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Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase ( IDH) –mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Genetics of recurrent PDX

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.

Figures S1-S8, Supplementary Methods, Supplementary Bibliography

Supplementary Figure 3 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 4 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Figure 1 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Figure 4 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 1 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 3 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 2 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Table 5 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Supplementary Figure 2 from Sleeping Beauty–Mediated Somatic Mutagenesis Implicates CSF1 in the Formation of High-Grade Astrocytomas

Knowledge about inherited and acquired genetics of adult diffuse glioma has expanded significantly over the past decade. Genomewide association studies (GWAS) stratified by histologic subtype identified six germline variants that were associated specifically with glioblastoma (GBM) and 12 that were associated with lower grade glioma. A GWAS performed using the 2016 WHO criteria, stratifying patients by IDH mutation and 1p/19q codeletion (as well as TERT promoter mutation), discovered that many of the known variants are associated with specific WHO glioma subtypes. In addition, the GWAS stratified by molecular group identified two additional novel regions: variants in D2HGDH that were associated with tumors that had an IDH mutation and a variant near FAM20C that was associated with tumors that had both IDH mutation and 1p/19q codeletion. The results of these germline associations have been used to calculate polygenic risk scores, from which to estimate relative and absolute risk of overall glioma and risk of specific glioma subtypes. We will review the concept of polygenic risk models and their potential clinical utility, as well as discuss the published adult diffuse glioma polygenic risk models. To date, these prior genetic studies have been done on European populations. Using the published glioma polygenic risk model, we show that the genetic associations published to date do not generalize across genetic ancestries, demonstrating that genetic studies need to be done on more diverse populations.

Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and frequent mitogen-activated protein kinase (MAPK) pathway activation. We molecularly profiled 13 cases with diagnostic histopathological features of PLNTY (10 female; median age, 16 years; range, 5–52). Patients frequently presented with seizures (9 of 12 with available history) and temporal lobe tumors (9 of 13). MAPK pathway activating alterations were identified in all 13 cases. Fusions were present in the 7 youngest patients: FGFR2-CTNNA3 (n = 2), FGFR2-KIAA1598 (FGFR2-SHTN1) (n = 1), FGFR2-INA (n = 1), FGFR2-MPRIP (n = 1), QKI-NTRK2 (n = 1), and KIAA1549-BRAF (n = 1). BRAF V600E mutation was present in 6 patients (17 years or older). Two fusion-positive cases additionally harbored TP53/RB1 abnormalities suggesting biallelic inactivation. Copy number changes predominantly involving whole chromosomes were observed in all 10 evaluated cases, with losses of chromosome 10q occurring with FGFR2-KIAA1598 (SHTN1)/CTNNA3 fusions. The KIAA1549-BRAF and QKI-NTRK2 fusions were associated respectively with a 7q34 deletion and 9q21 duplication. This study shows that despite its name, PLNTY also occurs in older adults, who frequently show BRAF V600E mutation. It also expands the spectrum of the MAPK pathway activating alterations associated with PLNTY and demonstrates recurrent chromosomal copy number changes consistent with chromosomal instability.

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (i) editing cultured cells; (ii) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, while cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, while detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and copy number alterations acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single nucleotide polymorphism rs55705857 (A>G), which confers a 6-fold increased risk of IDH-mutant low-grade glioma (LGG) and is amongst the highest genetic associations with cancer. By fine-mapping the locus, we reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. To functionally test rs55705857, we generated an IDH1R132H-driven LGG mouse model and show that mutating the highly conserved, orthologous mouse rs55705857 locus dramatically accelerated tumor development from 463 to 172 days and increased penetrance from 30% to 75%. Overall, our work generates new LGG models and reveals mechanisms of the heritable predisposition to lethal glioma in ∼40% of LGG-patients.

BackgroundTwenty-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.MethodsA 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.ResultsNine 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).ConclusionsPerforming a GWAS by molecular subtype identified 2 new regions and a candidate independent region near RTEL1, which were associated with specific glioma molecular subtypes.

BACKGROUND Determination of the causation of germline single nucleotide polymorphisms (SNPs) located in non-coding regions of the genome is challenging. The genomic region of 8q24 has been identified as important in many kinds of cancer, linked to a topologically associated domain (TAD) encompassing MYC; this TAD contains a GWAS SNP (rs55705857) associated with IDH-mutant glioma. METHODS Germline genotyping data from 622 IDH-mutant glioma and 668 controls were used to fine map the rs55705857 locus by detailed haplotype analysis. Chromatin immunoprecipitation sequencing (ChIP-seq) of histone markers H3K4me1, H3K4me3, H3K27ac and H3K36me3 was performed on normal brain samples (n=8) and human glioma samples (n=11 IDH-wt and 52 IDH-mut). RNAseq from 9 normal and 83 brain tumors (n=26 IDH-wt and 55 IDH-mut) were used to assess differential gene expression. RESULTS Fine-mapping identified rs55705857 SNP as the most likely causative allele (OR=8.69; p CONCLUSIONS Fine-mapping of the 8q24 locus provided strong evidence that rs55705857 is the causative 8q24 locus associated with IDH-mut glioma. Functional experiments suggest that IDH mutation facilitates rs55705857 interaction with MYC to alter downstream MYC targets.

Background IDH1/2 mutated glioma has been associated with a germline risk variant, the rs55705857 G allele. The Utah Population Database (UPDB), a computerized genealogy of people in Utah, is a unique resource to evaluate cancer risk in related individuals. Methods One hundred and two individuals with IDH1/2 mutant or 1p/19q co-deleted glioma were genotyped and linked to the UPDB. DNA came from blood (21), tumor tissue (43), or both (38). We determined congruence between somatic and germline samples and estimated the relative risk for developing cancer to first and second-degree relatives of G and A allele carriers at rs55705857. Results Somatic (glioma) DNA had 85.7% sensitivity (CI 57.2–98.2%) and 95.8% specificity (CI 78.9–99.89%) for germline rs55705857 G allele. Forty-one patients were linked to pedigrees in the UPDB with at least three generations of data. First-degree relatives of rs55705857 G allele carriers were at significantly increased risk for developing cancer (RR = 1.72, p = 0.045, CI 1.02–2.94), and specifically for oligodendroglioma (RR = 57.61, p = 0.017, CI 2.96–320.98) or prostate cancer (RR = 4.10, p = 0.008, CI 1.62–9.58); relatives of individuals without the G allele were not at increased risk. Second-degree relatives of G allele carriers also had significantly increased risk for developing cancer (RR = 1.50, p = 0.007, CI 1.15–2.01). Conclusions Tumor DNA may approximate genotype at the rs55705857 locus. We confirmed this locus confers an increased risk of all cancers and especially of oligodendroglioma. No increased cancer or brain tumor risk is seen in family members of individuals without the high-risk G allele. Electronic supplementary material The online version of this article (10.1186/s12885-019-5381-2) contains supplementary material, which is available to authorized users.

BACKGROUND: Twenty-five single nucleotide polymorphisms (SNPs) are associated with adult diffuse glioma risk. We hypothesized that the inclusion of these 25 SNPs with age at diagnosis and sex could estimate risk of glioma as well as identify glioma subtypes. METHODS: Case-control design and multinomial logistic regression were used to develop models to estimate the risk of glioma development while accounting for histologic and molecular subtypes. Case-case design and logistic regression were used to develop models to predict isocitrate dehydrogenase (IDH) mutation status. A total of 1273 glioma cases and 443 controls from Mayo Clinic were used in the discovery set, and 852 glioma cases and 231 controls from UCSF were used in the validation set. All samples were genotyped using a custom Illumina OncoArray. RESULTS: Patients in the highest 5% of the risk score had more than a 14-fold increase in relative risk of developing an IDH mutant glioma. Large differences in lifetime absolute risk were observed at the extremes of the risk score percentile. For both IDH mutant 1p/19q non-codeleted glioma and IDH mutant 1p/19q codeleted glioma, the lifetime risk increased from almost null to 2.3% and almost null to 1.7%, respectively. The SNP-based model that predicted IDH mutation status had a validation concordance index of 0.85. CONCLUSIONS: These results suggest that germline genotyping can provide new tools for the initial management of newly discovered brain lesions. Given the low lifetime risk of glioma, risk scores will not be useful for population screening; however, they may be useful in certain clinically defined high-risk groups.

BACKGROUND Molecular parameters have been incorporated into the 2016 WHO Classification of CNS Tumors and subsequent cIMPACT-NOW updates to facilitate clinical management of glioma patients. However, there have been few reports of the overall clinical utility of comprehensive genetic testing in adult glioma patients. We report the results of sequencing and chromosomal microarray analysis of adult gliomas seen at the Mayo Clinic and through the Mayo Clinic Laboratories (MCL) reference practice. METHODS A consecutive series of 379 Mayo Clinic adult glioma patients were consented to receive targeted next generation sequencing and chromosomal microarray analysis, regardless of standard clinical ordering practices. Known diagnostic, prognostic, and predictive alterations were annotated for each case. These results were compared to the larger MCL reference practice of 2400 adult glioma samples that received one or both tests clinically. RESULTS Of the consecutive Mayo Clinic cases, 67% had alterations that fell into at least one of the diagnostic, prognostic, and/or predictive categories. Molecular testing generated diagnostic, prognostic, and predictive information in 44%, 34%, and 36% of cases, respectively. Diagnostically, molecular testing mainly aided in arriving at a final integrated diagnosis, and only a small number of cases (n= 5; 1%) had a change in diagnosis. The consecutive cases represent the typical distribution of adult glioma types (47% IDH-wildtype glioblastoma/astrocytoma, 22% IDH-mutant astrocytoma/glioblastoma, 13% IDH-mutant 1p/19q-codeleted oligodendroglioma, and 18% other tumor types). The MCL practice is enriched in tumors with atypical molecular patterns (37% IDH-wildtype glioblastoma/astrocytoma, 17% IDH-mutant astrocytoma/glioblastoma, 18% IDH-mutant 1p/19q-codeleted oligodendroglioma, and 29% other tumor types). CONCLUSIONS Molecular testing provides useful diagnostic, prognostic, and predictive information for the clinical management of adult glioma patients. While the current guidelines for ordering molecular testing are effective for diagnostic purposes, additional prognostic and predictive information can be gleaned from comprehensive molecular testing of adult gliomas.

Background Large-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. Methods We 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. Results We 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. Conclusions The 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.

BackgroundOligodendroglioma 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.

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.

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.

Low-grade glioma (LGG) are generally slowly growing brain cancers, that frequently undergo malignant progression to aggressive, secondary glioblastoma with a dismal prognosis. By combining genetically engineered Idh1-mutant mice with in vivo CRISPR gene editing we generated a mouse model faithfully recapitulating the founder mutations of LGG. Clonal activation of the neomorphic Idh1 R132H mutation cooperates with Trp53 and Atrx mutations to trigger development of brain tumors but only with ~30% penetrance and very long latency. To elucidate the molecular mechanisms underlying the malignant progression of IDH1-mutant LGG, we devised and deployed a direct in vivo CRISPR screen targeting genes commonly mutated in human IDH-mutant secondary glioblastoma. Stereotaxic delivery of a lentiviral sgRNA library targeting the mouse orthologs of these genes into the brain of Idh1 R132H ;Trp53;Atrx;Cas9 and control Idh1 wt ;Trp53;Atrx;Cas9 compound mutant mice resulted in rapid formation of tumors that recapitulate human Idh1-mutant glioblastoma. Deconvoluting the screen showed that PI3K pathway members Pten and Pik3ca as well as Notch1, Smarca4 and Fat1 are preferentially enriched in Idh1 R132H-tumors, while Rb1 and NF2 were enriched in Idh1 wt tumors. Co-mutation analysis further identified additional co-occurring driver combinations such as Bcor-Met, Olig2-Met, Olig2-Med12 or Bcor-Olig2. We validated the tumor suppressive function of Notch1 and Pten using conventional floxed knock-out alleles and found that Notch1 functions in a haploinsufficient manner. Interestingly, Idh1 R132H did not alter tumor latency or pathology in a high grade p53;Pten;Rb1 mutant background, indicating that the neomorphic IDH-mutations can drive low but not high grade glioma development. Our study provides a functional landscape of gliomagenesis suppressors in vivo.

Abnormalities on brain MRI may be identified in ~15% of individuals aged 50-66. It can be difficult to discriminate glioma, CNS lymphoma, Inflammatory Demyelinating Disease (CNSIDD) and solitary metastasis, and misdiagnosis may expose patients to unnecessary anxiety, surgery, or radiotherapy. CNS lymphoma requires only biopsy, solitary metastasis may be resected and radiated or radiated empirically, and high-grade glioma requires maximal safe resection followed by chemoradiation. CNSIDD should only be biopsied when diagnostic uncertainty requires it, and resection and radiotherapy are unnecessary, introducing unwarranted morbidity. Polygenic risk models can identify patients at the highest risk of developing glioma; we hypothesized that these polygenic models could help with differential diagnosis of indeterminate brain lesions. We also hypothesized that race would be an important contributing factor in the models. In the initial discovery and validation European (EUR) cohorts the mean probability of glioma for IDHmut non-codeleted glioma was 0.55 and 0.52, respectively, and in healthy controls was 0.19 and 0.21, respectively. To further evaluate sensitivity, we analyzed additional genotype data from 867 gliomas (764 EUR, 54 AFR, 24 AMR, 15 EAS, 10 SAS) from The Cancer Genome Atlas (TCGA). Across 764 EUR IDHmut non-codeleted glioma, the mean probability 0.53, whereas across 54 AFR and 24 AMR the mean was 0.22 and 0.32, respectively. To evaluate specificity, we analyzed 3200 TCGA patients with primary tumor types that commonly metastasize to the brain (2676 EUR, 365 AFR, 46 AMR, 95 EAS, 18 SAS), and 236 AFR healthy controls. For patients with non-CNS primary tumors, the mean probability ranged from 0.09-0.18 for EUR and 0.04-0.07 for AFR. For AFR healthy controls, the mean was 0.05. Overall, race is a significant factor for polygenic risk models. Further work entails evaluating polygenic risk models in IDHwt glioma, CNS lymphoma and CNSIDD cohorts as well as in additional races.

Establishing causal links between genetic polymorphisms and increased heritable risk of developing brain cancer is a major challenge. The non-coding single nucleotide polymorphism rs55705857 (A >G) is associated with a ~6-fold increased risk to develop IDH-mutant low-grade glioma (LGG). The rs55705857 G allele has a minor allele frequency of only ~5% in the general population but is found in ~40% of patients with IDH-mutant LGG and patients carrying risk-alleles are diagnosed on average 7-12 years earlier than those carrying non-risk A alleles. This makes rs55705857 one of the highest reported genetic associations with cancer, comparable with inherited BRCA1 gene mutations and the risk of developing breast cancer or other familial glioma genes such as NF1/2, CDKN2A or p53. To generate a LGG mouse model, we combined clonal activation of IDH1R132H with mutations of Trp53 and Atrx, which resulted in the development of LGG-like brain tumors in 25% of mice. Mutating the highly conserved, orthologous mouse rs55705857 locus to mimic the human risk allele dramatically accelerated tumor development from 463 to 172 days and increased penetrance to 75%. The resulting tumors exhibit elevated R-2-hydroxyglutarate levels, well-differentiated fibrillary neoplastic histology and metabolic rewiring, recapitulating histopathological and molecular hallmarks of human LGG. Mechanistically, we show that the rs55705857 locus resides within a brain-specific enhancer, which shows enhanced activity in IDH-mutant tumors. In addition, we found that the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. The hyperactive chromatin status combined with the tissue specificity of this enhancer explains the cooperativity between mutant IDH and rs55705857 and why rs55705857 is associated specifically with IDH-mutant glioma, but not other cancers. Overall, we generated new LGG mouse models, which provide insights into the pathophysiology of this deadly disease and shed light into the heritable predisposition to LGG development.

BACKGROUND: Single-gene mutation syndromes account for some familial glioma (FG); however, they make up only a small fraction of glioma families. Gliomas can be classified into 3 major molecular subtypes based on isocitrate dehydrogenase (IDH) mutation and 1p/19q codeletion. We hypothesized that the prevalence of molecular subtypes might differ in familial versus sporadic gliomas and that tumors in the same family should have the same molecular subtype. METHODS: Participants in the FG study (Gliogene) provided samples for germline DNA analysis. Formalin-fixed, paraffin-embedded tumors were obtained from a subset of FG cases, and DNA was extracted. We analyzed tissue from 75 families, including 10 families containing a second affected family member. Copy number variation data were obtained using a first-generation Affymetrix molecular inversion probe (MIP) array. RESULTS: Samples from 62 of 75 (83%) FG cases could be classified into the 3 subtypes. The prevalence of the molecular subtypes was: 30 (48%) IDH-wildtype, 21 (34%) IDH-mutant non-codeleted, and 11 (19%) IDH-mutant and 1p/19q codeleted. This distribution of molecular subtypes was not statistically different from that of sporadic gliomas (P = 0.54). Of 10 paired FG samples, molecular subtypes were concordant for 7 (κ = 0.59): 3 IDH-mutant non-codeleted, 2 IDH-wildtype, and 2 IDH-mutant and 1p/19q codeleted gliomas. CONCLUSIONS: Our data suggest that within individual families, patients develop gliomas of the same molecular subtype. However, we did not observe differences in the prevalence of the molecular subtypes in FG compared with sporadic gliomas. These observations provide further insight into the distribution of molecular subtypes in FG.

// Matthias Holdhoff 1 , Gregory J. Cairncross 2 , Thomas M. Kollmeyer 3 , Ming Zhang 1 , Peixin Zhang 4 , Minesh P. Mehta 5 , Maria Werner-Wasik 6 , Luis Souhami 7 , Jean-Paul Bahary 8 , Young Kwok 5 , Alan C. Hartford 9 , Arnab Chakravarti 10 , Srinivasan Yegnasubramanian 1 , Bert Vogelstein 1 , Nickolas Papadopoulos 1 , Kenneth Kinzler 1 , Robert B. Jenkins 3 and Chetan Bettegowda 1 1 The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA 2 Charbonneau Cancer Institute at the University of Calgary, Calgary, AB, USA 3 Mayo Clinic, Rochester, MN, USA 4 NRG Oncology Statistics and Data Management Center, Philadelphia, PA, USA 5 University of Maryland Medical Center, Baltimore, MD, USA 6 Thomas Jefferson University Hospital, Philadelphia, PA, USA 7 McGill University Health Centre, Montreal, QC, Canada 8 Centre Hospitalier de l’Universite de Montreal, Montreal University, Montreal, QC, Canada 9 Darthmouth-Hitchcock Medical Center, Lebanon, NH, USA 10 The Ohio State University, Columbus, OH, USA Correspondence to: Matthias Holdhoff, email: // Chetan Bettegowda, email: // Keywords : oligodendroglioma, chemotherapy, survival, genomics, co-deletion 1p/19q Received : October 01, 2016 Accepted : March 21, 2017 Published : March 31, 2017 Abstract Background: The NRG Oncology RTOG 9402 trial showed significant survival benefit in patients with 1p/19q co-deleted anaplastic oligodendrogliomas (AO) who received both radiation (RT) and chemotherapy (PCV regimen) versus RT alone. Substantial separation of the survival curves was only seen after 7.3 years. We aimed to determine whether there are specific genetic alterations that distinguish co-deleted AO patients who benefit from the addition of PCV from those who do not. Methods: We performed whole exome sequencing on matched tumor and normal DNA from all available short-term (STS) and long-term survivors (LTS) who received RT+PCV. hTERT status and rs55705857 genotypes (G-allele) were analyzed in both cohorts. Results: Six STS (survival of

BACKGROUND Oligodendrogliomas are classified as either WHO grade II or III depending on histologic features. Grade often influences treatment decisions. However, there is variability in patient outcome within tumors of similar grade. We hypothesized that copy number burden (CNB) and specific copy number variants (CNV) might be associated with oligodendroglioma grade and prognosis. METHODS Copy number array analyses were performed on 285 molecular oligodendrogliomas (IDH-mutant, 1p/19q-whole arm-codeleted) from the Mayo Clinic internal and consult neuropathology practice and 167 TCGA molecular oligodendrogliomas. CNB was defined as the total number of copy number alterations. The association of CNB and CNV with grade and overall survival (when available) was assessed. All Mayo and TCGA data were evaluated using the ChAS software suite (Thermo- Fisher) and blindly reviewed by a clinical cytogeneticist (RBJ). RESULTS The mean CNB was 5.0 and 10.4 in the Mayo WHO grade II and III oligodendrogliomas, respectively (p = 5.4 x 10–17). Among the TCGA WHO grade II and III oligodendrogliomas the mean CNB was 4.4 and 5.3, respectively (p = 0.034). Common CNVs (occurring in at least 5% of cases) were -4/4q-, +8/8q+, -9/9p-/cnLOH 9p, +11/11q+, -14/14q-, -15 and -18/18q-. Of these, -9/9p-/cnLOH 9p was significantly associated with higher grade in both the Mayo and TCGA cohorts (p = 8.3 x 10-10 and 0.018, respectively). In the TCGA cohort the presence of >10 CNVs or +11/11q+ was associated with a poorer survival (p = 0.016 and 0.006, respectively). CONCLUSIONS CNB is significantly associated with WHO grade in IDH-mutant, 1p/19q co-deleted oligodendrogliomas. The presence of a significantly elevated CNB in some WHO grade II tumors may be suspicious for the presence of unappreciated grade III histologic features. Longer follow-up will be necessary to determine if CNB or single CNVs are associated with survival.

BackgroundGlioblastoma (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.

Molecular parameters were incorporated in the WHO classification of central nervous system (CNS) tumors. Despite advances in understanding the molecular biology of CNS tumors, targetable genomic abnormalities are lacking. To identify diagnostically, prognostically and potential therapeutically relevant abnormalities, we developed a next generation sequencing (NGS) assay for formalin-fixed paraffin-embedded (FFPE) tissue that evaluates 219 genes associated with adult and pediatric CNS tumors. This test consists of a DNA and an RNA subpanel for the detection of sequence alterations and gene rearrangements (known gene fusions and abnormal transcript variants, and novel fusion transcripts that contain any of the interrogated genes as a partner). The assay utilizes an amplicon-based approach with molecular barcode chemistry (to allow traceability of PCR artifacts/duplicates), Illumina sequencing and custom bioinformatics pipelines. Analytical validation included 175 samples. Overall concordance with alternative methods were 99% and 96% and success rates were 97% and 95% for the DNA and RNA subpanels, respectively. Inter and intra-assay reproducibility was 100% for both subpanels. The limit of detection (analytical sensitivity) for nucleic acid input and tumor content were 8.5 ng and 30%, and 10 ng and 10% for the DNA and RNA subpanels, respectively. The analytical specificity was high, with per base DNA sequencing false positive rate

Patient derived xenograft (PDX) models have shown great utility for pre-clinical and translational studies for a range of malignancies. We have established a comprehensive, publically available collection of 95 high-grade glioma flank PDX models. Viable PDX were derived from Glioblastoma, IDH-wildtype (n=91), Glioblastoma, IDH-mutant (n=2), Diffuse Midline Glioma, H3 K27M-mutant (n=1), and Anaplastic Oligodendroglioma (n=1) and include both primary (n=60) and recurrent (n=35) tumors. Comprehensive molecular characterization of PDX is ongoing and, to date, has included whole exome sequencing (WES, n=82), RNA-sequencing (n=40), and genome-wide methylation profiling (n=78) that included MGMT promoter, with data available in cBioPortal. PDX reflected the genetic characteristics of glioblastoma, with the majority harboring TERT promoter mutations, chromosomal gain +7, loss -10 and homozygous deletion of CKDN2A/B. EGFR alterations were frequent (~40%) and included amplification, point mutation, EGFRvIII, and other splice variants. Other common alterations, including amplifications of MET, CDK4, CDK6, MDM2, MDM4 and mutation of TP53, PTEN, NF1, RB1, PIK3CA, PIK3R1 were present at similar frequencies reported by TCGA, with the exception of PDGFRA alterations that were underrepresented in PDX. RNA-sequencing showed representation of each of the glioblastoma gene expression subtypes. To assess preservation of genetic features during xeongrafting, we performed WES on 20 matched patient tumors. The vast majority of genetic driver alterations were shared between patient and PDX, including EGFR, EGFRvIII, CDK4, MDM2, and MET amplifications. In 3 PDX, subclonal selection events were observed, including amplifications of MYCN, CDK6 and an EGFR splice variant, as well as selection against PDGFRA amplification. Overall, our large panel of PDX models reflects the genetic heterogeneity of glioblastoma and largely preserves the genetic features of the primary patient tumors. The PDX National Resource is a powerful tool for neuro-oncology research, with all PDX models and genomic data openly available (http://www.mayo.edu/research/labs/translational-neuro-oncology/mayo-clinic-brain-tumor-patient-derived-xenograft-national-resource).

Background Glioblastoma (GBM) exhibits profound intratumoral genetic heterogeneity. Each tumor comprises multiple genetically distinct clonal populations with different therapeutic sensitivities. This has implications for targeted therapy and genetically informed paradigms. Contrast-enhanced (CE)-MRI and conventional sampling techniques have failed to resolve this heterogeneity, particularly for nonenhancing tumor populations. This study explores the feasibility of using multiparametric MRI and texture analysis to characterize regional genetic heterogeneity throughout MRI-enhancing and nonenhancing tumor segments. Methods We collected multiple image-guided biopsies from primary GBM patients throughout regions of enhancement (ENH) and nonenhancing parenchyma (so called brain-around-tumor, [BAT]). For each biopsy, we analyzed DNA copy number variants for core GBM driver genes reported by The Cancer Genome Atlas. We co-registered biopsy locations with MRI and texture maps to correlate regional genetic status with spatially matched imaging measurements. We also built multivariate predictive decision-tree models for each GBM driver gene and validated accuracies using leave-one-out-cross-validation (LOOCV). Results We collected 48 biopsies (13 tumors) and identified significant imaging correlations (univariate analysis) for 6 driver genes: EGFR, PDGFRA, PTEN, CDKN2A, RB1, and TP53. Predictive model accuracies (on LOOCV) varied by driver gene of interest. Highest accuracies were observed for PDGFRA (77.1%), EGFR (75%), CDKN2A (87.5%), and RB1 (87.5%), while lowest accuracy was observed in TP53 (37.5%). Models for 4 driver genes (EGFR, RB1, CDKN2A, and PTEN) showed higher accuracy in BAT samples (n = 16) compared with those from ENH segments (n = 32). Conclusion MRI and texture analysis can help characterize regional genetic heterogeneity, which offers potential diagnostic value under the paradigm of individualized oncology.

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.

Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and genetic MAPK pathway activation through alterations such as BRAF V600E mutation, and FGFR2-KIAA1598/CTNNA3 and FGFR3-TACC3 fusions. We report the molecular profiling of PLNTY in 9 patients, 7 female: 2 male, median age at diagnosis of 16 years (range, 5–34). All tumors were supratentorial with diagnostic morphological features of PLNTY including oligodendroglioma-like areas and strong diffuse CD34 immunostaining. Four (of 9; 44%) tumors were positive for BRAF V600E immunostain, indicating the presence of a BRAF V600E mutation. The 6 cases evaluated for IDH status were negative by IDH1-R132H immunostain (n=5; 3 BRAF V600E-negative and 2 BRAF V600E-positive) or by NGS (n=1; BRAF V600E-negative). Oncoscan chromosomal microarray performed in 5 BRAF V600E-negative tumors showed recurrent copy number changes including gain of whole chromosomes 5, 7, 8, 9, 12, 18, 19, 20, 21 and X, and loss of chromosomes 1, 2, 10q, 13, 22 and Xq. The 10q losses (n=2) were highly suggestive of an FGFR2-KIAA1598 and of a potentially novel FGFR2 underlying fusion event in one case each. Custom targeted neuro-oncology DNA and RNA NGS panel performed in 2 cases revealed a fusion similar to fusions previously reported in pilocytic astrocytoma: one with a KIAA1549-BRAF (exon 15-exon 9) fusion associated with a 7q34 ~785 kilobase deletion rather than the characteristic ~2 megabase duplication seen in pilocytic astrocytoma, and another with a QKI-NTRK2 (exon 6-exon 15) fusion associated with a 9q21 ~191 kilobase duplication disrupting NTRK2. The KIAA1549-BRAF fusion-positive case also had a TP53 mutation with loss of whole chromosome 17, suggesting TP53 complete inactivation. This study confirms that PLNTY is a low-grade neuroepithelial tumor with frequent MAPK pathway activation and expands the spectrum of MAPK activating alterations observed in PLNTY.

© 2017, Springer-Verlag Berlin Heidelberg. The “integrated diagnosis” for infiltrating gliomas in the 2016 revised World Health Organization (WHO) classification of tumors of the central nervous system requires assessment of the tumor for IDH mutations and 1p/19q codeletion. Since TERT promoter mutations and ATRX alterations have been shown to be associated with prognosis, we analyzed whether these tumor markers provide additional prognostic information within each of the five WHO 2016 categories. We used data for 1206 patients from the UCSF Adult Glioma Study, the Mayo Clinic and The Cancer Genome Atlas (TCGA) with infiltrative glioma, grades II-IV for whom tumor status for IDH, 1p/19q codeletion, ATRX, and TERT had been determined. All cases were assigned to one of 5 groups following the WHO 2016 diagnostic criteria based on their morphologic features, and IDH and 1p/19q codeletion status. These groups are: (1) Oligodendroglioma, IDH-mutant and 1p/19q-codeleted; (2) Astrocytoma, IDH-mutant; (3) Glioblastoma, IDH-mutant; (4) Glioblastoma, IDH-wildtype; and (5) Astrocytoma, IDH-wildtype. Within each group, we used univariate and multivariate Cox proportional hazards models to assess associations of overall survival with patient age at diagnosis, grade, and ATRX alteration status and/or TERT promoter mutation status. Among Group 1 IDH-mutant 1p/19q-codeleted oligodendrogliomas, the TERT-WT group had significantly worse overall survival than the TERT-MUT group (HR: 2.72, 95% CI 1.05–7.04, p = 0.04). In both Group 2, IDH-mutant astrocytomas and Group 3, IDH-mutant glioblastomas, neither TERT mutations nor ATRX alterations were significantly associated with survival. Among Group 4, IDH-wildtype glioblastomas, ATRX alterations were associated with favorable outcomes (HR: 0.36, 95% CI 0.17–0.81, p = 0.01). Among Group 5, IDH-wildtype astrocytomas, the TERT-WT group had significantly better overall survival than the TERT-MUT group (HR: 0.48, 95% CI 0.27–0.87), p = 0.02). Thus, we present evidence that in certain WHO 2016 diagnostic groups, testing for TERT promoter mutations or ATRX alterations may provide additional useful prognostic information.

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Glioma, the most common central nervous system cancer in adults, has poor prognosis. Here we identify a new SNP associated with glioma risk, rs1920116 (near TERC), that reached genome-wide significance (Pcombined= 8.3 × 10-9) in a meta-analysis of genome-wide association studies (GWAS) of high-grade glioma and replication data (1,644 cases and 7,736 controls). This region has previously been associated with mean leukocyte telomere length (LTL). We therefore examined the relationship between LTL and both this new risk locus and other previously established risk loci for glioma using data from a recent GWAS of LTL (n = 37,684 individuals). Alleles associated with glioma risk near TERC and TERT were strongly associated with longer LTL (P = 5.5 × 10-20and 4.4 × 10-19, respectively). In contrast, risk-associated alleles near RTEL1 were inconsistently associated with LTL, suggesting the presence of distinct causal alleles. No other risk loci for glioma were associated with LTL. The identification of risk alleles for glioma near TERC and TERT that also associate with telomere length implicates telomerase in gliomagenesis.© 2014 Nature America, Inc. All rights reserved.

INTRODUCTION: Gliomas are typified by genetic variability and instability. The full potential of molecular genetics to classify tumors has not been realized. We previously proposed a novel glioma classification system that increases the sensitivity and specificity of diagnosis and improve prognostication. We have completed copy number array mutational analyses and a targeted next-generation sequencing 50-gene glioma panel on 148 Mayo Clinic formalin-fixed paraffin-embedded gliomas with subsequent data validation performed on 419 gliomas from the TCGA. RESULTS: Median age at resection was 49 years (range: 17–80; 95% CI: 46.5–51.6 years). There were 36 (24.3%) IDH-mutant, 1p/19q codeleted oligodendrogliomas; 46 (31.1%) IDH-mutant, 1p/19q non-codeleted astrocytomas and 66 (44.6%) IDH-wildtype gliomas. Small terminal alterations (STAs) defined as copy number variations (CNVs) IDH-mutant, 1p/19q non-codeleted tumors. Acquisition of (3)2 STAs was observed in 47% IDH-mutant, 1p/19q non-codeleted versus 27% IDH-wildtype tumors (P=1.8x10(-5)). In the TCGA dataset, these were seen in 67% versus 26% respectively (P=2.8x10(-16)). STAs were glioma subclass-specific and did not correlate with overall genetic instability as determined manually or with a computational method. ATRX loss was observed in 27 of 41 (65.9%) tumors with an ATRX mutation versus 20 of 106 (18.9%) ATRX-wildtype tumors (P=1.3x10(-7)). ATRX loss also correlated with the alternative lengthening of telomeres (ALT) phenotype and was seen in 25 of 31 (80.6%) ALT-positive versus 7 of 55 (12.7%) ALT-negative tumors (P=1.5x10(-9)). STAs were present in 19 of 32 (59.4%) ALT-positive cases and 8 of 35 (22.9%) ALT-negative cases (P=0.0061). CONCLUSION: IDH-mutant, 1p/19q non-codeleted astrocytomas have a high prevalence of STAs. This is associated with ATRX loss and the ALT. These alterations do not appear to have de novo implications for survival but may provide novel diagnostic tools and therapeutic targets.

To date, 25 single nucleotide polymorphisms (SNPs) have been shown to be associated with overall glioma risk or with risk of specific subtypes of glioma. We hypothesized that the inclusion of these 25 SNPs with patient age at diagnosis and sex could predict risk of glioma as well as predict IDH mutation status. Thus, case-control design and multinomial logistic regression were used to develop models to estimate the risk of glioma development while accounting for molecular subtypes. Case-case design and logistic regression were used to develop models to predict IDH mutation status. Each model included all 25 glioma risk SNPs, patient age at diagnosis and sex. A total of 1273 glioma cases and 443 controls from Mayo Clinic were used in the discovery set, and 852 glioma cases and 231 controls from UCSF were used in the validation set. All samples were genotyped using a custom Illumina OncoArray. We observed that patients in the highest 5% of the risk score had more than a 14-fold increased relative risk of developing an IDH-mutant glioma, compared to patients with median risk score. Large differences in lifetime absolute risk were observed at the extremes of the risk score percentile categories. For both IDH-mutated 1p/19q non-codeleted glioma and IDH-mutated 1p/19q-codeleted glioma, the lifetime risk increased from almost null to 2.3% and almost null to 1.7%, respectively. The SNP-based model that predicted IDH mutation status had a validation c-index of 0.85. These results suggest that germline genotyping has the potential to provide a new tool for clinicians for the initial management of newly-discovered brain lesions. Specifically, given the low lifetime risk of glioma, SNP-based risk scores should not be useful for general population screening. However, with further research these risk scores may be useful in certain clinically-defined high-risk groups.

Adult diffuse glioma that harbor IDH mutation and 1p/19q codeletion commonly have CIC mutations; however, the functional and prognostic significance of CIC mutations remains unclear. We hypothesized that when CIC is not mutated, other genes may be inactivated by hypermethylation. Identifying such genes may help to further understand the biology of 1p/19q-codeleted glioma. To test our hypothesis, we utilized a two-stage design. In the discovery stage, 72 IDH-mutated 1p/19q-codeleted tumors were utilized from TCGA (40 CIC mutated, 32 wild-type); all via Illumina 450K methylation array. In the validation stage, 43 IDH-mutated 1p/19q-codeleted tumors were utilized from Mayo Clinic (33 CIC mutated, 10 wild-type); all via Illumina 850K methylation array. All tumors also had TERT promoter mutation. Genome-wide significance (p≤5x10(-8)) was used in the discovery stage to identify differentially methylated probes between CIC mutated vs wild-type tumors. Seven probes reached significance in TCGA; four validated at pCIC. DLL3, located on 19q, had a mean change in beta value between CIC wild-type and mutated tumors of 0.28 (p=1.29x10(-8)) and 0.24 (p=0.0024) in TCGA and Mayo, respectively. ETV1, located on 7p, had a mean change in beta value of 0.30 (p=1.37x10(-8)) and 0.15 (p=0.00011) in TCGA and Mayo, respectively. In the 72 TCGA tumors, we observed an inverse correlation between DLL3 methylation and gene expression amongst CIC mutated and wild-type tumors; -0.42 (p=0.0063) and -0.33 (p=0.069), respectively. There was also an inverse correlation between ETV1 methylation and gene expression amongst CIC mutated and wild-type tumors; -0.68 (pDLL3 and ETV1 was downregulated in CIC wild-type vs mutated tumors (p=0.000027 and 0.0000028, respectively). We confirm that ETV1 has higher gene expression in CIC mutated vs wild-type tumors. Our results suggest that DLL3 expression is inactivated by promoter methylation in CIC wild-type 1p/19q-codeleted oligodendrogliomas; thus, DLL3 may be an alternative target gene on 19q.

Glioma, the most common central nervous system cancer in adults, generally has poor prognosis. Glioblastoma, the most common and most aggressive form of glioma, has a median patient survival time of just 15 months from diagnosis under current standard of care.1 While prognosis is better for low-grade astrocytic tumors and tumors with an oligodendroglial component, over time these tumors all progress to high-grade glioma.2 Gliomagenesis is a complex and multifaceted process influenced by both inherited and acquired genetic variation. Glioma risk loci in 7 genes have been confirmed in genome-wide association studies.3–6 Several of these risk loci are found in genes previously implicated in gliomagenesis due to their mutation in glioma-associated hereditary cancer syndromes (TP53, CDKN2B/ANRIL) or their alteration in glioma tumors (TP53, CDKN2B/ANRIL, EGFR).7–9 Risk loci in 2 genes involved in telomerase structure and function (TERT [telomerase reverse transcriptase] and RTEL1 [regulator of telomere elongation helicase 1]) had not been implicated in gliomagenesis prior to genome-wide association studies, but telomerase activation has been observed in ∼90% of all human cancers.10 Telomeres act as a protective cap at the end of chromosomes but are progressively shortened during mitotic divisions.11 Telomere depletion ultimately leads to replicative senescence, limiting the proliferative capacity of cells. With activation of telomerase, an enzyme that adds DNA sequence repeats to telomeres, dividing cells can replace lost telomeric DNA and continue proliferating.10 TERT is a key component of human telomerase, and RTEL1 is needed to allow telomerase-dependent telomere extension to proceed effectively.12,13 Of the tumors that do not maintain telomere length through activation of telomerase, a significant subset activates a secondary pathway: alternative lengthening of telomeres (ALT).14 Inheriting an increased number of glioma risk alleles might be expected to decrease age at diagnosis among glioma patients by reducing the number of somatic mutations an individual must acquire to initiate tumor formation or by facilitating the accumulation of such mutations. To investigate this, we examined the associations of known glioma risk loci with age at diagnosis in glioma patients from the University of California, San Francisco (UCSF) and the Mayo Clinic. Caucasian glioma patients were genotyped at single nucleotide polymorphisms (SNPs) in 7 genes associated with glioma risk in previous genome-wide association studies, including variants in TERT, EGFR, CCDC26, CDKN2B/ANRIL, PHLDB1, TP53, and RTEL1. Associations were also calculated within histologic subgroups, stratified by tumor IDH­-mutation status, and pooled across study sites. Interactions between age and risk SNPs were also examined in case-control comparisons.

We and others first reported several inherited variants that increase glioma risk in 2009.1,2 Additional confirmation and new risk loci have been identified since then.3–8 We9 and Simon et al.10 showed that the glioma risk loci in 8q24 and 11q23 first identified by Shete et al.1 were associated with risk of lower grade infiltrating gliomas but not with glioblastomas. It is now well established that mutation in the isocitrate dehydrogenase (IDH) 1 and 2 genes leads to genome-wide histone and DNA methylation changes that result in abnormal gene expression and gliomagenesis, defining a distinct subclass of gliomas.11,12 IDH mutations occur in ∼50%–80% of grade II-III gliomas and secondary glioblastomas but in fewer than 10% of primary glioblastomas.13–17 Tumor IDH mutations are associated with younger age of onset and better overall survival among patients with gliomas of all grades and histologies18,19 and are also associated with other somatic, genetic, and epigenetic alterations.17,20 Given these observations, we hypothesized that the 8q24 and 11q23 glioma risk loci might be specific to IDH-mutated, but not to IDH-wild-type gliomas. In a separate recent publication,21 we showed that the 8q24 locus is specifically associated with risk for oligodendroglial tumors and IDH-mutated astrocytomas of all grades. Here, we report that the 11q23 glioma risk variant rs498872 T allele is associated with risk of IDH-mutated gliomas but not with risk of IDH-wild-type gliomas, regardless of grade or histology.

Genomewide association studies (GWAS) and candidate-gene studies have implicated single-nucleotide polymorphisms (SNPs) in at least 45 different genes as putative glioma risk factors. Attempts to validate these associations have yielded variable results and few genetic risk factors have been consistently replicated. We conducted a case-control study of Caucasian glioma cases and controls from the University of California San Francisco (810 cases, 512 controls) and the Mayo Clinic (852 cases, 789 controls) in an attempt to replicate previously reported genetic risk factors for glioma. Sixty SNPs selected from the literature (eight from GWAS and 52 from candidate-gene studies) were successfully genotyped on an Illumina custom genotyping panel. Eight SNPs in/near seven different genes (TERT, EGFR, CCDC26, CDKN2A, PHLDB1, RTEL1, TP53) were significantly associated with glioma risk in the combined dataset (P < 0.05), with all associations in the same direction as in previous reports. Several SNP associations showed considerable differences across histologic subtype. All eight successfully replicated associations were first identified by GWAS, although none of the putative risk SNPs from candidate-gene studies was associated in the full case-control sample (all P values > 0.05). Although several confirmed associations are located near genes long known to be involved in gliomagenesis (e.g., EGFR, CDKN2A, TP53), these associations were first discovered by the GWAS approach and are in noncoding regions. These results highlight that the deficiencies of the candidate-gene approach lay in selecting both appropriate genes and relevant SNPs within these genes.