omega-3-fatty acid, and portions of human deoxyribonucleic acid. Despite its operational and scientific value, it appears that the DoD Serum Repository has been underutilized. Changes to policy and increased capacity for specimen processing could increase use of the repository without risking privacy or the availability of specimens for the healthcare of individual service members in the future.

Genome-wide association studies (GWASs) have identified multiple common genetic variants associated with an increased risk of testicular germ cell tumors (TGCTs). A previous GWAS reported a possible TGCT susceptibility locus on chromosome 1q23 in the UCK2 gene, but failed to reach genome-wide significance following replication. We interrogated this region by conducting a meta-analysis of two independent GWASs including a total of 940 TGCT cases and 1559 controls for 122 single-nucleotide polymorphisms (SNPs) on chromosome 1q23 and followed up the most significant SNPs in an additional 2202 TGCT cases and 2386 controls from four case-control studies. We observed genome-wide significant associations for several UCK2 markers, the most significant of which was for rs3790665 (PCombined = 6.0 × 10(-9)). Additional support is provided from an independent familial study of TGCT where a significant over-transmission for rs3790665 with TGCT risk was observed (PFBAT = 2.3 × 10(-3)). Here, we provide substantial evidence for the association between UCK2 genetic variation and TGCT risk.

An altered anti-Epstein-Barr virus (EBV) serologic profile preceding diagnosis is associated with an increased risk of Hodgkin lymphoma. It is unknown whether this atypical pattern predicts Hodgkin lymphoma risk further subdivided by determination of EBV in tumor cells. A nested case-control study of 128 incident Hodgkin lymphoma cases and 368 matched controls from active-duty military personnel with archived serum in the US Department of Defense Serum Repository was conducted to determine whether a panel of anti-EBV antibody titers differed in EBV(+) and EBV(-) Hodgkin lymphoma. Among 40 EBV(+) Hodgkin lymphoma cases and matched controls, statistically significant increased risks were associated with elevated anti-EBV VCA IgG antibody titers (relative risk = 3.1; 95% confidence interval [CI], 1.1-8.7), and an anti-EBNA-1/anti-EBNA-2 antibody ratio ≤ 1.0 versus > 1.0 (relative risk = 4.7; 95% CI, 1.6-13.8). In contrast, no significant associations were found among 88 EBV(-) Hodgkin lymphoma cases relative to their matched controls. In case-case analysis, EBV(+) disease was significantly associated with a low anti-EBNA-1/anti-EBNA-2 antibody ratio. This distinctive serologic response to EBV latent antigens, indicative of immune dysfunction in other clinical settings, is associated with an increased risk of developing EBV(+) but not EBV(-) Hodgkin lymphoma.

Abstract Background: Testicular germ cell tumors (TGCT), the most commonly occurring cancer among young men in the U.S., are thought to be endocrine-related tumors. The relationship between steroid hormone levels and development of TGCT, however, has been difficult to study given the rarity of the tumor in the general population and the paucity of pre-diagnostic serum from young men. The U.S. Servicemen's Testicular Tumor Environmental and Endocrine Determinants (STEED) study, however, was able to overcome these obstacles by enrolling military servicemen who had previously donated serum specimens to the Department of Defense Serum Repository. Methods: STEED participants were enrolled between 2002 and 2005. The associations between TGCT risk and log-transformed serum concentrations of testosterone, free testosterone, estradiol, free estradiol, 3α-androstanediol glucuronide (3α-diol-G), an indicator of peripheral androgen action, and sex hormone-binding globulin (SHBG) were examined in 517 case and 790 control participants. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression models, adjusting for date of serum donation, age, race/ethnicity, family history of testicular cancer, history of cryptorchidism, height, and body mass index. Results: The analysis found that while testosterone was not significantly associated with TGCT risk, there was an inverse association between 3α-diol-G and risk (OR=0.80, 95%CI=0.63-0.99). In addition, there was a significant inverse association with TGCT and the ratio of 3α-diol-G to testosterone (OR=0.76, 95%CI=0.61-0.93). There was also a significant direct association between estradiol and TGCT risk (OR=1.41, 95%CI=1.06-1.87). The relationships between the free forms of testosterone and estradiol and risk were the same as the relationships between the total forms of each hormone and risk. There was no association between TGCT and SHBG. Conclusion: These results suggest that the hormone metabolism of men who subsequently develop TGCT may vary from that of other men. In men who develop TGCT, more testosterone may be converted to estradiol and less to dihydrotestosterone, the most potent form of androgen, than in men who do not develop TGCT. Further examination of metabolites, both upstream and downstream in the steroid hormone pathway, may help elucidate the mechanism by which these exposures are related to development of TGCT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4468. doi:1538-7445.AM2012-4468

BACKGROUND Previously, we have shown that increasing adult height is associated with increased risk of testicular germ-cell tumor (TGCT). Recently, a number of single nucleotide polymorphisms (SNPs) have been found to be related to height. We examined whether these SNPs were associated with TGCT and whether they explained the relationship between height and TGCT. METHODS We genotyped 15 height-related SNPs in the US Servicemen's Testicular Tumor Environmental and Endocrine Determinants (STEED) case-control study. DNA was extracted from buccal cell samples and Taqman assays were used to type the selected SNPs. We used logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (95%CIs). RESULTS There were 561 cases and 676 controls for analysis. Two SNPs were found to be associated with risk of TGCT, rs6060373 (CC vs TT, OR = 1.51, 95% CI: 1.06-2.15) and rs143384 (CC vs TT, OR = 1.53, 95% CI: 1.09-2.15). rs6060373 is an intronic polymorphism of ubiquinol-cytochrome c reductase complex chaperone (UQCC), and rs143384 is a 5'UTR polymorphism of growth differentiation factor 5 (GDF5). No individual SNP attenuated the association between height and TGCT. Adjustment for all SNPs previously associated with adult height reduced the associations between adult height and TGCT by ~8.5%, although the P-value indicated only weak evidence that this difference was important (P = 0.26). CONCLUSIONS This novel analysis provides tentative evidence that SNPs which are associated with adult height may also share an association with risk of TGCT.

Exposure to endocrine-disrupting chemicals, such as polychlorinated biphenyls (PCB), may alter hormonal balance and thereby increase risk of testicular germ cell tumors (TGCT). To study the relationship of PCBs to TGCT, prediagnostic serum samples from 736 cases and 913 controls in the Servicemen's Testicular Tumor Environmental and Endocrine Determinants study were analyzed. Adjusted odds ratios and 95% confidence intervals were estimated using logistic regression. PCB levels were examined in association with all TGCT and, separately, with each histologic type (seminoma and nonseminoma). Risks associated with seven functional groupings of PCBs, as well as sum of PCBs, were also examined. There were significantly decreased risks of TGCT in association with eight PCBs (PCB-118, PCB-138, PCB-153, PCB-156, PCB-163, PCB-170, PCB-180, and PCB-187) and no association with the remaining three (PCB-99, PCB-101, and PCB-183). The same eight congeners were significantly associated with decreased risk of nonseminoma, whereas five (PCB-138, PCB-153, PCB-156, PCB-163, and PCB-170) were associated with decreased risk of seminoma. All functional groupings of PCBs were also associated with decreased risk of TGCT and of nonseminoma, whereas six of the seven functional groups were associated with decreased risk of seminoma. Sum of PCBs was significantly associated with decreased risk of TGCT (P(trend) = 0.006), nonseminoma (P(trend) = 0.007), and seminoma (P(trend) = 0.05). Overall, these data do not support the hypothesis that PCB exposure increases the risk of TGCT.

Risk factors for testicular germ cell tumors (TGCT) have not been well identified; however, data suggest that risks of cancer in family members of men with TGCT is elevated. Using family history data from 738 cases and 904 controls enrolled in the U.S. Servicemen's Testicular Tumor Environmental and Endocrine Determinants (STEED) Study from 2002 to 2005, the risk of cancer in first‐ and second‐degree family members of these men was examined. Relative risks (RRs) and 95% confidence intervals (CIs) were estimated using Cox proportional hazards models, adjusting for reference age of case or control, race/ethnicity of case or control, sex of family member and lineage (maternal vs. paternal). An increased risk of all cancer among first‐degree relatives of cases compared to controls was observed (RR = 1.17, 95% CI: 1.01–1.35). There were suggestions of differences in risk when stratifying all relatives by lineage. For maternal relatives, there was a statistically significant increased risk of all cancer (RR = 1.16, 95% CI: 1.04–1.30), digestive tract (RR = 1.52, 95% CI: 1.15–2.00) and male genital organ cancer (RR = 1.70, 95% CI: 1.15–2.51); there was also a suggestion of increased risks of hematopoetic cancers, cancers in the female genital organs and nonmelanoma skin cancer. For paternal relatives, there was a statistically significant association only with decreased risk of lung cancer (RR = 0.69, 95% CI: 0.51–0.94). Thus, this study suggests that there may be aggregation of cancer among families of men diagnosed with TGCT. Published 2008 Wiley‐Liss, Inc.

Studies have consistently shown that taller men are at increased risk of testicular germ-cell tumors. Thus, it is plausible that factors associated with height may also influence risk of these tumors. The authors examined associations between testicular germ-cell tumor risk and circulating concentrations of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor-binding protein 3 (IGFBP-3) among 517 cases and 790 controls from the US Servicemen's Testicular Tumor Environmental and Endocrine Determinants (STEED) Study (2002-2005). Odds ratios and 95% confidence intervals were estimated using logistic regression models, adjusting for age, race/ethnicity, height, and body mass index. All tests of significance were two-sided. Overall, there were no associations between IGF-1 or IGFBP-3 concentrations and risk of testicular germ-cell tumors (p > 0.05). However, when cases were separated by histologic type, there was a suggestion of a reduction in seminoma risk associated with the highest concentrations of IGF-1 as compared with the lowest concentrations (odds ratio = 0.66, 95% confidence interval: 0.40, 1.09). Although there were no overall associations with insulin-like growth factor, contrary to expectation, there was a suggestion that IGF-1 concentrations may be inversely associated with risk of seminoma.

AbstractGene-knockout studies in mice suggest that INHA, encoding a subunit of gonadotropin-regulating proteins known as inhibins, is a tumor suppressor for testicular stromal cell tumors. It is not known whether genetic variation in the inhibin pathway also influences susceptibility to testicular germ cell tumors (TGCT), the most common testicular cancer in young men. To address this question, we conducted a case-control analysis (577 cases; 707 controls) of single-nucleotide polymorphisms (SNP) in genes in the inhibin pathway among participants in the U.S. Servicemen's Testicular Tumor Environmental and Endocrine Determinants Study. Thirty-eight tagging SNPs in six genes (INHA, INHBA, INHBB, INHBC, INHBE, and SMAD4) were genotyped. Odds ratios (OR) and 95% confidence intervals (CI) relating variant genotypes to TGCT risk were calculated using unconditional logistic regression. Among White subjects, an elevated risk of TGCT was observed for carriers of the T allele of the INHA variant rs2059693 (CT genotype: OR, 1.33; 95% CI, 1.04–1.71; TT: OR, 1.60; 95% CI, 1.01–2.52; Ptrend = 0.008). The association with rs2059693 was stronger for nonseminomas, and for teratomas and teratocarcinomas in particular (N = 58; CT: OR, 1.63; 95% CI, 0.89–2.99; TT: OR, 4.54; 95% CI 2.00–10.3; Ptrend = 0.0008). We found no evidence of association with variants in the other investigated genes. These findings suggest that genetic variation in the INHA locus influences TGCT development. [Cancer Res 2008;68(8):3043–8]

Much evidence supports the premise that population genetic variation contributes significantly to the risk of testicular germ-cell tumor (TGCT). However, investigations of the association between genomic markers and TGCT susceptibility are scarce. Single nucleotide polymorphisms (SNPs) at the locus 8q24 have recently been found to be associated with prostate, colorectal and breast cancer. The US Servicemen's testicular tumor environmental and endocrine determinants (STEED) study was used to investigate the association of 15 specific 8q24 SNPs with TGCT and its two main histologic groups of seminoma and nonseminoma. Conditional and unconditional logistic regression models, adjusted for the matching variables of year of birth, race/ethnicity and serum date, were utilized to produce odds ratios (OR) and 95% confidence intervals (95%CI). The analysis included 680 controls and 568 TGCT cases. In the TGCT analysis, no SNP was associated with risk in both heterozygotes and variant homozygotes. When stratified by histology the seminoma analysis also showed no association with the 8q24 SNPs. Conversely, the analysis of nonseminomas had three tentative associations (rs6470494, ORgenotype AG = 1.15, 95%CI: 0.86-1.54; ORgenotype GG = 1.68, 95%CI: 1.04-2.73; p for trend = 0.04) (rs13254738, ORgenotype GT = 1.04, 95%CI: 0.77-1.40; ORgenotype TT = 1.62, 95%CI: 1.06-2.49; p for trend = 0.07) (rs10505476, ORgenotype CT = 0.67, 95%CI: 0.50, 0.91; ORgenotype TT = 0.81, 95%CI: 0.47-1.39; p for trend = 0.04). There was no linkage disequilibrium between any of the 8q24 SNPs analyzed in this population. In conclusion, this study has found little evidence for an association between the reported 8q24 SNPs and TGCTs, although the findings for nonseminoma may be worth further investigation.

Testicular germ cell tumors (TGCT) are the most common cancer among young men in the United States and Western Europe. Prior evidence suggests that TGCT may arise in perinatal life, although few risk factors have yet been identified. To study the etiology of TGCT, the US Servicemen's Testicular Tumor Environmental and Endocrine Determinants (STEED) case-control study enrolled participants and their mothers between 2002 and 2005. Five hundred twenty-seven mothers of cases and 561 mothers of controls provided information on perinatal variables. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (95%CI) associated with the candidate risk factors. Analyses were conducted for all TGCT together and for each histologic subgroup (seminoma and nonseminoma) separately. Young maternal age (<20 vs. 20-29 years, OR = 1.51, 95%CI: 1.09-2.10), young paternal age (<25 vs. 25-29 years, OR = 1.45, 95%CI: 1.08-1.94), maternal parity (3 vs. 1, OR = 0.63, 95%CI: 0.44-0.90) and breech birth (OR = 1.92, 95%CI: 1.03-3.56) were associated with risk of TGCT. For seminoma, young maternal age (<20 vs. 20-29 years, OR = 1.67, 95%CI: 1.10-2.54), young paternal age (<25 vs. 25-29 years, OR = 1.53, 95%CI: 1.03-2.27), maternal parity (3 vs. 1, OR = 0.58, 95%CI: 0.35-0.96) and low birth weight (<2,500 g vs. 2,500-4,000 g, OR = 1.82, 95%CI: 1.00-3.30) were risk factors. Nonseminoma was associated with breech birth (OR = 2.44, 95%CI: 1.25-4.78) and Cesarean section (OR = 2.10, 95%CI: 1.25-3.54). These results support the hypothesis that TGCT may originate in very early life.

Testicular germ cell tumors (TGCT) that arise in young men are composed of two histologic types, seminomas and nonseminomas. Risk patterns for the two types appear to be similar and may be related to either endogenous or exogenous hormonal exposures in utero. Why similar risk patterns would result in different histologic types is unclear, but could be related to varying genetic susceptibility profiles. Genetic variation in hormone metabolizing genes could potentially modify hormonal exposures, and thereby affect which histologic type a man develops. To examine this hypothesis, 33 single nucleotide polymorphisms (SNPs) in four hormone metabolism candidate genes (CYP1A1, CYP17A1, HSD17B1, HSD17B4) and the androgen receptor gene (AR) were genotyped. Associations with TGCT were evaluated among 577 TGCT cases (254 seminoma, 323 nonseminoma) and 707 controls from the US Servicemen's Testicular Tumor Environmental and Endocrine Determinants (STEED) study. There were no significant associations with TGCT overall based on a test using an additive model. However, compared to homozygotes of the most common allele, two nonredundant SNPs in CYP1A1 were inversely associated with nonseminoma: CYP1A1 promoter SNP rs4886605 OR = 0.75 (95% CI = 0.54-1.04) among the heterozygotes and OR = 0.37, 95% CI = 0.12-1.11 among the homozygotes with a p-value for trend = 0.02; rs2606345 intron 1 SNP, OR = 0.69 (95% CI = 0.51-0.93) among heterozygotes and OR = 0.70 (95% CI = 0.42-1.17) among homozygotes, with a p-value for trend = 0.02. Caution in interpretation is warranted until findings are replicated in other studies; however, the results suggest that genetic variation in CYP1A1 may be associated with nonseminoma.

Abstract A106 Although the incidence of testicular germ cell tumors (TGCT) has been rising worldwide, little known about the etiology. There is evidence, however, that TGCT is related to other male reproductive disorders, specifically, hypospadias, cryptorchidism and infertility. Because testosterone is responsible for the development and proliferation of testicular cells and testicular descent, it is plausible that testosterone and its binding protein, sex hormone binding globulin (SHBG), may be associated with TGCT risk. Using 512 case and 789 control participants from the U.S. Servicemen’s Testicular Tumor Environmental and Endocrine Determinants (STEED) Study, we assessed associations of pre-diagnostic serum SHBG and testosterone (measured total and calculated free) concentrations and risk of TGCT. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using adjusted logistic regression models. Among the controls, SHBG concentrations increased with age (p<0.01), while free testosterone concentrations decreased with age (p=0.02). Total testosterone concentrations, however, were not associated with age (p=0.84). SHBG and total testosterone concentrations decreased with increasing body mass index (p<0.01), however free testosterone did not (p=0.67). Overall, there were no statistically significant associations between SHBG and total testosterone concentrations and TGCT risk. There were suggestions that men had an increased risk of TGCT if they were in the highest quintile of SHBG (OR=1.41, 95% CI, 0.98-2.02) and free testosterone (OR=1.31, 95% CI, 0.91-1.88), but there were no statistically significant trends. Serum total testosterone concentrations were not associated with TGCT risk. There did not appear to be differences in risk by tumor subtype, seminoma versus nonseminoma, nor effect modification by body mass index. These results suggest that serum testosterone is not associated with risk of TGCT; other endogenous hormones, however, such as gonadotropins and estrogens, may provide additional clues to the etiology of TGCT. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A106.

BACKGROUND:Exposure to endocrine-disrupting chemicals, such as persistent organochlorine pesticides, has been suggested to increase the risk of testicular germ cell tumors (TGCTs).METHODS:To study the relationship of POP exposure to TGCT risk, prediagnostic serum samples from 754 case subjects and 928 control subjects enrolled in the Servicemen's Testicular Tumor Environmental and Endocrine Determinants Study were analyzed for cis-nonachlor, trans-nonachlor, oxychlordane, total chlordanes, beta-hexachlorocyclohexane, mirex, p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), and p,p'-dichlorodiphenyltrichloroethane. Adjusted odds ratios (ORs) and their associated 95% confidence intervals (CIs) for the risk of TGCT overall and for the histological subgroups, seminoma and nonseminoma, were estimated using multivariable logistic regression. All statistical tests were two-sided.RESULTS:TGCT risk was statistically significantly associated with higher plasma levels of p,p'-DDE (for highest quartile [Q4] vs lowest quartile [Q1], OR = 1.71, 95% CI = 1.23 to 2.38, P(trend) = .0002) and of two chlordane components, cis-nonachlor (Q4 vs Q1, OR = 1.56, 95% CI = 1.11 to 2.18, P(trend) = .009) and trans-nonachlor (Q4 vs Q1, OR = 1.46, 95% CI = 1.07 to 2.00, P(trend) = .026). Seminoma risk was statistically significantly associated with p,p'-DDE (Q4 vs Q1, OR = 1.91, 95% CI = 1.22 to 2.99, P(trend) = .0008), cis-nonachlor (Q4 vs Q1, OR = 1.93, 95% CI = 1.27 to 2.93, P(trend) = .0045), trans-nonachlor (Q4 vs Q1, OR = 1.72, 95% CI = 1.11 to 2.67, P(trend) = .033), and a chlordane metabolite, oxychlordane (Q4 vs Q1, OR = 1.64, 95% CI = 1.04 to 2.60, P(trend) = .048), whereas nonseminoma risk showed a statistically significant association with p,p'-DDE only (Q4 vs Q1, OR = 1.63, 95% CI = 1.10 to 2.42, P(trend) = .0044).CONCLUSIONS:Increased exposure to p,p'-DDE may be associated with the risk of both seminomatous and nonseminomatous TGCTs, whereas exposure to chlordane compounds and metabolites may be associated with the risk of seminoma. Because evidence suggests that TGCT is initiated in very early life, it is possible that exposure to these persistent organic pesticides during fetal life or via breast feeding may increase the risk of TGCT in young men.