Wenche Kristiansen

Identification of 19 new risk loci and potential regulatory mechanisms influencing susceptibility to testicular germ cell tumor

Genome-wide association studies (GWAS) have transformed understanding of susceptibility to testicular germ cell tumors (TGCTs), but much of the heritability remains unexplained. Here we report a new GWAS, a meta-analysis with previous GWAS and a replication series, totaling 7,319 TGCT cases and 23,082 controls. We identify 19 new TGCT risk loci, roughly doubling the number of known TGCT risk loci to 44. By performing in situ Hi-C in TGCT cells, we provide evidence for a network of physical interactions among all 44 TGCT risk SNPs and candidate causal genes. Our findings implicate widespread disruption of developmental transcriptional regulators as a basis of TGCT susceptibility, consistent with failed primordial germ cell differentiation as an initiating step in oncogenesis. Defective microtubule assembly and dysregulation of KIT–MAPK signaling also feature as recurrently disrupted pathways. Our findings support a polygenic model of risk and provide insight into the biological basis of TGCT.

CYP1A1, CYP3A5 and CYP3A7 polymorphisms and testicular cancer susceptibility

Testicular cancer (TC) incidence is increasing worldwide, but the aetiology remains largely unknown. An unbalanced level of oestrogens and androgens in utero is hypothesized to influence TC risk. Polymorphisms in genes encoding cytochrome P450 (CYP) enzymes involved in metabolism of reproductive hormones, such as CYP1A1, CYP3A5 and CYP3A7, may contribute to variability of an individuals susceptibility to TC. The aim of this case-control study was to investigate possible associations between different CYP genotypes and TC, as well as histological type of TC. The study comprised 652 TC cases and 199 controls of Norwegian Caucasian origin. Genotyping of the CYP1A1*2A (MspI), CYP1A1*2C (I462V), CYP1A1*4 (T461N), CYP3A5*3C (A6986G) and CYP3A7*2 (T409R) polymorphisms was performed using TaqMan allelic discrimination or sequencing. The CYP1A1*2A allele was associated with 44% reduced risk of TC with each polymorphic allele [odds ratio (OR) = 0.56, 95% confidence interval (CI) = 0.40-0.78, p(trend) = 0.001], whereas the CYP1A1*2C allele was associated with 56% reduced risk of TC with each polymorphic allele (OR = 0.44, 95% CI = 0.25-0.75, p(trend) = 0.003). The decreased risk per allele was significant for seminomas (OR = 0.46, 95% CI, 0.31-0.70, p(trend) < 0.001 and OR = 0.31, 95% CI = 0.14-0.66, p(trend) = 0.002, respectively), but only borderline significant for non-seminomas (OR = 0.65, 95% CI = 0.45-0.95, p(trend) = 0.027 and OR = 0.55, 95% CI = 0.30-1.01, p(trend) = 0.052, respectively). There were no statistically significant differences in the distribution of the CYP3A5*3C and CYP3A7*2 polymorphic alleles between TC cases and controls. This study suggests that polymorphisms in the CYP1A1 gene may contribute to variability of individual susceptibility to TC.

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.

Genetic variation in AKT1, PTEN and the 8q24 locus, and the risk of testicular germ cell tumor

STUDY QUESTION Is there an association between testicular germ cell tumor (TGCT) and genetic polymorphisms in AKT1, PTEN and the 8q24 locus? SUMMARY ANSWER Our findings suggest that genetic variation in PTEN may influence the risk of TGCT. WHAT IS KNOWN ALREADY There is strong evidence that genetic variation influences the risk of TGCT. The oncogene, AKT1, the tumor suppressor gene, PTEN and the chromosome 8q24 locus play important roles in cancer development in general. STUDY DESIGN, SIZE, DURATION We have conducted a population-based Norwegian-Swedish case-parent study, based on cases diagnosed in 1990-2008, including 831 triads (TGCT case and both parents), 474 dyads (TGCT case and one parent) and 712 singletons (only the TGCT case). In addition we expanded the study to include 3922 unrelated male controls from the TwinGene project. PARTICIPANTS/MATERIALS, SETTING, METHODS We genotyped 26 single nucleotide polymorphisms (SNPs) in AKT1, PTEN and the 8q24 locus. First, triads and dyads were included in a likelihood-based association test. To increase the statistical power, case singletons and controls from the TwinGene project were included in a single test for association. We examined if the allelic effect on TGCT risk differed by histological subgroup, country of origin or parent of origin. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated with Bonferroni correction (P bonf) for multiple testing. MAIN RESULTS AND THE ROLE OF CHANCE In the case-parent analyses, none of the 26 SNPs were significantly associated with TGCT. Of the 23 SNPs investigated in the combined study, one SNP in PTEN (rs11202586) remained associated with TGCT risk after adjusting for multiple testing (OR = 1.16, 95% CI = 1.06-1.28, P bonf = 0.040). We found no difference in risk according to histological subgroup, parent of origin or between countries. LIMITATIONS, REASONS FOR CAUTION Our study is strengthened by the population-based design and large sample size, which gives high power to detect risk alleles. The reported association was not highly significant, and although it was based on an a priori hypothesis of this tumor suppressor gene being implicated in the etiology of TGCT, replication studies, as well as functional studies of this polymorphism, are warranted. WIDER IMPLICATIONS OF THE FINDINGS We report, to our knowledge, a novel association between TGCT and a marker in the tumor suppressor gene PTEN. Previous studies have linked PTEN to TGCT etiology, and there is also a link between PTEN and KITLG, which contains TGCT susceptibility loci revealed through recent genome-wide studies.

Investigation of six testicular germ cell tumor susceptibility genes suggests a parent-of-origin effect in SPRY4

Recent genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) associated with testicular germ cell tumor (TGCT) risk in the genes ATF7IP, BAK1, DMRT1, KITLG, SPRY4 and TERT. In the present study, we validate these associations in a Scandinavian population, and explore effect modification by parental sex and differences in associations between the major histological subtypes seminoma and non-seminoma. A total of 118 SNPs in the six genes were genotyped in a population-based Swedish-Norwegian sample comprising 831 TGCT case-parent triads, 474 dyads, 712 singletons and 3919 population controls. Seven hundred and thirty-four additional SNPs were imputed using reference haplotypes from the 1000 genomes project. SNP-TGCT association was investigated using a likelihood-based association test for nuclear families and unrelated subjects implemented in the software package UNPHASED. Forward stepwise regression within each gene was applied to determine independent association signals. Effect modifications by parent-of-origin and effect differences between histological subtypes were explored. We observed strong association between SNPs in all six genes and TGCT (lowest P-value per gene: ATF7IP 6.2 × 10(-6); BAK1 2.1 × 10(-10); DMRT1 6.7 × 10(-25); KITLG 2.1 × 10(-48); SPRY4 1.4 × 10(-29); TERT 1.8 × 10(-18)). Stepwise regression indicated three independent signals for BAK1 and TERT, two for SPRY4 and one each for DMRT1, ATF7IP and KITLG. A significant parent-of-origin effect was observed for rs10463352 in SPRY4 (maternal odds ratio = 1.72, paternal odds ratio = 0.99, interaction P = 0.0013). No significant effect differences between seminomas and non-seminomas were found. In summary, we validated previously reported genetic associations with TGCT in a Scandinavian population, and observed suggestive evidence of a parent-of-origin effect in SPRY4.

Gene variations in sex hormone pathways and the risk of testicular germ cell tumour: a case–parent triad study in a Norwegian–Swedish population

BACKGROUND Testicular germ cell tumour (TGCT) is the most common cancer in young men, and an imbalance between the estrogen and androgen levels in utero is hypothesized to influence TGCT risk. Thus, polymorphisms in genes involved in the action of sex hormones may contribute to variability in an individuals susceptibility to TGCT. METHODS We conducted a Norwegian-Swedish case-parent study. A total of 105 single-nucleotide polymorphisms (SNPs) in 20 sex hormone pathway genes were genotyped using Sequenom MassArray iPLEX Gold, in 831 complete triads and 474 dyads. To increase the statistical power, the analysis was expanded to include 712 case singletons and 3922 Swedish controls, thus including triads, dyads and the case-control samples in a single test for association. Analysis for allelic associations was performed with the UNPHASED program, using a likelihood-based association test for nuclear families with missing data, and odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. False discovery rate (FDR) was used to adjust for multiple testing. RESULTS Five genetic variants across the ESR2 gene [encoding estrogen receptor beta (ERβ)] were statistically significantly associated with the risk of TGCT. In the case-parent analysis, the markers rs12434245 and rs10137185 were associated with a reduced risk of TGCT (OR = 0.66 and 0.72, respectively; both FDRs <5%), whereas rs2978381 and rs12435857 were associated with an increased risk of TGCT (OR = 1.21 and 1.19, respectively; both FDRs <5%). In the combined case-parent/case-control analysis, rs12435857 and rs10146204 were associated with an increased risk of TGCT (OR = 1.15 and 1.13, respectively; both FDRs <5%), whereas rs10137185 was associated with a reduced risk of TGCT (OR = 0.79, FDR <5%). In addition, we found that three genetic variants in CYP19A1 (encoding aromatase) were statistically significantly associated with the risk of TGCT in the case-parent analysis. The T alleles of the rs2414099, rs8025374 and rs3751592 SNPs were associated with an increased risk of TGCT (OR = 1.30, 1.30 and 1.21, respectively; all FDRs <5%). We found no statistically significant differences in allelic effect estimates between parental inherited genetic variation in the sex hormone pathways and TGCT risk in the offspring, and no evidence of heterogeneity between seminomas and non-seminomas, or between the Norwegian and the Swedish population, in any of the SNPs examined. CONCLUSIONS Our findings provide support for ERβ and aromatase being implicated in the aetiology of TGCT. Exploring the functional role of the TGCT risk-associated SNPs will further elucidate the biological mechanisms involved.

Variations in testosterone pathway genes and susceptibility to testicular cancer in Norwegian men.

Imbalance between the oestrogen and androgen levels in utero is hypothesized to influence testicular cancer (TC) risk. Thus, variation in genes involved in the action of sex hormones may contribute to variability of an individuals susceptibility to TC. Mutations in testosterone pathway genes may alter the level of testosterone in vivo and hypothetically the risk of developing TC. Luteinizing hormone receptor (LHR), 5α-reductase II (SRD5A2) and androgen receptor (AR) are key elements in androgen action. A case-control study comprising 651 TC cases and 313 controls in a Norwegian population was conducted for investigation of polymorphisms in the LHR, SRD5A and AR genes and their possible association with TC. A statistical significant difference was observed in patients being heterozygous for the LHR Asn312Ser polymorphism when comparing genotypes between all TC cases and controls (OR = 0.66, 95% CI = 0.48-0.89, p(adj) = 0.049). No statistically significant difference between the histological subtypes seminoma and non-seminoma was observed. Our results may suggest a possible association between genetic variation in the LHR gene and the risk of developing TC.

Genetic variations associated with the effect of testicular cancer treatment on gonadal hormones

STUDY QUESTION Do genetic variations in the testosterone pathway genes modify the effect of treatment on the levels of testosterone and LH in long-term testicular cancer (TC) survivors (TCSs)? SUMMARY ANSWER Variations in LH receptor (LHR) and in 5α-reductase II (SRD5A2) genes may modify the effect of TC treatment on testosterone levels, whereas genetic variations in the androgen receptor (AR) may modify the effect on LH levels. WHAT IS KNOWN ALREADY TCSs experience variable degrees of long-term reduction in gonadal function after treatment. This variability can in part be explained by treatment intensity, but may also be due to individual variations in genes involved in the function and metabolism of reproductive hormones. STUDY DESIGN, SIZE, DURATION Cross-sectional study on testosterone and LH levels in 637 Norwegian TCSs in relation to genetic variants and TC treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS The single nucleotide polymorphisms LHR Asn291Ser (rs12470652) and Ser312Asn (rs2293275), as well as SRD5A2 Ala49Thr (rs9282858) and Val89Leu (rs523349) were analyzed by allele-specific PCR. The insertion polymorphism LHR InsLQ (rs4539842) was analyzed by sequencing. The numbers of AR CAG and GGN repeats were determined by capillary electrophoresis. Blood samples were collected 5-21 years after diagnosis (median 11 years) and serum total testosterone and LH were analyzed by commercial immunoassays. The TCSs were divided into four groups according to their treatment; surgery only, radiotherapy and chemotherapy with ≤850 or >850 mg of cisplatin. Polymorphisms presenting P < 0.1 for the interaction term with treatment in an initial two-way analysis of covariance (ANCOVA) were investigated further in two consecutive one-way ANCOVA analyses to elucidate the interaction between treatment and genotype. MAIN RESULTS AND THE ROLE OF CHANCE For the whole group of TCSs, there were no significant differences between the hormone levels in homozygotes for the wild type and carriers of at least one polymorphic allele for the investigated polymorphisms. Three of the polymorphisms showed signs of interaction with treatment, i.e. LHR InsLQ, SRD5A2 A49T and the AR CAG repeat. Follow-up analyses revealed three situations where only one of the genotypes of the polymorphism where associated with significantly different hormone levels after surgery compared with after additional cytotoxic treatment: For LHR InsLQ, only the wild-type allele was associated with lower testosterone levels after cisplatin > 850 mg compared with after surgery (24% lower, P < 0.001). For SRD5A2 A49T, testosterone levels were lower after radiotherapy compared with after surgery, but only for the heterozygotes for the polymorphism (39% lower, P = 0.001). In comparison, the testosterone levels were just slightly lower after radiotherapy (6% lower, P = 0.039) or cisplatin ≤ 850 mg (7% lower, P = 0.041), compared with surgery, independent of genotypes. For AR CAG, only the reference length of CAG = 21-22 had significantly higher LH levels after cisplatin ≤ 850 mg compared with after surgery (70% higher, P < 0.001). Independent of genotypes, however, LH levels after cisplatin ≤ 850 mg were only 26% higher than after surgery (P = 0.005). LIMITATIONS, REASONS FOR CAUTION Unadjusted P-values are presented. For analysis involving genotypes, the level of statistical significance was adjusted for the total number of polymorphisms tested, n = 7, i.e. to P < 0.007 (0.5/7). The rather weak associations indicate that additional polymorphisms are involved in the modulation. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this is the first study supporting the notion that polymorphisms may explain at least some of the inter-individual differences in endocrine response to TC treatment. Our findings suggest that individuals with certain genotypes may be more vulnerable to certain treatments. Knowledge on genetic predisposition concerning treatment-related endocrine gonadotoxicity to different treatment regimens may help tailoring TC therapy when possible. STUDY FUNDING/COMPETING INTERESTS This study was supported by the Research Council of Norway (Grant No. 160619). There were no competing interests.

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

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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 < 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 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

Abstract 1348: Association between genetic variation at the AKT1 and PTEN genes, and in the 8q24 locus, and the risk of testicular germ cell tumor.

Background There is strong evidence that genetic variation influences the risk of testicular germ cell tumor (TGCT). The oncogene AKT1, tumor suppressor gene PTEN and the 8q24 locus play important roles in cancer development, and investigation of their role in TGCT is warranted. Methods We investigated the association between risk of TGCT and genetic variation in the AKT1 and PTEN genes, and the 8q24 locus in a Norwegian-Swedish case-parent study. We included 831 triads, 474 dyads and 712 singletons, and 26 single nucleotide polymorphisms (SNPs) were genotyped. First, triads and dyads were included in a likelihood-based association test. To increase the statistical power, case singletons and 3922 unrelated controls from the Swedish TwinGene project were included in a single test for association. Interaction terms were included in the statistical model to examine if the allelic effect on TGCT risk was modified by histological subgroup, country of origin or parent of origin. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated and Bonferroni correction (P bonf ) was used to adjust for multiple testing. Results In the case-parent triad analyses, none of the 26 SNPs were significantly associated with TGCT. Of the 23 SNPs investigated in the combined study, one SNP in the PTEN gene, the rs11202586, remained associated with TGCT risk after adjusting for multiple testing (OR=1.16, 95% CI=1.06-1.28, P bonf= 0.040). We found no difference in risk according to histological subgroup, parent of origin or between countries. Conclusions Our findings suggest that genetic variation in the PTEN gene influences the risk of TGCT. Citation Format: Kristine E. Andreassen, Wenche Kristiansen, Robert Karlsson, Elin L. Aschim, Olav Dahl, Sophie D. Fossa, Hans-Olov Adami, Fredrik Wiklund, Trine B. Haugen, Tom Grotmol. Association between genetic variation at the AKT1 and PTEN genes, and in the 8q24 locus, and the risk of testicular germ cell tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1348. doi:10.1158/1538-7445.AM2013-1348