Deceptive morphologic and epigenetic heterogeneity in diffuse intrinsic pontine glioma

Abstract

Historically, the diagnosis of diffuse intrinsic pontine glioma (DIPG) was based on typical imaging findings and clinical characteristics instead of pathology. However, the discovery of mutations in histone H3 variants, and the availability of tumor material for molecular analysis, has led to a paradigm shift in DIPG research and clinical practice. Using data from whole-brain autopsies in a series of nine DIPG patients with known histone mutational status, we here aim to review histopathological characteristics with special focus on intratumoral heterogeneity (ITH) and histone 3 K27 trimethylation (H3 K27me3). All DIPGs showed marked histologic ITH, with 56% even showing focal areas resembling a WHO grade I phenotype. As expected, H3 K27me3 immunoreactivity was lost in the tumors that were H3 K27M-mutated (seven patients; 67% H3.3, 11% H3.1). Strikingly, the H3K27 wildtype tumors (two patients; 22%) also contained H3 K27me3-immunonegative areas. Our study underscores the importance of the choice of the biopsy site, as ITH in DIPGs could theoretically lead to erroneous histological diagnoses with small biopsies. New in this respect is our finding that a substantial number of otherwise typical DIPGs has areas resembling WHO grade I tumors (esp. pilocytic astrocytoma, subependymoma). Furthermore, our study shows that negative H3 K27me3 immunohistochemistry in a DIPG does not imply a H3 K27-mutant tumor.

Keywords: H3 K27M; diffuse intrinsic pontine glioma; histone 3; intratumoral heterogeneity; trimethylation.

Figure 1. Intratumoral heterogeneity of DIPG (patient VUMC-DIPG-6)

(A) axial T2-weighted MR-image; letters correspond to tumor areas illustrated. (B) Gemistocitic morphology of tumor cells and calcifications. (C) High-grade component with necrosis surrounded by pseudopalissading of tumor cells. (C') In the same area, tumor cells expressing the stem cell markers GFAPδ and CD44. (D) Subependymoma-like component showing negligible mitotic activity (D'), dot-like cytoplasmic immunopositivity for the epithelial membrane antigen (EMA, D''), indicating ependymal differentiation, and immunopositivity for olig2 (D'''). (E) High-grade component with microvascular proliferation. (F) Tumor cells infiltrating the gray matter of the pontine nuclei. (G) High-grade component with small cell morphology and brisk mitotic activity (G'). (H) Low-grade component with isolated tumor cells infiltrating the distant white matter and expressing olig2 (H'). In the same area, perivascular clustering of tumor cells immunopositive for neurofilament (NF) 70-200kDA (H''). (I) Isolated tumor cells in the distant cerebellar cortex. Bars: 100μm, C' 200μm.

Figure 2. Immunohistochemistry for the H3 K27M-mutant protein and H3 K27me3

Patient VUMC-DIPG-6 (H3. 3 K27M-mutant, left panels) is immunopositive for the mutant protein in tumor nuclei and shows H3 K27me3-loss in both high-grade (left) and low-grade (middle-left) components. Patient VUMC-DIPG-1 (H3 K27 wildtype, right panels) is immunonegative for the mutant protein in tumor cells, with aspecific microglia staining. Stain against H3 K27me3 shows H3 K27me3-conservation in some areas (middle-right), whereas other areas display H3 K27me3-loss (right). Bars: 100μm.