Kollmeyer, Thomas

AbstractBackgroundLarge-scale genome-wide association studies (GWAS) have implicated thousands of germline genetic variants in modulating individuals’ risk to various diseases, including cancer. At least 25 risk loci have been identified for low-grade gliomas (LGGs), but their molecular functions remain largely unknown.MethodsWe hypothesized that GWAS loci contain causal single nucleotide polymorphisms (SNPs) that reside in accessible open chromatin regions and modulate the expression of target genes by perturbing the binding affinity of transcription factors (TFs). We performed an integrative analysis of genomic and epigenomic data from The Cancer Genome Atlas and other public repositories to identify candidate causal SNPs within linkage disequilibrium blocks of LGG GWAS loci. We assessed their potential regulatory role via in silico TF binding sequence perturbations, convolutional neural network trained on TF binding data, and simulated annealing–based interpretation methods.ResultsWe built an interactive website (http://education.knoweng.org/alg3/) summarizing the functional footprinting of 280 variants in 25 LGG GWAS regions, providing rich information for further computational and experimental scrutiny. We identified as case studies PHLDB1 and SLC25A26 as candidate target genes of rs12803321 and rs11706832, respectively, and predicted the GWAS variant rs648044 to be the causal SNP modulating ZBTB16, a known tumor suppressor in multiple cancers. We showed that rs648044 likely perturbed the binding affinity of the TF MAFF, as supported by RNA interference and in vitro MAFF binding experiments.ConclusionsThe identified candidate (causal SNP, target gene, TF) triplets and the accompanying resource will help accelerate our understanding of the molecular mechanisms underlying genetic risk factors for gliomas.

Abstract Background: Genome-wide association studies (GWAS) revealed that 25 regions in 24 genes are associated with adult diffuse glioma development. These regions were identified by performing GWAS of glioma overall and by pathology (GBM and nonGBM). The 2016 WHO Classification of Tumors of the Central Nervous System utilizes two somatic alterations to molecularly-classify adult diffuse glioma: IDH mutation and 1p/19q codeletion. We hypothesized that germline variants may increase susceptibility to, or interact with, these somatic alterations to accelerate the development of specific molecular subtypes of glioma. We further hypothesize that germline variants associated with IDH-mutated glioma might be associated with other IDH-mutated tumors, namely, cholangiocarcinoma, acute myeloid leukemia (AML) and melanoma. Methods: We performed a GWAS by glioma molecular subtype - as defined by presence or absence of IDH somatic mutation and 1p/19q codeletion. A total of 1320 glioma cases and 1889 controls were used in the discovery set, and 799 glioma cases and 808 controls in the validation set. A meta-analysis was performed with a genome-wide p-value threshold of 5 × 10−8. GTEx data were used to perform an expression quantitative trait loci (eQTL) analysis. For germline variants that were significantly associated with IDH-mutated glioma, we evaluated pleiotropy with cholangiocarcinoma, AML and melanoma using TCGA and Mayo Biobank controls. Results: Variants in or near D2HGDH on chromosome 2 were genome-wide significant in IDH-mutated glioma (meta p-value = 2.82 × 10−10). TCGA reported that the D2HGDH region was commonly deleted in IDH-mutated gliomas that do not have 1p/19q codeletion. In TCGA data for IDH-mutated, non-codeleted glioma, we observed that the D2HGDH variant was inversely associated with tumor deletions of D2HGDH (odds ratio=0.57, p-value=0.015). The eQTL analyses demonstrated significant associations between D2HGDH germline variant and expression of D2HGDH (p=2.2 × 10−11). Further stratifying IDH-mutated glioma by 1p/19q codeletion status, one variant near FAM20C on chromosome 7 was genome-wide significant in gliomas that have IDH mutation and 1p/19q codeletion (meta p-value=9.56 × 10−9). Analyses are currently underway to evaluate pleiotropy of these IDH-mutated glioma germline variants with other IDH-mutated tumors including cholangiocarcinoma, AML and melanoma. Variants in or near GMEB2 on chromosome 20 were genome-wide significant in IDH wild-type glioma (meta p-value=2.60 × 10−10). The most significant variant in the GMEB2 region remained significant after adjustment for the known RTEL1 glioma risk variant nearby on chromosome 20 (p=0.029). Conclusions: We identified and validated novel germline variants in two genes that are associated with etiology of IDH-mutated and one gene that is associated with IDH wild-type adult diffuse glioma. Citation Format: Jeanette E. Eckel-Passow, Kristen L. Drucker, Thomas M. Kollmeyer, Matthew L. Kosel, Paul A. Decker, Annette M. Molinaro, Terri Rice, Corrine E. Praska, Lauren E. Clark, Alissa A. Caron, Alexej Abyzov, Anthony Batzler, Jun S. Song, Melike Pekmezci, Helen M. Hansen, Lucie S. McCoy, Paige M. Bracci, Joseph Wiemels, John K. Wiencke, Stephen Francis, Terence C. Burns, Caterina Giannini, Daniel H. Lachance, Margaret Wrensch, Robert B. Jenkins. Adult diffuse glioma GWAS by molecular subtype identifies variants in D2HGDH, FAM20C and GMEB2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1193.

AbstractBackgroundOligodendroglioma is genetically defined by concomitant IDH (IDH1/IDH2) mutation and whole-arm 1p/19q codeletion. Codeletion of 1p/19q traditionally evaluated by fluorescence in situ hybridization (FISH) cannot distinguish partial from whole-arm 1p/19q codeletion. Partial 1p/19q codeletion called positive by FISH is diagnostically a “false-positive” result. Chromosomal microarray (CMA) discriminates partial from whole-arm 1p/19q codeletion. Herein, we aimed to estimate the frequency of partial 1p/19q codeletion that would lead to a false-positive FISH result.MethodsFISH 1p/19q codeletion test probe coordinates were mapped onto Oncoscan CMA data to determine the rate of partial 1p/19q codeletion predicted to be positive by FISH. Diffuse astrocytic gliomas with available CMA data (2015–2018) were evaluated and classified based on IDH1-R132H/ATRX/p53 immunohistochemistry, IDH/TERT promoter targeted sequencing, and/or CMA according to classification updates. Predicted false-positive cases were verified by FISH whenever possible.ResultsThe overall estimated false-positive FISH 1p/19q codeletion rate was 3.6% (8/223). Predicted false positives were verified by FISH in 6 (of 8) cases. False-positive rates did not differ significantly (P = .49) between IDH-mutant (4.6%; 4/86) and IDH-wildtype (2.9%; 4/137) tumors. IDH-wildtype false positives were all WHO grade IV, whereas IDH-mutant false positives spanned WHO grades II-IV. Testing for 1p/19q codeletion would not have been indicated for most false positives based on current classification recommendations.ConclusionSelective 1p/19q codeletion testing and cautious interpretation for conflicting FISH and histopathological findings are recommended to avoid potential misdiagnosis.

Abstract Background Twenty-five germline variants are associated with adult diffuse glioma, and some of these variants have been shown to be associated with particular subtypes of glioma. We hypothesized that additional germline variants could be identified if a genome-wide association study (GWAS) were performed by molecular subtype. Methods A total of 1320 glioma cases and 1889 controls were used in the discovery set and 799 glioma cases and 808 controls in the validation set. Glioma cases were classified into molecular subtypes based on combinations of isocitrate dehydrogenase (IDH) mutation, telomerase reverse transcriptase (TERT) promoter mutation, and 1p/19q codeletion. Logistic regression was applied to the discovery and validation sets to test for associations of variants with each of the subtypes. A meta-analysis was subsequently performed using a genome-wide P-value threshold of 5 × 10−8. Results Nine variants in or near D-2-hydroxyglutarate dehydrogenase (D2HGDH) on chromosome 2 were genome-wide significant in IDH-mutated glioma (most significant was rs5839764, meta P = 2.82 × 10−10). Further stratifying by 1p/19q codeletion status, one variant in D2HGDH was genome-wide significant in IDH-mutated non-codeleted glioma (rs1106639, meta P = 4.96 × 10−8). Further stratifying by TERT mutation, one variant near FAM20C (family with sequence similarity 20, member C) on chromosome 7 was genome-wide significant in gliomas that have IDH mutation, TERT mutation, and 1p/19q codeletion (rs111976262, meta P = 9.56 × 10−9). Thirty-six variants in or near GMEB2 on chromosome 20 near regulator of telomere elongation helicase 1 (RTEL1) were genome-wide significant in IDH wild-type glioma (most significant was rs4809313, meta P = 2.60 × 10−10). Conclusions Performing a GWAS by molecular subtype identified 2 new regions and a candidate independent region near RTEL1, which were associated with specific glioma molecular subtypes.

Abstract Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Abstract Genome-wide association studies (GWAS) have revealed that 25 regions in 24 genes are associated with adult diffuse glioma development. These regions were identified by performing GWAS of glioma overall and GWAS by pathology (GBM and nonGBM). Subsequently, these regions have been evaluated for associations with specific molecular subtypes. The 2016 WHO Classification of Tumors of the Central Nervous System utilizes two somatic alterations to molecularly-classify adult diffuse glioma: IDH mutation and 1p/19q codeletion. TERT promoter mutation has also been shown to be associated with age at diagnosis and patient outcome. We hypothesized that germline variants may increase susceptibility to, or interact with, these somatic alterations to accelerate the development of specific molecular subtypes of glioma. To test our hypothesis, we performed a GWAS by glioma molecular subtype – as defined by presence or absence of IDH and TERT somatic mutation and 1p/19q codeletion – utilizing a two-stage design and subsequent meta analysis that included 3001 total glioma cases and 2697 total controls. Data were imputed using the Michigan Server and logistic regression was used, adjusting for age and sex. The Cancer Genome Atlas (TCGA) data were used to perform an expression quantitative trait loci (eQTL) analysis on candidate germline variants. Variants in 2q37 and 7p22 were associated with IDH-mutated glioma (meta analysis p< 5x10-8). The eQTL analyses demonstrated significant associations between 2q37 variants and expression of nearby genes as well as associations between 7p22 variants and nearby genes (p< 0.0001). In conclusion, we identified and validated novel germline variants in two genes that are associated with etiology of IDH-mutated adult diffuse glioma.

AbstractBackgroundGlioblastoma (GBM) represents an aggressive cancer type with a median survival of only 14 months. With fewer than 5% of patients surviving 5 years, comprehensive profiling of these rare patients could elucidate prognostic biomarkers that may confer better patient outcomes. We utilized multiple molecular approaches to characterize the largest patient cohort of isocitrate dehydrogenase (IDH)–wildtype GBM long-term survivors (LTS) to date.MethodsRetrospective analysis was performed on 49 archived formalin-fixed paraffin embedded tumor specimens from patients diagnosed with GBM at the Mayo Clinic between December 1995 and September 2013. These patient samples were subdivided into 2 groups based on survival (12 LTS, 37 short-term survivors [STS]) and subsequently examined by mutation sequencing, copy number analysis, methylation profiling, and gene expression.ResultsOf the 49 patients analyzed in this study, LTS were younger at diagnosis (P = 0.016), more likely to be female (P = 0.048), and MGMT promoter methylated (UniD, P = 0.01). IDH-wildtype STS and LTS demonstrated classic GBM mutations and copy number changes. Pathway analysis of differentially expressed genes showed LTS enrichment for sphingomyelin metabolism, which has been linked to decreased GBM growth, invasion, and angiogenesis. STS were enriched for DNA repair and cell cycle control networks.ConclusionsWhile our findings largely report remarkable similarity between these LTS and more typical STS, unique attributes were observed in regard to altered gene expression and pathway enrichment. These attributes may be valuable prognostic markers and are worth further examination. Importantly, this study also underscores the limitations of existing biomarkers and classification methods in predicting patient prognosis.