Risk of Testicular Germ Cell Tumors and Polymorphisms in the Insulin-Like Growth Factor Genes

With the exception of cryptorchidism, family history, and prior diagnosis of testicular germ cell tumors (TGCT), other risk factors for TGCT are not well identified (1). Several studies, however, implicate increased adult stature as a risk factor (2-4), suggesting that determinants of stature may also be related to TGCT. One possible mechanism may be the insulin-like growth factor (IGF) pathway (5), which contributes to many regulatory processes, including somatic growth and cellular proliferation (6).

Critical components of the IGF pathway include two ligand proteins (IGF-1 and IGF-2), their receptors, binding proteins, and an acid labile subunit (IGFALS). IGF-1, a mitogen that initiates signaling cascades via binding to its receptor, IGF-1R (6), may also have antiapoptotic properties (7). Alterations in the structure of IGF-1R and IGFALS, which binds IGF-1 to IGF-binding protein-3, may affect availability of bioavailable IGF-1 concentrations. Although there are no studies on relationships with TGCT risk, IGF-1 concentrations have been positively associated with prostate, colon, and premenopausal breast cancer risk (8).

Associations between IGF-1 polymorphisms and serum concentrations are not as clear, as relationships have been observed in some (9, 10) but not all (11, 12) studies. There is evidence, however, that IGF polymorphisms are associated with both cancer (12, 13) and childhood height (14). No published studies have examined the relationship between these polymorphisms and TGCT risk. A case-control study was conducted to determine associations of polymorphisms in IGF-1, IGF-2, IGF-1R, and IGFALS and risk of TGCT; relationships between these polymorphisms and adult height were also explored.

Study participants, enrolled from 2002 to 2005 in the U.S. Servicemen's Testicular Tumor Environmental and Endocrine Determinants Study, have been described previously in detail (2). Briefly, all men who had at least one serum sample stored in the Department of Defense Serum Repository, were ages ≤45 years, and were on active duty from 1988 to 2003 were eligible for the study. Cases included men who developed TGCT after serum sample donation, and diagnoses were limited to classic seminomas or nonseminomas. Eligible controls, men who did not develop TGCT, were pair matched to cases based on age (<1 year), ethnicity (White, Black, other), and date of serum sample donation (<30 days). Of the eligible cases (n = 853) and controls (n = 1,182), 767 (90%) cases and 928 (79%) controls agreed to participate and completed the study. The study was approved by the institutional review boards of the National Cancer Institute and the Walter Reed Army Institute for Research.

Through a computer-assisted telephone interview, each participant completed a questionnaire, eliciting information on demographic factors, including height and weight, and on known or suspected risk factors for TGCT. Buccal cell samples for DNA were also provided by 590 cases and 711 controls.

Forty-three single nucleotide polymorphisms (SNP) in four genes (IGF-1, IGF-2, IGF-1R, and IGFALS) in the IGF pathway were examined (Supplementary Table). SNPs were selected if they were reported previously as functionally relevant, had been identified as a haplotype-tagging SNP, had a minor allele frequency ≥0.05, and if there was a valid genotyping assay available at the National Cancer Institute Core Genotyping Facility. TaqMan assays (Applied Biosystems) were optimized on the ABI 7900 HT detection system and were 100% concordant with the sequence analysis of 102 individuals from the SNP500Cancer Web site (15). Success rates for genotyping ranged from 96% to 100%, and concordance for 99 quality-control samples was ≥96% for all SNPs, except IGF-1 rs5742714 (94%), IGF-2 rs2373721 (91%), IGF-1R rs2272037 (94%), and IGFALS rs3751893 (94%). Accuracy of these assays was confirmed by rechecking the quality-control data. All SNPs were found to be in Hardy-Weinberg equilibrium in White controls.

Of the 577 cases and 707 controls that were successfully genotyped, 547 were matched case-control pairs. For all analyses, the most common genotype or haplotype was considered the reference group. Data analysis was conducted for all TGCT together and for each histologic type [seminoma (n = 254) and nonseminoma (n = 323)], separately.

General linear models were used to assess the relationship between IGF polymorphisms and height among controls. Using logistic regression and adjusting for age at reference date, ethnicity, and serum date, odds ratios (OR) and 95% confidence intervals (95% CI) were used to estimate log-additive associations between IGF polymorphisms and TGCT risk. Conditional logistic regression models for matched-pairs data were also conducted and yielded similar results; these data are not shown in the current article. Trends across genotype were evaluated by including the categorical genotype as an ordinal integer valued variable, specifying the number of minor alleles, in the regression model. Separate analyses were also conducted for seminomas and nonseminomas compared with controls. Associations stratified by height (in cm) at the median cut point (177.8 cm) for controls were also examined; tests of effect modification were determined by including a cross-product of height and genotype. All genotype analyses were conducted using SAS (SAS Institute).

For White men, haplotypes were constructed and relationships with TGCT risk were assessed. Haplotypes among men of other ethnicities were not constructed due to small numbers. Haplotype block construction for IGF-1 was determined by the Gabriel method (16) in Haploview (Broad Institute, MIT). There was no strong linkage disequilibrium between SNPs in IGF-1R, IGF-2, and IGFALS; thus, haplotype blocks were not assessed for these genes. Haplotypes, within blocks for IGF-1 and overall for the others, were inferred by maximum likelihood using HaploStats (17) in R (University of Auckland), and adjusted ORs and 95% CIs were estimated. Only results for haplotypes with control frequencies greater than 5% are presented. All tests of significance were two sided.

Selected characteristics of cases and controls are presented in Table 1. Cases and controls had a mean age of 28 years. As anticipated, >85% of participants were White. Compared with controls, cases had a higher frequency of prior cryptorchidism and family history of testicular cancer. There were some differences between the ethnic groups in minor allele frequencies of the polymorphisms (Supplementary Table).

When examining relationships between IGF polymorphisms and height, there were significant trends in height associated with increasing number of minor alleles for four IGF-1R polymorphisms. For IVS2-89673 (rs907806), height increased with number of minor alleles (P_trend = 0.03); mean height (cm) was 174.0, 177.5, and 178.6 cm for the AA, AG, and GG genotypes, respectively. Similar significant trends were seen for the A allele of IVS5+289 (rs3743258). For 3,019 bp 3′ of STP (rs9282714), the inverse effect was observed (P_trend = 0.01); mean height (cm) was 180.2, 178.7, and 177.8 cm for the TT, T-, and - - genotypes, respectively. Similar but weaker associations were seen for an increasing number of A alleles in Ex16-58 (rs229765).

Overall, no associations were observed between IGF-1 polymorphisms and TGCT risk (Table 2). Similarly, there were no associations when stratified by ethnicity or height. When separating histologic types (seminoma versus nonseminoma), however, there was a suggestion that three intronic SNPs in IGF-1 might be associated with nonseminoma risk only. For IVS3-5895 (rs978458), there was a suggestion that the A allele was associated with a reduced nonseminoma risk (OR, 0.84; 95% CI, 0.63-1.11 for the GA genotype and OR, 0.60; 95% CI, 0.35-1.05 for the AA genotype; P_trend = 0.05). There was no relationship with seminomas. Similar associations were observed for the IVS2-10010 (rs5742667) and IVS2-13577 (rs2373721) polymorphisms, which were in strong linkage disequilibrium with rs978458.

For the IGF-1 polymorphisms, five regions of strong linkage disequilibrium were identified: block A (size, 6 kb), block B (size, 6 kb), block C (size, 47 kb), block D (size, 10 kb), and block E (size, 4 kb). Overall, there were no associations between the inferred haplotypes for each block and TGCT risk in White men (Table 3). However, when stratified by height, there was a suggestion of differences in TGCT risk associated with a haplotype in block C. For shorter men (≤177.8 cm), there was a 50% increase in risk (95% CI, 1.06-2.12) associated with the CGACCCGGGA haplotype compared with the most common haplotype (CGACCGGGGA). For taller men (>177.8 cm), these associations were not present; tests for effect modification, however, yielded no statistically significant differences.

No associations between SNPs or haplotypes in the IGF-1R, IGF-2, and IGFALS genes and TGCT risk were observed. There were also no associations when stratified by ethnicity, height, or histology.

In this study, no associations between IGF polymorphisms and TGCT risk were identified. Similarly, there were no relationships when cases were stratified by ethnicity, height, or histology. There was a suggestion of an interaction, as an IGF-1 block C haplotype was associated with an increased risk of TGCT among shorter, but not taller, White men. Polymorphisms or haplotypes in IGF-1R, IGF-2, and IGFALS were not associated with risk.

In 2004, Zavos et al. hypothesized that perturbations in the IGF pathway might be related to TGCT (5) in part based on reported associations between greater height and increased TGCT risk (2-4). Among men in the U.S. Servicemen's Testicular Tumor Environmental and Endocrine Determinants as reported previously (2), there was a significantly increased TGCT risk, particularly seminomas, associated with greater height (≥172.73 cm). Four IGF-1R polymorphisms were associated with height; however, they were not associated with TGCT risk.

Although this study found no association with IGF polymorphisms, it is possible that relationships between TGCT risk and serum IGF concentrations exist. For other cancers, evidence for relationships with IGF-1 and IGFALS polymorphisms (12, 13) has been inconsistent. In contrast, associations between IGF-1 concentrations and risk of premenopausal breast (8), prostate (8), and colorectal cancers (18) have been observed. Other evidence to support a role of IGFs in TGCT risk are observations that White men have significantly higher IGF-1 concentrations than men of other ethnicities (19), and White men have a higher TGCT incidence than men of other ethnicities (20). These differences, however, could also be explained by environmental differences between ethnic groups.

In addition to height, other possible risk factors for TGCT may be related to IGF concentrations, including perinatal factors. There is some evidence that smaller birth size, measured as small-for-gestational-age and lower birth weight, is associated with lower IGF-1 concentrations (21) and increased risk of TGCT (22). Thus, it might be anticipated that lower IGF-1 concentrations in early life would be correlated with increased TGCT risk in adulthood. However, the association of TGCT with both smaller birth size and increased adult stature suggests that the relationships between TGCT and body size at various ages are likely to be complex.

Other genetic factors that may underlie the association between height and increased TGCT risk include variation in the growth hormone (GH), vitamin D receptor (VDR), and cytochrome P450 19 (CYP19) genes. Vitamin D metabolites regulate cellular differentiation and proliferation and are associated with risk of breast, prostate, and colorectal cancers (23). Several polymorphisms in the VDR gene have been reported to be associated with height (24). Likewise, increased height has been associated with polymorphisms in CYP19, which produces aromatase, a catalyst to convert androgens to estrogens (25). Thus, the vitamin D or aromatase pathways may explain associations between height and risk of TGCT. Although circulating concentrations of androgens and estrogens may be related to TGCT risk, assessment of steroid hormones in prediagnostic TGCT samples have rarely been reported.

Strengths of the study include being one of the largest case-control investigations of testicular cancer etiology reported and being drawn from a well-defined population (military servicemen). Histologic data were available and all diagnoses were confirmed. However, despite the large sample size, there was limited power to assess associations with weak effects or rare variants and associations within strata of height and histology; indeed, the suggested finding of differences in IGF-1 haplotype associations by height may be due to chance. In addition, the small number of non-White participants precluded an examination of differences in risk by ethnicity.

In conclusion, the study results indicate there are no associations between IGF polymorphisms and TGCT risk; however, suggestions of differences in relationships between TGCT risk and IGF-1 haplotypes by height may warrant further investigation. Additional investigations examining associations with polymorphisms in other IGF genes or other genes shown to be associated with height or with circulating IGF-1 concentrations could be informative.

We thank Emily Steplowski (IMS) for contributions to data management.