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The integrated diagnosis for infiltrating gliomas requires demonstration of IDH1 or IDH2 mutation and 1p/19q codeletion in the tumor to diagnose oligodendroglioma or anaplastic oligodendroglioma, IDH-mutant and 1p/19q-codeleted. IDH1 R132H, ATRX and p53 immunohistochemistry may help triaging cases for 1p/19q testing [3]. Recently, Filipski et al. [2] have reported that loss of H3K27me3 (trimethylation of lysine 27 of histone 3) stain could potentially stratify IDH-mutant diffuse gliomas based on cell type, recommending H3K27me3 stain as an additional marker in the immunohistochemical workup. A separate study reported differential loss of H3K27me3 staining in oligodendrogliomas and astrocytomas, which was only true using the monoclonal antibody [1]. We have therefore assembled a validation cohort of genetically well-characterized adult diffuse gliomas to assess the clinical utility of H3K27me3 immunohistochemistry in this setting. We identified 117 adult gliomas initially diagnosed at UCSF and Izmir KCU Ataturk EAH, in which sufficient tissue to be included in tissue microarrays in duplicate or triplicate was available. Three neuropathologists (MP, FD and TT) performed morphologic classification based on H&E-stained sections without immunohistochemistry or molecular results. Immunohistochemistry was performed on five-micron sections using standard techniques, and all stains were independently scored by two neuropathologists (MP and TT) with subsequent consensus on discrepant cases. All cases with positive immunohistochemistry for IDH1 R132H mutant protein (H09, Dianova GmbH, Hamburg, Germany) were classified as IDH mutant. Immunonegative cases were classified as mutant or wild type based on Sanger sequencing of IDH1 and IDH2 genes covering exon 4 regions. Chromosome 1p/19q status was separately assessed by fluorescence in situ hybridization using the Vysis 1p36/19q13 Dual Color Probe Kit (Abbott Laboratories, Abbott Park, IL). Complete loss of ATRX (HPA001906, Sigma-Aldrich, St. Louis, MO) in neoplastic cells in the presence of internal positive control was considered as ATRX-altered. Immunohistochemistry for p53 (DO-7, Dako, Agilent, Santa Clara, CA) was considered positive when more than 50% of tumor nuclei showed strong staining. Only complete loss of H3K27me3 (clone C36B11, 1:50 dilution, Cell Signaling Technology, Danvers, MA) staining in the presence of positive internal control was considered significant. Patchy/mosaic staining was recorded, but was considered as retained expression. The study cohort included 102 cases after exclusion of four IDH1R132H-negative cases without sequencing results, two IDH-mutant gliomas without 1p/19q codeletion status, two H3K27M-mutant diffuse midline gliomas and seven gliomas with failed H3K27me3 staining (one oligodendroglioma, three IDH-mutant and three IDH-wildtype astrocytomas). Among oligodendrogliomas, 21 (75%) showed complete loss of H3K27me3 staining, whereas 7 Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0040 1-019-02123 -8) contains supplementary material, which is available to authorized users.