Testicular Germ Cell Tumor Susceptibility Research Articles

Epigenetics and Testicular Cancer: Bridging the Gap Between Fundamental Biology and Patient Care.

Testicular cancer is the most common solid tumor affecting young males. Most testicular cancers are testicular germ cell tumors (TGCTs), which are divided into seminomas (SGCTs) and non-seminomatous testicular germ cell tumors (NSGCTs). During their development, primordial germ cells (PGCs) undergo epigenetic modifications and any disturbances in their pattern might lead to cancer development. The present study provides a comprehensive review of the epigenetic mechanisms–DNA methylation, histone post-translational modifications, bivalent marks, non-coding RNA–associated with TGCT susceptibility, initiation, progression and response to chemotherapy. Another important purpose of this review is to highlight the recent investigations regarding the identification and development of epigenetic biomarkers as powerful tools for the diagnostic, prognostic and especially for epigenetic-based therapy.

Identification of 22 susceptibility loci associated with testicular germ cell tumors

Testicular germ cell tumors (TGCT) are the most common tumor in young white men and have a high heritability. In this study, the international Testicular Cancer Consortium assemble 10,156 and 179,683 men with and without TGCT, respectively, for a genome-wide association study. This meta-analysis identifies 22 TGCT susceptibility loci, bringing the total to 78, which account for 44% of disease heritability. Men with a polygenic risk score (PRS) in the 95th percentile have a 6.8-fold increased risk of TGCT compared to men with median scores. Among men with independent TGCT risk factors such as cryptorchidism, the PRS may guide screening decisions with the goal of reducing treatment-related complications causing long-term morbidity in survivors. These findings emphasize the interconnected nature of two known pathways that promote TGCT susceptibility: male germ cell development within its somatic niche and regulation of chromosomal division and structure, and implicate an additional biological pathway, mRNA translation.

Between a Rock and a Hard Place: An Epigenetic-Centric View of Testicular Germ Cell Tumors.

Simple SummaryThis minireview focuses on the role of epigenetics in testicular cancer. A working model is developed that postulates that epigenetic features that drive testicular cancer malignancy also enable these tumors to be cured at a high rate with chemotherapy. Chemoresistance may occur by epigenetic uncoupling of malignancy and chemosensitivity, a scenario that may be amenable to epigenetic-based therapies.Compared to many common solid tumors, the main genetic drivers of most testicular germ cell tumors (TGCTs) are unknown. Decades of focus on genomic alterations in TGCTs including awareness of a near universal increase in copies of chromosome 12p have failed to uncover exceptional driver genes, especially in genes that can be targeted therapeutically. Thus far, TGCT patients have missed out on the benefits of targeted therapies available to treat most other malignancies. In the past decade there has been a greater appreciation that epigenetics may play an especially prominent role in TGCT etiology, progression, and hypersensitivity to conventional chemotherapy. While genetics undoubtedly plays a role in TGCT biology, this mini-review will focus on the epigenetic “states” or features of testicular cancer, with an emphasis on DNA methylation, histone modifications, and miRNAs associated with TGCT susceptibility, initiation, progression, and response to chemotherapy. In addition, we comment on the current status of epigenetic-based therapy and epigenetic biomarker development for TGCTs. Finally, we suggest a unifying “rock and a hard place” or “differentiate or die” model where the tumorigenicity and curability of TGCTs are both dependent on common but still ill-defined epigenetic states.

Association between KITLG Gene Polymorphisms and Testicular Germ Cell Tumors: A Systematic Review and Meta-analysis.

It has been reported that c-KIT ligand (KITLG) gene polymorphisms may be associated with testicular germ cell tumors (TGCT). Owing to mixed and inconclusive results, we conducted a systematic review and meta-analysis to summarize and clarify this association. A systematic search of studies on the association between KITLG gene polymorphisms and TGCT susceptibility was conducted in databases. Odds ratios and 95% confidence intervals were used to pool the effect size. Six articles were included in our systematic review and meta-analysis. Compared with adenine (A), KITLG rs995030 guanine (G) might be associated with increased risk of TGCT. There are insufficient data to fully confirm the association between KITLG rs4474514 and TGCT susceptibility. Well-designed studies with larger sample size and more subgroups are required to validate the risk identified in the current meta-analysis.

Abstract 2684: Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility

Abstract Testicular germ cell tumors (TGCT) are ideally suited to agnostic genome-wide association methods because of their known high heritability and homogeneous cell of origin. Here we report the most updated results from the international Testicular Cancer Consortium (TECAC) that extends published findings from our recent meta-analysis, which included 3,558 cases and 13,970 controls from five centers. To these existing samples, we added summary statistics from 300 cases and 151,991 controls provided by deCODE combined with imputed genotypes from an additional 5,602 cases and 5,006 controls assembled from 15 TECAC centers, almost all (97%) of which were centrally genotyped using the Illumina Human Core array. A total of 9,458 TGCT cases were included in our current meta-analysis, representing a 66% increase since our last publication. We identified 14 new and confirmed 41 previously reported loci that surpassed genome-wide significance (5 × 10-8). Fifteen previously reported loci did not reach this threshold, although one did so in subgroup analysis. In addition, 57 new loci attained nominal significance (5 × 10-8 ≤ p ≤ 1 × 10-5). As expected, a substantial proportion of top hits continue to map to genetic regions belonging to biological pathways essential to male germ cell development, sex determination, and chromosomal segregation. For example, newly identified genes include anaphase promoting complex subunit 2 (ANAPC2), BCL2 like 11 (BCL211), anti-Mullerian hormone receptor type 2 (AMHR2), and DEP domain containing mTOR interacting protein (DEPTOR). Our findings also further implicate genes located on the X chromosome, specifically the androgen receptor (AR). In analyses stratified by tumor subtype, we identified GATA-binding protein 4 (GATA4) as a susceptibility locus among men diagnosed with seminoma. Despite a similar effect size observed among men diagnosed solely with non-seminoma (i.e. excluding cases with a mixture of both seminoma and non-seminoma tumors), this marker did not surpass genome-wide significance in this subtype likely because of the smaller case sample size. GATA4 previously has been associated with overall risk of TGCT (Litchfield et al., Nat Genet. 2017). Biological mechanisms for all genome-wide significant loci are currently being explored using publicly available data sets, ATAC-seq of four TGCT cell lines, and SPATIaL-Seq (a novel high-resolution chromatin conformation capture (3C) assay) of the NTERA2 pluripotent human embryonal carcinoma cell line. Additional genotyping of top hits in an independent sample set of 1,103 TGCT cases and 1,210 controls is currently ongoing. Citation Format: Louise C. Pyle, John Pluta, Kevin T. Nead, Nandita Mitra, Javier Benitez, D. Timothy Bishop, Victoria Cortessis, Alberto Ferlin, Jourik Gietema, Mark Greene, Tom Grotmol, Ramneek Gupta, Rob Hamilton, Michelle A. Hildebrandt, Trine B. Haugen, Lambertus Kiemeney, Christian Kubisch, Davor Lessel, Paloma Martin, Thorunn Rafnar, Lorenzo Richiardi, Rolf Skotheim, Clare Turnbull, Fredrik Wiklund, Tongzhang Zheng, Ewa Rajpert-De Meyts, Stephen M. Schwartz, Katherine A. McGlynn, Peter A. Kanetsky, Katherine L. Nathanson, Testicular Cancer Consortium (TECAC). Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2684.

Genetics of testicular germ cell tumors.

Understanding the molecular basis underlying testicular germ cell tumors (TGCTs) may help improve patient outcomes, particularly for patients with poorer risk or chemoresistant disease. Here, we review the major contemporary advances in elucidating TGCT genetics by discussing patterns of TGCT inheritance, recent genomic and transcriptomic discoveries in TGCT, and the role of genetics in predicting therapeutic resistance and in guiding treatment. In the absence of a major high-penetrance TGCT susceptibility gene, inheritance is likely driven by a complex polygenic model with considerable variation. The most common genomic alterations found in TGCTs include gains in chromosome 12p and mutations in KIT, KRAS, and NRAS, particularly in seminomas. Sensitivity to cisplatin-based chemotherapy likely relies on intact TP53, reciprocal loss of heterozygosity, and high mitochondrial priming. Targetable mutations are uncommon in TGCTs, however, posing a challenge for the development of effective personalized therapies. Consistent with the characteristically low tumor mutational burden, immune checkpoint inhibitors do not appear to be effective for most TGCTs. Refinements in next-generation sequencing techniques over the last few years have enabled considerable advances in elucidating the genomic, transcriptomic, and epigenetic landscape of TGCTs. Future efforts focused on developing novel treatment modalities are needed.

MiRNA-302s may act as oncogenes in human testicular germ cell tumours

Testicular germ cell tumour (TGCT) represents the most common malignancy in young men in large parts of the world, but the aetiology is yet unclear. Multiple TGCT susceptibility loci have been identified, and we have shown that one of these, SPRY4, may act as a TGCT oncogene. Furthermore, many of the loci are in non-coding regions of the genome. miRNAs, a class of non-coding RNAs may play a crucial role in cell proliferation, differentiation, and apoptosis, and alteration in their expression may lead to oncogenesis. Differential expression of miRNAs in TGCT and normal testis has been reported in previous studies. In this study, we used qPCR to analyse, in normal and malignant testis tissue, the expression of the ten miRNAs that we had previously identified by sequencing to be the most upregulated in TGCT. We found high expression of these miRNAs also by qPCR analysis. The levels of miR-302a-3p, miR-302b-3p, and miR-302c-3p were downregulated after treatment of the TGCT cell lines NT2-D1 and 833 K with the chemotherapy drug cisplatin. By using miRNA inhibitor-mediated transient transfection, we inhibited the expression of the three members of miR-302 family (miR-302s). Inhibition of miR-302s resulted in a decreased cell proliferation in NT2-D1 cells, but not in 833 K cells. In both cell lines, inhibition of miR-302s resulted in decreased expression of SPRY4, which we have previously shown to regulate MAPK/ERK and PI3K/Akt signalling pathways in these cells. Inhibition of miR-302b-3p and miR-302c-3p decreased phosphorylation of ERK1/2, whereas inhibition of miR-302a-3p and miR-302b-3p led to decreased expression of the apoptosis inhibitor, survivin. Our findings suggest that miR-302s act as TGCT oncogenes by inducing the expression of SPRY4 and activating MAPK/ERK pathway while inhibiting apoptosis via increased survivin expression.

Association between androgen receptor gene polymorphisms and testicular germ cell tumor: A systematic review and meta-analysis.

To estimate association between androgen receptor (AR) gene polymorphisms and testicular germ cell tumor (TGCT) susceptibility. Systematic search of studies on the association between AR gene polymorphisms and TGCT susceptibility was conducted. Odds ratios and 95% confidence intervals were used to pool effect size. For CAG repeat, no evidence was found for association between (>25 vs. ≤25), (>25 vs. 21-25), (<21 vs. 21-25), (others vs. 21-25), (>23 vs. ≤23), (<21 vs. ≥21), (<21 vs. ≥21)'s some subgroups and TGCT susceptibility, which showed stability. In (>24 vs. ≤24), (>24 vs. 21-24), (<21 vs. 21-24), and (others vs. 21-24) and almost all of their subgroups, increased TGCT risk was found without sensitivity analysis. For GGN, no statistical change of TGCT risk was found in (<23 vs. ≥23), (<23 vs. 23), which showed stability. For single nucleotide polymorphism (SNP) rs6152 G > A, rs1204038 G > A and rs2361634 A > G, no statistical change was found without sensitivity analysis. GGN repeat number <23 may not be associated with TGCTs susceptibility. However, there was insufficient data to fully confirm association in GGN repeat number >23, CAG repeat number, SNP rs6152, rs1204038, and rs2361634.

Association of Inherited Pathogenic Variants in Checkpoint Kinase 2 (CHEK2) With Susceptibility to Testicular Germ Cell Tumors

ImportanceApproximately 50% of the risk for the development of testicular germ cell tumors (TGCTs) is estimated to be heritable, but no mendelian TGCT predisposition genes have yet been identified. It is hypothesized that inherited pathogenic DNA repair gene (DRG) alterations may drive susceptibility to TGCTs.ObjectiveTo systematically evaluate the enrichment of germline pathogenic variants in the mendelian cancer predisposition DRGs in patients with TGCTs vs healthy controls.Design, Setting, and ParticipantsA case-control enrichment analysis was performed from January 2016 to May 2018 to screen for 48 DRGs in 205 unselected men with TGCT and 27 173 ancestry-matched cancer-free individuals from the Exome Aggregation Consortium cohort in the discovery stage. Significant findings were selectively replicated in independent cohorts of 448 unselected men with TGCTs and 442 population-matched controls, as well as 231 high-risk men with TGCTs and 3090 ancestry-matched controls. Statistical analysis took place from January to May 2018.Main Outcomes and MeasuresGene-level enrichment analysis of germline pathogenic variants in individuals with TGCTs relative to cancer-free controls.ResultsAmong 205 unselected men with TGCTs (mean [SD] age, 33.04 [9.67] years), 22 pathogenic germline DRG variants, one-third of which were in CHEK2 (OMIM 604373), were identified in 20 men (9.8%; 95% CI, 6.1%-14.7%). Unselected men with TGCTs were approximately 4 times more likely to carry germline loss-of-function CHEK2 variants compared with cancer-free individuals from the Exome Aggregation Consortium cohort (odds ratio [OR], 3.87; 95% CI, 1.65-8.86; nominal P = .006; q = 0.018). Similar enrichment was also seen in an independent cohort of 448 unselected Croatian men with TGCTs (mean [SD] age, 31.98 [8.11] years) vs 442 unselected Croatian men without TGCTs (at least 50 years of age at time of sample collection) (OR, >1.4; P = .03) and 231 high-risk men with TGCTs (mean [SD] age, 31.54 [9.24] years) vs 3090 men (all older than 50 years) from the Penn Medicine Biobank (OR, 6.30; 95% CI, 2.34-17.31; P = .001). The low-penetrance CHEK2 variant (p.Ile157Thr) was found to be a Croatian founder TGCT risk variant (OR, 3.93; 95% CI, 1.53-9.95; P = .002). Individuals with the pathogenic CHEK2 loss-of-function variants developed TGCTs 6 years earlier than individuals with CHEK2 wild-type alleles (5.95 years; 95% CI, 1.48-10.42; P = .009).Conclusions and RelevanceThis multicenter case-control analysis of men with or without TGCTs provides evidence for CHEK2 as a novel moderate-penetrance TGCT susceptibility gene, with potential clinical utility. In addition to highlighting DNA-repair deficiency as a potential mechanism driving TGCT susceptibility, this analysis also provides new avenues to explore management strategies and biological investigations for high-risk individuals.

Update on epidemiologic considerations and treatment trends in testicular cancer

We aim to give an overview of the epidemiology and treatment trends of testicular germ cell tumors (TGCTs), with an emphasis on recent trends. The incidence of TGCT appears to be increasing, particularly in developed countries, although the reasons are not well understood. There is evidence of racial differences in predisposition to TGCT, with white men having highest risk and men of African or Asian descent having lower risk. In the United States, the incidence of TGCT among Hispanics appears to be rising most quickly. A recent genomic analysis indicates there is no highly penetrant major TGCT susceptibility gene. Incorporation of multidisciplinary care has led to excellent long-term cure rates; however, access to care and insurance remains barriers in young men. Recent treatment trends have centered on maximizing oncologic outcomes while minimizing long-term morbidity. Emerging population-level data provide critical insight into the evolving demographics of TGCT, which may allow for elucidation of biologic and environmental determinants of TGCT. Further, identification of socioeconomic barriers to excellent clinical outcomes will allow for targeted interventions to patients with unique demographic and socioeconomic considerations. Treatment trend analyses suggest that the field is moving toward minimizing treatment-related morbidity.

Large-scale Analysis Demonstrates Familial Testicular Cancer to have Polygenic Aetiology

Testicular germ cell tumour (TGCT) is the most common cancer in young men. Multiplex TGCT families have been well reported and analyses of population cancer registries have demonstrated a four- to eightfold risk to male relatives of TGCT patients. Early linkage analysis and recent large-scale germline exome analysis in TGCT cases demonstrate absence of major high-penetrance TGCT susceptibility gene(s). Serial genome-wide association study analyses in sporadic TGCT have in total reported 49 independent risk loci. To date, it has not been demonstrated whether familial TGCT arises due to enrichment of the same common variants underpinning susceptibility to sporadic TGCT or is due to shared environmental/lifestyle factors or disparate rare genetic TGCT susceptibility factors. Here we present polygenic risk score analysis of 37 TGCT susceptibility single-nucleotide polymorphisms in 236 familial and 3931 sporadic TGCT cases, and 12 368 controls, which demonstrates clear enrichment for TGCT susceptibility alleles in familial compared to sporadic cases (p=0.0001), with the majority of familial cases (84–100%) being attributable to polygenic enrichment. These analyses reveal TGCT as the first rare malignancy of early adulthood in which familial clustering is driven by the aggregate effects of polygenic variation in the absence of a major high-penetrance susceptibility gene. Patient summaryTo date, it has been unclear whether familial clusters of testicular germ cell tumour (TGCT) arise due to genetics or shared environmental or lifestyle factors. We present large-scale genetic analyses comparing 236 familial TGCT cases, 3931 isolated TGCT cases, and 12 368 controls. We show that familial TGCT is caused, at least in part, by presence of a higher dose of the same common genetic variants that cause susceptibility to TGCT in general.

Subphenotype meta-analysis of testicular cancer genome-wide association study data suggests a role for RBFOX family genes in cryptorchidism susceptibility

Can subphenotype analysis of genome-wide association study (GWAS) data from subjects with testicular germ cell tumor (TGCT) provide insight into cryptorchidism (undescended testis, UDT) susceptibility? Suggestive intragenic GWAS signals common to UDT, TGCT case-case and TGCT case-control analyses occur in genes encoding RBFOX RNA-binding proteins (RBPs) and their neurodevelopmental targets. UDT is a strong risk factor for TGCT, but while genetic risk factors for TGCT are well-known, genetic susceptibility to UDT is poorly understood and appears to be more complex. We performed a secondary subphenotype analysis of existing GWAS data from the Testicular Cancer Consortium (TECAC) and compared these results with our previously published UDT GWAS data, and with data previously acquired from studies of the fetal rat gubernaculum. Studies from the National Cancer Institute (NCI), United Kingdom (UK) and University of Pennsylvania (Penn) that enrolled white subjects were the source of the TGCT GWAS data. We completed UDT subphenotype case-case (TGCT/UDT vs TGCT/non-UDT) and case-control (TGCT/UDT vs control), collectively referred to as 'TECAC' analyses, followed by a meta-analysis comprising 129 TGCT/UDT cases, 1771 TGCT/non-UDT cases, and 3967 unaffected controls. We reanalyzed our UDT GWAS results comprising 844 cases and 2718 controls by mapping suggestive UDT and TECAC signals (defined as P < 0.001) to genes using Ingenuity Pathway Analysis (IPA®). We compared associated pathways and enriched gene categories common to all analyses after Benjamini-Hochberg multiple testing correction, and analyzed transcript levels and protein expression using qRT-PCR and rat fetal gubernaculum confocal imaging, respectively. We found suggestive signals within 19 genes common to all three analyses, including RBFOX1 and RBFOX3, neurodevelopmental paralogs that encode RBPs targeting (U)GCATG-containing transcripts. Ten of the 19 genes participate in neurodevelopment and/or contribute to risk of neurodevelopmental disorders. Experimentally predicted RBFOX gene targets were strongly overrepresented among suggestive intragenic signals for the UDT (117 of 628 (19%), P = 3.5 × 10-24), TECAC case-case (129 of 711 (18%), P = 2.5 × 10-27) and TECAC case-control (117 of 679 (17%), P = 2 × 10-21) analyses, and a majority of the genes common to all three analyses (12 of 19 (63%), P = 3 × 10-9) are predicted RBFOX targets. Rbfox1, Rbfox2 and their encoded proteins are expressed in the rat fetal gubernaculum. Predicted RBFOX targets are also enriched among transcripts differentially regulated in the fetal gubernaculum during normal development (P = 3 × 10-31), in response to in vitro hormonal stimulation (P = 5 × 10-45) and in the cryptorchid LE/orl rat (P = 2 × 10-42). GWAS data included in this study are available in the database of Genotypes and Phenotypes (dbGaP accession numbers phs000986.v1.p1 and phs001349.v1p1). These GWAS data did not reach genome-wide significance for any individual analysis. UDT appears to have a complex etiology that also includes environmental factors, and such complexity may require much larger sample sizes than are currently available. The current methodology may also introduce bias that favors false discovery of larger genes. Common suggestive intragenic GWAS signals suggest that RBFOX paralogs and other neurodevelopmental genes are potential UDT risk candidates, and potential TGCT susceptibility modifiers. Enrichment of predicted RBFOX targets among differentially expressed transcripts in the fetal gubernaculum additionally suggests a role for this RBP family in regulation of testicular descent. As RBFOX proteins regulate alternative splicing of Calca to generate calcitonin gene-related peptide, a protein linked to development and function of the gubernaculum, additional studies that address the role of these proteins in UDT are warranted. The Eunice Kennedy Shriver National Institute for Child Health and Human Development (R01HD060769); National Center for Research Resources (P20RR20173), National Institute of General Medical Sciences (P20GM103464), Nemours Biomedical Research, the Testicular Cancer Consortium (U01CA164947), the Intramural Research Program of the NCI, a support services contract HHSN26120130003C with IMS, Inc., the Abramson Cancer Center at Penn, National Cancer Institute (CA114478), the Institute of Cancer Research, UK and the Wellcome Trust Case-Control Consortium (WTCCC) 2. None of the authors reports a conflict of interest.

Large-scale Sequencing of Testicular Germ Cell Tumour (TGCT) Cases Excludes Major TGCT Predisposition Gene

Testicular germ cell tumour (TGCT), the most common cancer in young men, has a significant heritable basis that has long raised questions as to the existence of underlying major high-penetrance susceptibility gene(s). To determine the contribution of rare gene mutations to the inherited risk of TGCT, we analysed germline whole-exome data for 919 TGCT cases and 1609 cancer-free controls. We compared frequencies between TGCT cases and controls of rare (<1%) and low-frequency (1-5%) coding variants (1) individually and (2) collapsed at the gene level via burden testing (T1, disruptive; T2, all deleterious; and T3, all nonsynonymous) using Fisher's exact test with Bonferroni correction of significance thresholds. No individual variant or individual gene showed a significant association with TGCT after correction for multiple testing. In the largest whole-exome sequencing study of testicular cancer reported to date, our findings do not support the existence of a major high-penetrance TGCT susceptibility gene (of odds ratio >10 and allele frequency [combined]>0.01%). Owing to its power, this study cannot exclude the existence of susceptibility genes responsible for occasional TGCT families or of rare mutations that confer very modest relative risks. In concert with findings from genome-wide association studies, our data support the notion that inherited susceptibility is largely polygenic with substantial contribution from common variation. Patient summaryIn the largest study of its kind, we sequenced ∼20 000 genes in 919 men with testicular germ cell tumour (TGCT) and 1609 TGCT-free individuals and found no evidence of a single major gene underlying predisposition to TGCT (in the manner of BRCA1 for breast cancer). Instead, familial risk of TGCT is likely to be due to varying dosages of hundreds of minor genetic factors.

Copy number variations of E2F1: a new genetic risk factor for testicular cancer.

Testicular germ cell tumor (TGCT) is one of the most heritable forms of cancer. In last years, many evidence suggested that constitutional genetic factors, mainly single nucleotide polymorphisms, can increase its risk. However, the possible contribution of copy number variations (CNVs) in TGCT susceptibility has not been substantially addressed. Indeed, an increasing number of studies have focused on the effect of CNVs on gene expression and on the role of these structural genetic variations as risk factors for different forms of cancer. E2F1 is a transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis and response to DNA damage. Therefore, deficiency or overexpression of this protein might significantly influence fundamental biological processes involved in cancer development and progression, including TGCT. We analyzed E2F1 CNVs in 261 cases with TGCT and 165 controls. We found no CNVs in controls, but 17/261 (6.5%) cases showed duplications in E2F1 Blot analysis demonstrated higher E2F1 expression in testicular samples of TGCT cases with three copies of the gene. Furthermore, we observed higher phosphorylation of Akt and mTOR in samples with E2F1 duplication. Interestingly, normal, non-tumoral testicular tissue in patient with E2F1 duplication showed lower expression of E2F1 and lower AKT/mTOR phosphorylation with respect to adjacent tumor tissue. Furthermore, increased expression of E2F1 obtained in vitro in NTERA-2 testicular cell line induced increased AKT/mTOR phosphorylation. This study suggests for the first time an involvement of E2F1 CNVs in TGCT susceptibility and supports previous preliminary data on the importance of AKT/mTOR signaling pathway in this cancer.

Parent-of-origin effects of A1CF and AGO2 on testicular germ-cell tumors, testicular abnormalities, and fertilization bias

Testicular tumors, the most common cancer in young men, arise from abnormalities in germ cells during fetal development. Unconventional inheritance for testicular germ cell tumor (TGCT) risk both in humans and mice implicates epigenetic mechanisms. Apolipoprotein B mRNA-editing enzyme complex 1 (APOBEC1) cytidine deaminase and Deadend-1, which are involved in C-to-U RNA editing and microRNA-dependent mRNA silencing, respectively, are potent epigenetic modifiers of TGCT susceptibility in the genetically predisposed 129/Sv inbred mouse strain. Here, we show that partial loss of either APOBEC1 complementation factor (A1CF), the RNA-binding cofactor of APOBEC1 in RNA editing, or Argonaute 2 (AGO2), a key factor in the biogenesis of certain noncoding RNAs, modulates risk for TGCTs and testicular abnormalities in both parent-of-origin and conventional genetic manners. In addition, non-Mendelian inheritance was found among progeny of A1cf and Ago2 mutant intercrosses but not in backcrosses and without fetal loss. Together these findings suggest nonrandom union of gametes rather than meiotic drive or preferential lethality. Finally, this survey also suggested that A1CF contributes to long-term reproductive performance. These results directly implicate the RNA-binding proteins A1CF and AGO2 in the epigenetic control of germ-cell fate, urogenital development, and gamete functions.

Prospectively Identified Incident Testicular Cancer Risk in a Familial Testicular Cancer Cohort.

Human testicular germ cell tumors (TGCT) have a strong genetic component and a high familial relative risk. However, linkage analyses have not identified a rare, highly penetrant familial TGCT (FTGCT) susceptibility locus. Currently, multiple low-penetrance genes are hypothesized to underlie the familial multiple-case phenotype. The observation that two is the most common number of affected individuals per family presents an impediment to FTGCT gene discovery. Clinically, the prospective TGCT risk in the multiple-case family context is unknown. We performed a prospective analysis of TGCT incidence in a cohort of multiple-affected-person families and sporadic-bilateral-case families; 1,260 men from 140 families (10,207 person-years of follow-up) met our inclusion criteria. Age-, gender-, and calendar time-specific standardized incidence ratios (SIR) for TGCT relative to the general population were calculated using SEER*Stat. Eight incident TGCTs occurred during prospective FTGCT cohort follow-up (versus 0.67 expected; SIR = 11.9; 95% CI, 5.1-23.4; excess absolute risk = 7.2/10,000). We demonstrate that the incidence rate of TGCT is greater among bloodline male relatives from multiple-case testicular cancer families than that expected in the general population, a pattern characteristic of adult-onset Mendelian cancer susceptibility disorders. Two of these incident TGCTs occurred in relatives of sporadic-bilateral cases (0.15 expected; SIR = 13.4; 95% CI, 1.6-48.6). Our data are the first to indicate that despite relatively low numbers of affected individuals per family, members of both multiple-affected-person FTGCT families and sporadic-bilateral TGCT families comprise high-risk groups for incident testicular cancer. Men at high TGCT risk might benefit from tailored risk stratification and surveillance strategies.

Abstract 843: Imputation and meta-analysis of five genome-wide association studies identify multiple new loci associated with testicular germ cell tumor

Abstract Genome-wide association studies (GWAS) have already identified 21 genetic loci associated with testicular germ cell tumor (TGCT). Many of the loci contain biologically plausible genes that function in male germ cell maturation and differentiation, as well as KIT-MAPK signaling and chromosomal segregation. We recently formed the international TEsticular CAncer Consortium (TECAC), and pooled data from the three published and one unpublished GWAS (3,556 TGCT cases and 13,969 controls) to identify additional novel susceptibility loci. We imputed across the data set using the 1,000 Genomes Project version 3 and conducted a fixed effects meta-analysis, including the first analysis of the X chromosome. Eleven new loci achieved genome-wide significance level (p&amp;lt;5e-8), mapping to the following regions: 2q14.2, 3q26.2, 4q35.2, 7q36.3, 10q26.13, 15q21.3, 15q22.31, 16q24.2, 17q12, 19p12 and Xq28, most of which harbor biologically plausible genes. The per allele odds ratios associated with these SNPs continue to be higher than those associated with other cancer types, ranging from 1.2 to 1.6. The signal at 17q12 maps to a region which includes HNF1B, a locus already associated with risk of endometrial and prostate cancer. Two of the signals are in the introns of genes known to be involved in embryonal stem cell pluripotency (TFCLP1 - 2q14.2 and ZFPA2/REX1 - 4q35.2), one in the intron of a chromosomal segregation gene (NCAPG - 27q36.3), and one proximate to MEK1 (MAP2K1 - 15q22.31). The identification of these loci provides additional evidence of the importance of the previously implicated biological pathways. Heritability analysis indicated that these eleven new loci explain approximately 7% more familial (father-to-son) risk in addition to ∼26% explained by previously established 21 loci. Our new findings substantially increase the number of known TGCT susceptibility alleles, thus moving the field closer to a comprehensive understanding of the underlying genetic architecture of TGCT, and providing further clues into the biological etiology of TGCT. Citation Format: Zhaoming Wang, Peter A. Kanetsky, Katherine A. McGlynn, D. Timothy Bishop, Charles C. Chung, Marlene D. Dalgaard, Tom Grotmol, Mark H. Greene, Ramneek Gupta, Trine B. Haugen, Kevin Litchfield, Jennifer T. Loud, Nandita Mitra, Kasper Nielsen, Clare Turnbull, Ewa Rajpert-DeMeyts, Saran Vardhanabhuti, Fredrik Wiklund, Stephen Schwartz, Stephen J. Chanock, Katherine L. Nathanson, TECAC consortium. Imputation and meta-analysis of five genome-wide association studies identify multiple new loci associated with testicular germ cell tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2015-843

Abstract 842: Two new loci and gene sets related to sex determination and cancer progression are associated with susceptibility to testicular germ cell tumor

Abstract Genome-wide association (GWA) studies have reported 19 distinct susceptibility loci for testicular germ cell tumor (TGCT). The aim of the present study was to identify additional loci and gene sets associated with susceptibility to TGCT. A GWA study for TGCT was performed by genotyping 610,240 single nucleotide polymorphisms (SNPs) in 1,326 cases and 6,687 controls from Sweden and Norway. We put forward 34 SNPs from 17 novel regions and 11 SNPs previously reported, for replication in 710 case-parent triads and 289 cases and 290 controls. Predefined biological pathways and processes, in addition to a custom-built sex determination gene set, were subject to enrichment analyses using Meta-Analysis Gene Set Enrichment of Variant Associations (M) and Improved Gene Set Enrichment Analysis for Genome-wide Association Study (I). In the combined meta-analysis, we observed genome-wide significant association for rs7501939 on chromosome 17q12 (OR = 1.29, 95% CI = 1.19-1.40, P = 1.1 × 10-9) and rs2195987 on chromosome 19p12 (OR = 1.31, 95% CI: 1.19-1.45, P = 3.2 × 10-8). The marker rs7501939 on chromosome 17q12 is located in an intron of the HNF1B gene, encoding a member of the homeodomain-containing superfamily of transcription factors. The sex determination gene set (FDRM &amp;lt; 0.001, FDRI &amp;lt; 0.001) and pathways related to NF-κB, glycerophospholipid and ether lipid metabolism, as well as cancer and apoptosis, was associated with TGCT (FDR &amp;lt; 0.1). In addition to revealing two new TGCT susceptibility loci, our results support the notion that genes governing normal germ cell development in utero are implicated in the development of TGCT. Citation Format: Tom Grotmol, Wenche Kristiansen, Robert Karlsson, Trine B. Rounge, Thomas Whitington, Bettina K. Andreassen, Patrik KE Magnusson, Hans-Olov Adami, Clare Turnbull, Trine B. Haugen, Fredrik Wiklund. Two new loci and gene sets related to sex determination and cancer progression are associated with susceptibility to testicular germ cell tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 842. doi:10.1158/1538-7445.AM2015-842

Two new loci and gene sets related to sex determination and cancer progression are associated with susceptibility to testicular germ cell tumor.

Genome-wide association (GWA) studies have reported 19 distinct susceptibility loci for testicular germ cell tumor (TGCT). A GWA study for TGCT was performed by genotyping 610 240 single-nucleotide polymorphisms (SNPs) in 1326 cases and 6687 controls from Sweden and Norway. No novel genome-wide significant associations were observed in this discovery stage. We put forward 27 SNPs from 15 novel regions and 12 SNPs previously reported, for replication in 710 case-parent triads and 289 cases and 290 controls. Predefined biological pathways and processes, in addition to a custom-built sex-determination gene set, were subject to enrichment analyses using Meta-Analysis Gene Set Enrichment of Variant Associations (M) and Improved Gene Set Enrichment Analysis for Genome-wide Association Study (I). In the combined meta-analysis, we observed genome-wide significant association for rs7501939 on chromosome 17q12 (OR = 0.78, 95% CI = 0.72-0.84, P = 1.1 × 10(-9)) and rs2195987 on chromosome 19p12 (OR = 0.76, 95% CI: 0.69-0.84, P = 3.2 × 10(-8)). The marker rs7501939 on chromosome 17q12 is located in an intron of the HNF1B gene, encoding a member of the homeodomain-containing superfamily of transcription factors. The sex-determination gene set (false discovery rate, FDRM < 0.001, FDRI < 0.001) and pathways related to NF-κB, glycerophospholipid and ether lipid metabolism, as well as cancer and apoptosis, was associated with TGCT (FDR < 0.1). In addition to revealing two new TGCT susceptibility loci, our results continue to support the notion that genes governing normal germ cell development in utero are implicated in the development of TGCT.

Testis Cancer: Genes, Environment, Hormones

Testicular cancer (TC) is the most common cancer in males aged 20-40 years, with a worldwide incidence of 7.5 per 100,000, but the rates vary considerably between countries and ethnic groups and there is evidence also for an increasing incidence in last decades. About 95% of all TCs are represented by testicular germ cell tumors (TGCTs), which include seminoma and non-seminoma histological types. It is generally assumed that the development of TGCT is under endocrine control. In particular, unbalanced androgen/estrogen levels and/or activity are believed to represent the key events for TGCT development and progression. Furthermore, recent evidence has suggested genetic association of TGCT with variations in genes involved in hypothalamic-pituitary-testicular axis and steroidogenic enzymes. This recent evidence expands the current knowledge on the role of genetic contribution in testicular cancer susceptibility, and supports the hypothesis that variations in hormone metabolism genes might change the hormonal environment implicated in testicular carcinogenesis. Therefore, hormonal carcinogenesis is an important and controversial area of current research in TGCT, and further attention is given to genetic factors influencing hormone-related cancer risk. The genetic component to TGCT is in general strong. In fact, although environmental factors clearly contribute to TGCT development (and probably to its increasing incidence in some geographical areas), the proportion of TGCT susceptibility accounted for by the genetic effects is estimated at 25%. TGCT has high familial risks compared with most other cancer types that are generally no more than two-fold: brothers of individuals with TGCT have an 8- to 12-fold increased risk of disease, and sons of affected individuals have a 4- to 6-fold increased risk. Despite this strong familial relative risk, early results from linkage studies identified a limited relationship with genetic factors, suggesting that TGCT is a genetically complex trait. However, more recently, four genome-wide association studies (GWAS) from the UK and USA have reported association of TGCTs with six new loci (KITLG, SPRY4, BAK1, DMRT1, TERT, and ATF7IP). The strongest association for TGCT susceptibility was found for SNPs in KITLG (ligand for the membrane-bound receptor tyrosine kinase KIT) gene with a greater than 2.5-fold increased risk of disease per major allele, which is the highest reported for any cancer to date. These studies are being now replicated by other researches and attention is given to the relationship between these genetic variations, TGCT risk and frequently associated anomalies of the reproductive tract, such as cryptorchidism and infertility. Finally, over the past few decades, TCGT research has focused also on external environmental causes acting mainly as endocrine disrupters of androgen and oestrogen pathways, even during the foetal development of the testis. It is well known that the testicular dysgenesis syndrome (TDS) hypothesis, proposed ten years ago, suggests that disturbed testicular development in fetal life may result in one or more of four disorders postnatally, named cryptorchidism, hypospadias, poor semen quality, and TGCT. These four disorders are therefore considered as one clinical entity and are linked together by epidemiological and pathophysiological relations. The relative contribution of genetics and environment in TGCT development, and the interactions between endocrine disruptors and variations in genes involved in hormonal carcinogenesis is therefore another interesting area of research.