This study was undertaken based on a longstanding etiologic model (5
) proposing two hormone-dependent phases of TGCT development: the first occurring early in development when inappropriate hormonal stimulation interferes with normal maturation of gonocytes leading to formation of CIS/ITGCNU, the presumed TGCT precursor (7
), and the second following puberty, when endocrine mechanisms drive CIS/ITGCNU cells that were dormant throughout childhood to progress into invasive TGCT. Features of the model, together with elevated risk of TGCT reported among men with androgen insensitivity, led us to hypothesize that longer CAG repeats and thus lesser AR transactivation would be associated with TGCT risk.
Although we found no association between CAG repeat length and overall TGCT risk, a result in agreement with four previously published hospital-based case–control studies (8
), longer CAG repeats were associated with risk of nonseminoma, though non-significantly. Unexpectedly, we also found risk of seminoma to be associated with shorter CAG repeats, a highly significant result further supported by a significant trend of increasing seminoma risk with shorter repeat length and significant heterogeneity between trends for seminoma and nonseminoma. Published data on AR
CAG repeat length among TGCTs of specific histologic types are limited. Consistent with our results, a Swedish study found a significantly greater percentage of CAG >25 in nonseminoma cases than seminoma cases (19
), although no such difference was reported in two other studies (8
). The most recent of these (21
) used different cutpoints to categorize CAG repeat length, so results could not be compared directly to those reported here.
Results for the individual histologic types may suggest biological distinctions between seminomas and nonseminomas, which differ in histologic appearance, age at presentation (supplementary Figure 1
is available at Carcinogenesis
Online) and clinical prognosis. Seminomas are similar histologically to CIS/ITGCNU cells, and a postulated default pathway of testicular carcinogenesis is for seminoma to arise from CIS/ITGCNU, with activation of pluripotency additionally required for development of nonseminoma (7
). Factors-driving progression of CIS/ITGCNU to invasive TGCT remains unclear, but AR expression has been demonstrated in CIS/ITGCNU cells (11
). Genome-wide association studies (22
) recently identified other loci associated with risk of both seminoma and nonseminoma, but the present study is the first to identify a genetic polymorphism for which TGCT risk associations vary significantly by histologic type. This is an important distinction because mechanisms explaining progression of CIS/ITGCNU to seminoma versus nonseminoma have not yet been identified.
The unanticipated finding of association of seminoma risk with shorter CAG repeat length, and thus presumably with greater AR transactivation, indicates that the role of the AR in testicular carcinogenesis may be more complex than simply mediating classical effects of androgens in gonocytes, CIS/ITGCNU and adult Sertoli cells. For example, differences in AR
methylation status of seminomas versus nonseminomas may provide an explanation for our discordant findings between these histologic types. In vitro
research has linked methylation of sites in the AR
minimal promoter with loss of AR expression in hormone-independent prostate cancer cell lines (25
). A separate study of TGCT tissue identified two Hha1 sites in the AR
that were methylated in differentiated nonseminomas but unmethylated in all seminomas (26
). If AR
methylation is associated with AR silencing in TGCTs, this suggests that AR activity would be more common in seminomas. It would then be plausible that during the adult phase of TGCT development, shorter CAG repeat length, and thus greater AR transactivation, could drive progression of CIS/ITGCNU to seminoma but be irrelevant in nonseminomas with silenced AR. A second possible role for the polymorphic AR in testicular carcinogenesis may be indirect influence through steroid hormone levels. Observational studies demonstrate lower levels of serum testosterone and estradiol among adult men with shorter CAG repeat length (16
). This phenomenon, postulated to reflect a feedback loop-moderating androgen action through the classical AR-mediated pathway (16
), would on expectation make CAG repeat length a proxy measure for circulating levels of these hormones, which may have unrecognized effects on malignant potential of the male germ cell lineage. Such effects could plausibly arise, for example, if men with shorter CAG repeats and thus lower testosterone levels experienced suboptimal signaling though a nonclassical androgen pathway. In contrast to the classic genomic pathway, in which the androgen–AR complex modulates gene transcription by binding specific DNA sequence elements, more recently described nonclassical actions do not depend on AR–DNA interactions and proceed on a far more rapid time frame though additional effectors (27
). Understanding of this pathway’s signaling requirements for normal male development and spermatogenesis is just beginning to emerge (28
The observed association between shorter CAG repeat length and seminoma risk also suggests that environmental agents, such as exogenous androgens or androgen agonists (6
), may act jointly with polymorphic AR variants to influence malignant potential of germ cells.
Chance seems an unlikely explanation for our findings for several reasons. Firstly, we observed both a monotonic trend of increasing seminoma risk over decreasing CAG repeat length and statistically significant heterogeneity between trends for risk of seminoma and nonseminoma. Secondly, the association between repeat length and seminoma risk persisted in all sensitivity analyses (excluding additional pairs from the same family as well as pairs in which cases had anaplastic or spermatocytic seminoma, personal history of cryptorchidism and/or family history of TGCT or cryptorchidism; supplementary Results, available at Carcinogenesis Online). Finally, mothers are an ideal reference group for testing association between a male-limited disease such as TGCT and an X-chromosome variant (17
). However, the possibility remains that other X-chromosome variants in linkage disequilibrium with CAG repeat genotypes could influence TGCT risk to some extent.
Strengths of our analysis include that cases analyzed were similar to cases from the base population on features that we did not oversample, and among features upon which we oversampled by design (personal history of cryptorchidism and family history of TGCT or cryptorchidism; supplementary Results, available at Carcinogenesis Online), sensitivity analyses revealed consistency of results. Moreover, comparing cases to mothers allowed us to estimate genotype–disease associations in a manner free of confounding by population structure, often a concern in studying conditions, like TGCT, that vary greatly by race/ethnicity. One consideration regarding the case–parent design is that parental genotypes associated with disease risk may interfere with reproductive ability. However, there is no evidence that CAG repeat length influences female fertility, thus mothers’ untransmitted alleles seem an appropriate control in this investigation. A limitation is that we enrolled only surviving TGCT cases, which may have excluded cases with most severe disease. However, survival rates are exceedingly high, so this concern seems minor.
In summary, we found shorter AR CAG repeat length is associated with risk of seminoma, suggesting the AR may be involved in progression from CIS/ITGCNU to seminoma, or that the activity of androgens, possibly through non-genomic mechanisms, may influence testicular carcinogenesis in utero. Mechanistic insights may be provided by investigating both AR expression and AR methylation in TGCT tissue of distinct histologic types and the role of AR transactivation and varying testosterone levels on the development and malignant potential of gonocytes. Further association studies are warranted to confirm these findings, including associations of shorter AR CAG repeats with seminoma risk, longer AR CAG repeats with nonseminoma risk and an apparently null association with mixed GCT risk. Such confirmation would further implicate molecules of androgen action and response in TGCT etiology, establishing two new TGCT research priorities: epidemiologic investigation of potential joint effects of functional AR variants and exposure to hormonally active compounds of both endogenous and exogenous origin, and exploration of cellular mechanisms underlying AR variant–TGCT associations.