LAUSR

mutant protein, and discuss the implications with regard to classification and grading. The first patient is a 30-year-old woman who presented with headaches and symptoms of increased intracranial pressure. Imaging demonstrated a heterogeneously enhancing mass centered in the right thalamus (Fig. 1a and Supplemental Fig. 1), and subtotal resection was performed. Sections demonstrated a highly cellular infiltrative astrocytic neoplasm with WHO grade IV histologic features including frequent mitoses, marked nuclear pleomorphism, palisading necrosis, and microvascular proliferation (Fig. 1c and Supplemental Fig. 2a, b). The tumor was negative for IDH1 R132H mutant protein immunostaining and showed somatic loss of ATRX expression diffusely in all tumor nuclei (Supplemental Fig. 2c). Immunohistochemistry for histone H3 K27M mutant protein revealed a mosaic staining pattern with expression seen in some but not all tumor cells (Fig. 1e and Supplemental Fig. 2d). While the majority of tumor cells lacked H3 K27M mutant protein expression, there were scattered large groups, small clusters, and admixed single cells that were positive. Immunohistochemistry for trimethylation of lysine-27 of histone H3 (H3K27me3) revealed an Diffuse midline gliomas are aggressive tumors centered in midline structures of the brain that most commonly occur in children and young adults [6]. They are genetically defined by the presence of K27M mutation in either the H3F3A or HIST1H3B genes, which encode the histone H3 variants H3.3 and H3.1, respectively [1]. Given their poor prognosis, the 2016 WHO Classification includes “Diffuse midline glioma, H3 K27M-mutant” as a WHO grade IV entity, even in cases where only lower-grade histologic features are present [4]. In all cases reported to date, H3 K27M mutation has been a clonal alteration in all regions of tumors assessed by sequencing, and immunostaining with antibodies against H3 K27M mutant protein has revealed uniform expression in all tumor cells [2, 5, 6]. Thus, it has been hypothesized that H3 K27M mutation is the earliest or initiating genetic alteration during gliomagenesis in these tumors. Here we describe two cases of diffuse midline glioma with subclonal H3F3A K27M mutation and mosaic expression of H3.3 K27M