Hardcastle, Jayson

Abstract Diffuse hemispheric glioma, H3 G34‐mutant (DHG‐H3 G34) has been primarily molecularly characterized by methylation profiling and sequencing studies. We describe an integrated histomolecular evaluation including high‐resolution copy number profiling of a series of 60 DHG‐H3 G34 to further our understanding of the spectrum of genetic changes associated with this tumor type. Cases were clinically tested using an 187‐gene mutation and fusion targeted neuro‐oncology next‐generation sequencing panel ( n = 60) and Oncoscan chromosomal microarray ( n = 26) by a single laboratory (2018–2022). A subset of cases had immunohistochemical results for OLIG2 ( n = 42), p53 ( n = 48), and ATRX ( n = 46), and methylation array data ( n = 8). Median age at testing was 21 years (range, 12–50). No significant difference was noted in clinical, histopathological, and mutational profile between pediatric and adult patients. H3‐3A G34 mutations included G34R ( n = 56; 94%), G34V ( n = 3), and a non‐canonical G34E ( n = 1). Concurrent mutations most often involved TP53 ( n = 55; 92%), ATRX ( n = 50; 83%), and PDGFRA ( n = 34; 57%). A reportedly primary tumor was confirmed to be hypermutant and had a PMS2 mutation. A single case also showed an FGFR3::FAM184B fusion. All cases with available chromosomal microarray data had unbalanced genomes, which were often complex (14/26; 54%). The most frequent recurrent copy number abnormalities were losses involving 3q, 4q, 10q, 13q, and 18q, and 17p copy‐neutral loss of heterozygosity (cnLOH) encompassing TP53 . This copy number profile was reminiscent of that seen in Grade 4 IDH‐mutant astrocytomas. Collectively, a TP53 abnormality at copy number (12/26, all cnLOH), sequence (55/60) and protein expression (46/48) level was detected in all 60 cases. In conclusion, integrated high‐resolution copy number and histomolecular analysis expanded the spectrum of genetic changes associated with DHG‐H3 G34, including the presence of universal TP53 abnormalities with frequent cnLOH—a copy number abnormality that has been largely unrecognized—for this new 2021 World Health Organization central nervous system tumor type.

Abstract Most TERT promoter (TERTp) clinically relevant sequence variants in CNS tumors are the canonical C228T and C250T mutations. Non-canonical TERTp mutations have been reported mainly in non-CNS tumors. We describe 12 CNS tumors with non-canonical TERTp variants interpreted as clinically relevant. Using clinical targeted NGS panels (2020-2024), we detected c.-100_-79dup (n=2), c.-92_-91insGGCGGCCCCGCCCCTTCCTTTC (n=1), c.-118_-117insTCCCCGGCCCAGCCCCTTCCGGG (n=1), c.-123_-76dup (n=1), c.-110_-89dup (n=2), and c.-57A>C (n=5). Most cases had chromosomal microarray (n=11) and/or methylation array data analyzed using the NCI/Bethesda v.2 classifier (n=9). All non-canonical TERTp variants occurred at a variant allele frequency suggestive of somatic clonal origin (mean, 34%; range, 18-46), and in adults (mean age, 60 years; range, 36-88). Except for the c.-57A>C variant, all non-canonical TERTp variants occurred in tumors with histological and/or molecular diagnostic features of glioblastoma, IDH-wildtype (GBM), often epigenetically aligning to this tumor type. All five tested cases matched to superfamily glioblastoma (score ≥ 0.9), with three to class GBM_RTK_I (>0.9 in 2, 0.86 in 1) and two to GBM_RTK_II (>0.9). The size and sequence of such variants suggest that these may be functionally equivalent to canonical mutations or generate favorable TERTp activation motif conformations like previously reported activating duplications. The c.-57A>C variant is a well-established oncogenic mutation observed in other cancer types but has not been reported in CNS tumors. We describe five cases with the c.-57A>C variant: one GBM matching to methylation class GBM_MES_TYP (0.99); two oligodendroglioma, IDH-mutant and 1p/19q-codeleted, one of which had methylation data matching to class O_IDH (0.99); one diffuse glioma, IDH-mutant, which was partially 1p/19q-codeleted and matched to class O_IDH (0.99); and one anaplastic meningioma, aligning to superfamily meningioma (0.99)/MNG_MAL (0.79). Our findings show that non-canonical TERTp variants typically occur in tumor types that often harbor TERTp canonical mutations, expanding the spectrum of TERTp clinically relevant variants that are molecular biomarkers in CNS tumors.

Abstract Purpose: Oncolytic herpes simplex viruses (oHSV) represent a promising therapy for glioblastoma (GBM), but their clinical success has been limited. Early innate immune responses to viral infection reduce oHSV replication, tumor destruction, and efficacy. Here, we characterized the antiviral effects of macrophages and microglia on viral therapy for GBM. Experimental Design: Quantitative flow cytometry of mice with intracranial gliomas (±oHSV) was used to examine macrophage/microglia infiltration and activation. In vitro coculture assays of infected glioma cells with microglia/macrophages were used to test their impact on oHSV replication. Macrophages from TNFα-knockout mice and blocking antibodies were used to evaluate the biologic effects of TNFα on virus replication. TNFα blocking antibodies were used to evaluate the impact of TNFα on oHSV therapy in vivo. Results: Flow-cytometry analysis revealed a 7.9-fold increase in macrophage infiltration after virus treatment. Tumor-infiltrating macrophages/microglia were polarized toward a M1, proinflammatory phenotype, and they expressed high levels of CD86, MHCII, and Ly6C. Macrophages/microglia produced significant amounts of TNFα in response to infected glioma cells in vitro and in vivo. Using TNFα-blocking antibodies and macrophages derived from TNFα-knockout mice, we discovered TNFα-induced apoptosis in infected tumor cells and inhibited virus replication. Finally, we demonstrated the transient blockade of TNFα from the tumor microenvironment with TNFα-blocking antibodies significantly enhanced virus replication and survival in GBM intracranial tumors. Conclusions: The results of these studies suggest that FDA approved TNFα inhibitors may significantly improve the efficacy of oncolytic virus therapy. Clin Cancer Res; 21(14); 3274–85. ©2015 AACR.