Anthony J. Hooten

Associations between variants in KITLG, SPRY4, BAK1, and DMRT1 and pediatric germ cell tumors

Recent genome wide association studies have identified susceptibility loci for adult testicular germ cell tumors (GCT) near KITLG, SPRY4, BAK1, and DMRT1. We evaluated variants in these four genes to determine whether these are also susceptibility loci for pediatric GCTs. DNA was isolated from 52 pediatric GCTs (ages 0–21 years) obtained from the Cooperative Human Tissue Network. Control DNA was isolated from de‐identified dried blood spots from 141 white newborns. Genotyping was conducted using TaqMan assays (rs4474514) or by PCR and sequencing (rs4324715, rs210138, and rs755383). Associations between variants and GCT were evaluated using logistic regression with adjustment for sex. We also evaluated whether the associations differed by age at GCT diagnosis (0–9 years, 10–21 years), sex, and tumor location (gonadal, non‐gonadal). We observed a significant association for rs210138 (BAK1) and pediatric GCT overall (odds ratio (OR) = 1.80, 95% confidence interval (CI) 1.10–2.95, P = 0.02) with non‐significant associations similar in magnitude in both the pediatric (P = 0.09) and adolescent (P = 0.06) age groups. The KITLG (rs4474514) and SPRY4 (rs4324715) variants were significantly associated with GCT only in the adolescent age group (rs4474514: OR = 2.28, 95% CI 1.09–4.79, P = 0.03 and rs4324715: OR = 2.40, 95% CI 1.19–4.83, P = 0.01). Associations were mostly similar when stratified by sex. This is the first study to suggest that these loci may also be important in susceptibility to GCTs in the adolescent (KITLG, SPRY4, and BAK1) and pediatric (BAK1) age groups.

DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

BackgroundAberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development.MethodsWe used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing.ResultsMethylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location.ConclusionUnderstanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy.

Ontogeny of Gene Expression: A Changing Environment for Malignancy

More than 13,000 children are estimated to be newly diagnosed with cancer each year in the United States ([1][1]). Despite substantial improvements in survival over the last several decades, cancer is still the leading cause of death due to disease in children ages 1 to 14 years ([2][2]). The

Cross platform analysis of methylation, miRNA and stem cell gene expression data in germ cell tumors highlights characteristic differences by tumor histology

BackgroundAlterations in methylation patterns, miRNA expression, and stem cell protein expression occur in germ cell tumors (GCTs). Our goal is to integrate molecular data across platforms to identify molecular signatures in the three main histologic subtypes of Type I and Type II GCTs (yolk sac tumor (YST), germinoma, and teratoma).MethodsWe included 39 GCTs and 7 paired adjacent tissue samples in the current analysis. Molecular data available for analysis include DNA methylation data (Illumina GoldenGate Cancer Methylation Panel I), miRNA expression (NanoString nCounter miRNA platform), and stem cell factor expression (SABiosciences Human Embryonic Stem Cell Array). We evaluated the cross platform correlations of the data features using the Maximum Information Coefficient (MIC).ResultsIn analyses of individual datasets, differences were observed by tumor histology. Germinomas had higher expression of transcription factors maintaining stemness, while YSTs had higher expression of cytokines, endoderm and endothelial markers. We also observed differences in miRNA expression, with miR-371-5p, miR-122, miR-302a, miR-302d, and miR-373 showing elevated expression in one or more histologic subtypes. Using the MIC, we identified correlations across the data features, including six major hubs with higher expression in YST (LEFTY1, LEFTY2, miR302b, miR302a, miR 126, and miR 122) compared with other GCT.ConclusionsWhile prognosis for GCTs is overall favorable, many patients experience resistance to chemotherapy, relapse and/or long term adverse health effects following treatment. Targeted therapies, based on integrated analyses of molecular tumor data such as that presented here, may provide a way to secure high cure rates while reducing unintended health consequences.

Variants in BAK1, SPRY4, and GAB2 are associated with pediatric germ cell tumors: A report from the children's oncology group

Germ cell tumors (GCT) are a rare form of childhood cancer that originate from the primordial germ cell. Recent genome‐wide association studies (GWAS) have identified susceptibility alleles for adult testicular GCT (TGCT). We test whether these SNPs are associated with GCT in pediatric and adolescent populations. This case‐parent triad study includes individuals with GCT diagnosed between ages 0 and 19. We evaluated 26 SNPs from GWAS of adult TGCT and estimated main effects for pediatric GCT within complete trios (N = 366) using the transmission disequilibrium test. We used Estimation of Maternal, Imprinting and interaction effects using Multinomial modelling to evaluate maternal effects in non‐Hispanic white trios and dyads (N = 244). We accounted for multiple comparisons using a Bonferroni correction. A variant in SPRY4 (rs4624820) was associated with reduced risk of GCT (OR [95% CI]: 0.70 [0.57, 0.86]). A variant in BAK1 (rs210138) was positively associated with GCT (OR [95% CI]: 1.70 [1.32, 2.18]), with a strong estimated effect for testis tumors (OR [95% CI]: 3.31 [1.89, 5.79]). Finally, a SNP in GAB2 (rs948662) was associated with increased risk for GCT (OR [95% CI]: 1.56 [1.20, 2.03]). Nominal associations (P < 0.05) were noted for eight additional loci. A maternal effect was observed for KITLG SNP rs4474514 (OR [95% CI]: 1.66 [1.21, 2.28]) and a paternal parent‐of‐origin effect was observed for rs7221274 (P = 0.00007), near TEX14, RAD51C, and PPM1E. We observed associations between SNPs in SPRY4, BAK1, and GAB2 and GCTs. This analysis suggests there may be common genetic risk factors for GCT in all age groups.

An exploratory analysis of mitochondrial haplotypes and allogeneic hematopoietic cell transplantation outcomes.

Certain mitochondrial haplotypes (mthaps) are associated with disease, possibly through differences in oxidative phosphorylation and/or immunosurveillance. We explored whether mthaps are associated with allogeneic hematopoietic cell transplantation (HCT) outcomes. Recipient (n = 437) and donor (n = 327) DNA were genotyped for common European mthaps (H, J, U, T, Z, K, V, X, I, W, and K2). HCT outcomes for mthap matched siblings (n = 198), all recipients, and all donors were modeled using relative risks (RR) and 95% confidence intervals and compared with mthap H, the most common mitochondrial haplotypes. Siblings with I and V were significantly more likely to die within 5 years (RR = 3.0; 95% confidence interval [CI], 1.2 to 7.9; and RR = 4.6; 95% CI, 1.8 to 12.3, respectively). W siblings experienced higher acute graft-versus-host disease (GVHD) grades II to IV events (RR = 2.1; 95% CI, 1.1 to 2.4) with no events for those with K or K2. Similar results were observed for all recipients combined, although J recipients experienced lower GVHD and higher relapse. Patients with I donors had a 2.7-fold (1.2 to 6.2) increased risk of death in 5 years, whereas few patients with K2 or W donors died. No patients with K2 donors and few patients with U donors relapsed. Mthap may be an important consideration in HCT outcomes, although validation and functional studies are needed. If confirmed, it may be feasible to select donors based on mthap to increase positive or decrease negative outcomes.

Perigestational Dietary Folic Acid Deficiency Protects Against Medulloblastoma Formation in a Mouse Model of Nevoid Basal Cell Carcinoma Syndrome

Hereditary nevoid basal cell carcinoma syndrome (NBCCS) is caused by PTCH1 gene mutations that result in diverse neoplasms including medulloblastoma (MB). Epidemiological studies report reduced pediatric brain tumor risks associated with maternal intake of prenatal vitamins containing folic acid (FA) and FA supplements specifically. We hypothesized that low maternal FA intake during the perigestational period would increase MB incidence in a transgenic NBCCS mouse model, which carries an autosomal dominant mutation in the Ptch1 gene. Female wild-type C57BL/6 mice (n = 126) were randomized to 1 of 3 diets with differing FA amounts: 0.3 mg/kg (low), 2.0 mg/kg (control), and 8.0 mg/kg (high) 1 mo prior to mating with Ptch1 +/− C57BL/6 males. Females were maintained on the diet until pup weaning; the pups were then aged for tumor development. Compared to the control group, offspring MB incidence was significantly lower in the low FA group (Hazard Ratio = 0.47; 95% confidence interval 0.27–0.80) at 1 yr. No significant difference in incidence was observed between the control and high FA groups. Low maternal perigestational FA levels may decrease MB incidence in mice genetically predisposed to tumor development. Our results could have implications for prenatal FA intake recommendations in the presence of cancer syndromes.

Association between mitochondrial DNA haplogroup and myelodysplastic syndromes

Polymorphisms in mitochondrial DNA (mtDNA) are used to group individuals into haplogroups reflecting human global migration and are associated with multiple diseases, including cancer. Here, we evaluate the association between mtDNA haplogroup and risk of myelodysplastic syndromes (MDS). Cases were identified by the Minnesota Cancer Surveillance System. Controls were identified through the Minnesota State drivers license/identification card list. Because haplogroup frequencies vary by race and ethnicity, we restricted analyses to non‐Hispanic whites. We genotyped 15 mtSNPs that capture common European mitochondrial haplogroup variation. We used SAS v.9.3 (SAS Institute, Cary, NC) to calculate odds ratios (OR) and 95% confidence intervals (CI) overall and stratified by MDS subtype and IPSS‐R risk category. We were able to classify 215 cases with confirmed MDS and 522 controls into one of the 11 common European haplogroups. Due to small sample sizes in some subgroups, we combined mt haplogroups into larger bins based on the haplogroup evolutionary tree, including HV (H + V), JT (J + T), IWX (I + W + X), UK (U + K), and Z for comparisons of cases and controls. Using haplogroup HV as the reference group, we found a statistically significant association between haplogroup JT and MDS (OR = 0.58, 95% CI 0.36, 0.92, P = 0.02). No statistically significant heterogeneity was observed in subgroup analyses. In this population‐based study of MDS, we observed an association between mtDNA haplogroup JT and risk of MDS. While previously published studies provide biological plausibility for the observed association, further studies of the relationship between mtDNA variation and MDS are warranted in larger sample sizes.

Abstract LB-374: Variants in BAK1 and SPRY4 are associated with pediatric germ cell tumors:

Introduction. Pediatric germ cell tumors (GCT) are a rare form of childhood cancer that originate from the primordial germ cell. Little is known about etiology and few risk factors have been identified. Both pediatric GCT and adult testicular GCT (TGCT) appear to initiate in utero and thus may share etiologic factors. Several SNPs have been associated with adult TGCT in genome-wide association studies (GWAS). Here we test whether these SNPs are associated with pediatric GCT. Methods. This case-parent triad study includes cases diagnosed with GCT at age 0-19 years between July 1, 2008 and December 31, 2013 who were identified through the Children9s Cancer Research Network (CCRN). Buccal cell DNA was collected from cases and their biological parents. We identified 21 SNPs at 15 independent loci from GWAS of adult TGCT and estimated main effects for pediatric GCT using the transmission disequilibrium test (TDT). We used Estimation of Maternal, Imprinting and interaction effects using Multinomial modelling (EMIM) to evaluate maternal effects in non-Hispanic white trios. A Bonferroni correction was used to account for multiple comparisons. Results. DNA was available for 364 complete trios. The SPRY4 SNP rs4624820 was significantly associated with reduced risk of GCT (OR [95% CI]: 0.69 [0.56, 0.85]; Table). BAK1 SNP rs210138 was associated with an increased risk estimate for GCT (OR [95% CI]: 1.70 [1.32, 2.18]), with a particularly strong estimated effect for testis tumors (OR [95% CI]: 3.31 [1.89, 5.79]). Nominal associations (p Conclusions. We observed significant associations between SNPs in SPRY4 and BAK1 and pediatric GCTs, with a particularly strong association between the SNP in BAK1 and testis tumors. These SNPs may impact risk of GCT through apoptosis pathways. The results of this analysis support that there are common genetic risk factors for pediatric and adult GCT. Citation Format: Erin L. Marcotte, Nathan Pankratz, James Amatruda, A. Lindsay Frazier, Mark Krailo, Stella Davies, Jacqueline Starr, Erica Langer, Caroline Hallstrom, Anthony Hooten, Jenny N. Poynter. Variants in BAK1 and SPRY4 are associated with pediatric germ cell tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-374.

Abstract 812: Maternal folic acid supplementation and risk of medulloblastoma in offspring

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Epidemiological studies can give important insights into factors that modulate disease risk. However, the inherent limitations of observational studies make causal relationships difficult to infer. For example, several case-control studies have indicated that prenatal vitamins may protect against childhood brain tumors, including medulloblastoma, and folic acid (FA) is speculated to be the modulating factor. Using a murine model, we are testing whether low maternal dietary FA during the peri-gestational period increases medulloblastoma risk in offspring. We are using a well-defined transgenic mouse model of Gorlin syndrome, which is characterized by an autosomal dominant mutation in the PTCH1 gene. Heterozygous C57BL/6 strain Ptc1+/− mice have a medulloblastoma incidence of ∼40% at one year, making this transgenic model highly suitable for childhood brain cancer etiologic studies. Methods. A total of 126 female wild-type C57BL/6 mice were randomized to one of three amino acid defined FA diets: 1) 0.3 mg/kg (low), 2) 2.0 mg/kg (control), and 3) 8.0 mg/kg (high), one month prior to mating with Ptc1+/− C57BL/6 males and maintained on their respective diets until weaning of their pups. Red blood cell (RBC) folate measurements were obtained from the dams at weaning and their association with the assigned dietary FA dose was determined using one-way ANOVA. The offspring have been genotyped and weaned heterozygotes are being followed for tumor development for one year. Interim hazard ratios (HRs) and 95% confidence intervals (CIs) were computed using Cox proportional hazards regression to examine the association between the assigned maternal dietary FA dose and offspring tumor incidence. Results. In a total of 381 offspring, the overall Ptc1+/− genotype frequency is similar to previous reports with no significant differences between dietary groups (low: 40%, control: 40%, high: 42%). RBC folate concentrations in the dams at weaning increased significantly with increasing FA dose (p<0.0001). Ptc1+/− offspring from each of the dietary groups have been followed for a mean of ∼6 months. To date, 25%, 35%, and 37% of Ptc1+/− offspring have developed tumors in the low, control, and high FA groups, respectively.Compared to the control group, the hazard for tumor development was non-significantly decreased in offspring in the low FA group (HR=0.7; 95% CI 0.3-1.4) and similar in the high FA group (HR=1.0; 95% CI 0.5-1.8). Conclusions. In contrast to our hypothesis, these preliminary results indicate that higher doses of maternal dietary FA may increase offspring brain tumor incidence. We speculate that higher dietary FA levels during the peri-gestational period may influence brain tumor progression in mice predisposed to tumor development. The implications of these findings with respect to human populations will be discussed. Supported by R03CA141440, T32CA099936, and the Childrens Cancer Research Fund, Minneapolis, MN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 812. doi:10.1158/1538-7445.AM2011-812