Co-occurrence of histone H3 K27M and BRAF V600E mutations in paediatric midline grade I ganglioglioma

Abstract

Ganglioglioma (GG) is a grade I tumor characterized by alterations in the MAPK pathway, including BRAF V600E mutation. Recently, diffuse midline glioma with an H3 K27M mutation was added to the WHO 2016 classification as a new grade IV entity. As co-occurrence of H3 K27M and BRAF V600E mutations has been reported in midline tumors and anaplastic GG, we searched for BRAF V600E and H3 K27M mutations in a series of 54 paediatric midline grade I GG (midline GG) to determine the frequency of double mutations and its relevance for prognosis. Twenty-seven patients (50%) possessed the BRAF V600E mutation. The frequency of the co-occurrence of H3F3A/BRAF mutations at diagnosis was 9.3%. No H3 K27M mutation was detected in the absence of the BRAF V600E mutation. Double-immunostaining revealed that BRAF V600E and H3 K27M mutant proteins were present in both the glial and neuronal components. Immunopositivity for the BRAF V600E mutant protein correlated with BRAF mutation status as detected by massARRAY or digital droplet PCR. The median follow-up of patients with double mutation was 4 years. One patient died of progressive disease 8 years after diagnosis, whereas the four other patients were all alive with stable disease at the last clinical follow-up (at 9 months, 1 year and 7 years) without adjuvant therapy. We demonstrate in this first series of midline GGs that the H3 K27M mutation can occur in association with the BRAF V600E mutation in grade I glioneuronal tumors. Despite the presence of H3 K27M mutations, these cases should not be graded and treated as grade IV tumors because they have a better spontaneous outcome than classic diffuse midline H3 K27M-mutant glioma. These data suggest that H3 K27M cannot be considered a specific hallmark of grade IV diffuse gliomas and highlight the importance of integrated histomolecular diagnosis in paediatric brain tumors.

Keywords: BRAF V600E; H3 K27M; ganglioglioma; midline.

Figure 1

Clinical characteristics, histological features and genetic alterations in 54 pediatric grade I midline GGs.

Figure 2

Radiological, histological and immunohistochemical findings of midline GG38. Serial sections showing mixed glial (arrowhead) and neuronal ganglion cell elements (arrow) (H&E) (A) with strong, diffuse immunostaining for BRAF V600E in both the glial and neuronal components (B), positive immunostaining for H3 K27M in both the glial and neuronal components (C) and a loss of H3K27me3 trimethylation in both the glial and neuronal components (D). Double‐immunostaining for BRAF V600E (diaminobenzidine in brown)/H3 K27M (AP Red in red) depicting colocalization in the glial and ganglion cell components (E and F). Very high magnification depicting a double‐mutated ganglion cell (E) and glial cell (arrowhead, F). Note that the ganglion cell could only be positive for H3 K27M (F). A–D. Magnification ×400, E and F magnification ×600. Coronal plane tomography (G) and axial plane MRI T2‐FLAIR (H). Images depict a well‐circumscribed solid mass arising from the pontine area. The computed tomography images show hyperdensity and calcification. MR images depict a heterogeneous but well‐circumscribed lesion.

Figure 3

Double immunofluorescence staining of BRAF V600E and H3 K27M in midline GG38. The upper row depicts endothelial cells without labeling for H3 K27M (red) and BRAF V600E (green). Imaging revealed evidence of H3 K27M and BRAF V600E colocalization in glial cells (second row) and neurons (third row) in nuclear and cytoplasmic areas, respectively. Note that neurons could only be positive for H3 K27M (lower row).