Purpose: Isocitrate dehydrogenase (IDH)-mutant glioma is a distinct glioma molecular subtype for which no effective molecularly directed therapy exists. Low-grade gliomas, which are 80%-90% IDH-mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by IHC in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody-drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH-mutant glioma. Experimental Design: We evaluated DLL3 expression by RNA using TCGA data and by IHC in a discovery set of 63 gliomas and 20 nontumor brain tissues and a validation set of 62 known IDH wild-type and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH-mutant glioma tumorspheres was determined by cell viability assay. Results: Compared to IDH wild-type glioblastoma, IDH-mutant gliomas have significantly higher DLL3 RNA (P < 1 Â 10 À15) and protein by IHC (P ¼ 0.0014 and P < 4.3 Â 10 À6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH-mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 nontumor brains. Patient-derived IDH-mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner. Conclusions: DLL3 is selectively and homogeneously expressed in IDH-mutant gliomas and can be targeted with Rova-T in patient-derived IDH-mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.

Pleomorphic xanthoastrocytoma (PXA) is a rare type of brain tumor that affects children and young adults. Molecular prognostic markers of PXAs remain poorly established. Similar to gangliogliomas, PXAs show prominent immune cell infiltrate, but its composition also remains unknown. In this study, we correlated DNA methylation and BRAF status with clinical outcome and explored the tumor microenvironment. We performed DNA methylation in 21 tumor samples from 18 subjects with a histological diagnosis of PXA. MethylCIBERSORT was used to deconvolute the PXA microenvironment by analyzing the associated immune cell-types. Median age at diagnosis was 16 years (range 7–32). At median follow-up of 30 months, 3-year and 5-year overall survival was 73% and 71%, respectively. Overall survival ranged from 1 to 139 months. Eleven out of 18 subjects (61%) showed disease progression. Progression-free survival ranged from 1 to 89 months. Trisomy 7 and CDKN2A/B (p16) homozygous deletion did not show any association with overall survival (p = 0.67 and p = 0.74, respectively). Decreased overall survival was observed for subjects with tumors lacking the BRAF V600E mutation (p = 0.02). PXAs had significantly increased CD8 T-cell epigenetic signatures compared with previously profiled gangliogliomas (p = 0.0019). The characterization of immune cell-types in PXAs may have implications for future development of immunotherapy.

Molecular studies have identified distinct genomic drivers providing insights in biology of brain tumors. Advances in genetic and epigenetic analysis, as well as development of mutation-specific antibodies enable more accurate classification of histologically indistinguishable tumors. Compared with histopathologic grading, molecular biomarkers are also superior in predicting natural behavior of tumors and therapeutic response. Diffuse gliomas can be separated in astrocytoma and oligodendroglioma based on IDH1/2, ATRX, and TP53 mutational status. Pediatric gliomas are molecularly distinct from adult tumors and molecular drivers include histone H3 genes and fusions involving the MAPK pathway. Using genetic and epigenetic profiling, ependymal tumors, medulloblastomas, and atypical teratoid/rhabdoid tumors can be separated in biologically and clinically distinct entities. Identification of novel gene fusions and matched DNA methylation signatures enable accurate diagnosis of primitive neuroectodermal tumors, which were previously misdiagnosed. Genomic classification of central nervous system tumors is being readily translated into the clinical practice and will enable molecularly based patient management and clinical trials.

<div>AbstractPurpose:<p>Isocitrate dehydrogenase (<i>IDH</i>)-mutant glioma is a distinct glioma molecular subtype for which no effective molecularly directed therapy exists. Low-grade gliomas, which are 80%–90% <i>IDH</i>-mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by IHC in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody–drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in <i>IDH</i>-mutant glioma.</p>Experimental Design:<p>We evaluated <i>DLL3</i> expression by RNA using TCGA data and by IHC in a discovery set of 63 gliomas and 20 nontumor brain tissues and a validation set of 62 known <i>IDH</i> wild-type and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous <i>IDH</i>-mutant glioma tumorspheres was determined by cell viability assay.</p>Results:<p>Compared to <i>IDH</i> wild-type glioblastoma, <i>IDH</i>-mutant gliomas have significantly higher <i>DLL3</i> RNA (<i>P</i> < 1 × 10⁻¹⁵) and protein by IHC (<i>P</i> = 0.0014 and <i>P</i> < 4.3 × 10⁻⁶ in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in <i>IDH</i>-mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 nontumor brains. Patient-derived <i>IDH</i>-mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner.</p>Conclusions:<p>DLL3 is selectively and homogeneously expressed in <i>IDH</i>-mutant gliomas and can be targeted with Rova-T in patient-derived <i>IDH</i>-mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.</p></div>

<p>Summary of IHC of glioma subtypes</p>