Kollmeyer, Thomas M.

Abstract 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.

Abstract 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.

Abstract TP53 mutations are frequent in IDH-mutant astrocytomas but unusual in oligodendroglioma and the clinical significance of TP53 mutations in oligodendroglioma are not well characterized. We reviewed genetically defined oligodendroglioma (i.e., IDH-mutant, whole-arm 1p/19q-codeleted diffuse glioma) cases that were molecularly profiled (2017-2020) at our institution and identified 7 cases with TP53 mutation (9%; n=76). Molecular testing was performed using targeted neuro-oncology NGS panel (50-gene mutation and/or 187-gene mutation/rearrangement) and OncoScan™ microarray. Four (of 7) patients were female. Median age at diagnosis was 43 years (range, 23-63). Most common presenting symptom was seizures (3 of 7). All tumors were supratentorial. Histologically, 3 tumors were WHO grade II and 4 were WHO grade III. Two (of 3) patients with a WHO grade II tumor underwent biopsy and radiotherapy at diagnosis followed by temozolomide at recurrence (progression at 67 and 157 months after diagnosis; overall survival of 124 and 201 months). Three (of 4) patients with a WHO grade III tumor were diagnosed within the last two years and are currently progression-free after standard therapy. Molecularly, in addition to TP53 mutation(s), all cases had an IDH1 and TERT promoter mutation as well as other gene mutation(s) including FUBP1 (n=5), SETD2 (n=4), PIK3R1 (n=4), PIK3CA (n=3), NF1 (n=3) and CIC (n=3). In 3 (of 7) cases, the mutational profile with high mutation count enriched for C >T/G >A transitions was highly suggestive of a hypermutation phenotype (2 cases were recurrent tumors treated with temozolomide; a recurrent and a treatment-naïve tumor had mismatch repair gene mutation). Five (of 7) cases, including the 3 hypermutant cases, lacked functional TP53 (1 case with 2 mutations, 2 cases with 1 mutation plus loss of other copy, 2 cases with 1 mutation plus copy neutral loss-of-heterozygosity). TP53 mutations are uncommon in oligodendroglioma and appear enriched in hypermutant tumors.

Abstract 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.

Abstract 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.

Abstract 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.

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.