A new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered: a clinico-radiological and histomolecular characterisation

Abstract

Diffuse midline gliomas (DMG) H3 K27-altered are incurable grade 4 gliomas and represent a major challenge in neuro-oncology. This tumour type is now classified in four subtypes by the 2021 edition of the WHO Classification of the Central Nervous System (CNS) tumours. However, the H3.3-K27M subgroup still appears clinically and molecularly heterogeneous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tended to have a better prognosis. To better study the role of these co-driver alterations, we assembled a large paediatric and adult cohort of 29 tumours H3K27-altered with co-occurring activating mutation in BRAF or FGFR1 as well as 31 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylation analysis. Interestingly, unsupervised t-distributed Stochastic Neighbour Embedding (tSNE) analysis of DNA methylation profiles regrouped BRAF^V600E and all but one FGFR1^MUT DMG in a unique methylation cluster, distinct from the other DMG subgroups and also from ganglioglioma (GG) or high-grade astrocytoma with piloid features (HGAP). This new DMG subtype harbours atypical radiological and histopathological profiles with calcification and/or a solid tumour component both for BRAF^V600E and FGFR1^MUT cases. The analyses of a H3.3-K27M BRAF^V600E tumour at diagnosis and corresponding in vitro cellular model showed that mutation in H3-3A was the first event in the oncogenesis. Contrary to other DMG, these tumours occur more frequently in the thalamus (70% for BRAF^V600E and 58% for FGFR1^MUT) and patients have a longer overall survival with a median above three years. In conclusion, DMG, H3 K27 and BRAF/FGFR1 co-altered represent a new subtype of DMG with distinct genotype/phenotype characteristics, which deserve further attention with respect to trial interpretation and patient management.

Keywords: Adult glioma; BRAF-V600E mutation; DNA methylation profiling; FGFR1 mutation; Midline glioma; Paediatric-type high-grade glioma.

Fig. 1

Clinical and molecular characteristics of the patient cohort of DMG H3-K27M with BRAF and FGFR1 mutations. Overview of the clinical and molecular annotations of 60 paediatric and adult DMG H3-K27 patients presenting BRAF or FGFR1 mutations. Cases are presented in columns and genes status in rows. Age is reported in years. All molecular information is derived from DNA or RNA sequencing analyses, except immuno-histological data (including histological profile, EZHIP and ATRX expression). For survival, patients still alive at last follow-up are indicated by a half-filled square

Fig. 2

Multiple histopathological profiles of DMG H3-K27 with BRAF or FGFR1 mutations. Case 14 a A glioneuronal proliferation with ganglion cells, eosinophilic granular bodies and some microcalcifications (HPS, magnification × 400). Case 11 b A glioneuronal proliferation with numerous ganglion cells (HPS, magnification × 400). Case 13 c A glioneuronal proliferation with numerous ganglion cells and lymphocytic infiltrates (HPS, magnification × 400). Case 31 d A mainly circumscribed proliferation (neurofilament, magnification × 30). Case 32 e A mainly circumscribed proliferation (neurofilament, magnification × 400) with a diffuse component at the periphery of the tumour f (neurofilament, magnification × 400). g Diffuse chromogranin A immunoreactivity staining neuron cells (magnification × 400). h BRAF^V600E expression in all tumour cells including ganglion cells (magnification × 400). i H3K27M expression in all tumour cells including ganglion cells (magnification × 400). Case 7 j A glial proliferation with oligo-like features and microcalcifications (magnification × 400). Case 11 k Global loss of H3K27me3 (magnification × 400). (l) Loss of ATRX in tumour cells (magnification × 400). m Whorls of gliofibrillary processes (HPS, magnification × 20). n Whorls of gliofibrillary processes (HPS, magnification × 400), stained using GFAP antibody o, magnification × 400). Black scale bars represent 50 μm (a–c, e–l and n–o), 100 µm (m), and 500 µm (d)

Fig. 3

Radiological specificities of DMG H3-K27M BRAF^V600E or FGFR1^MUT. a T2-FLAIR (Fluid-attenuated inversion recovery) MR images sequences or CT scans (computed tomography) of DMG H3-K27M according to BRAF or FGFR1 mutation status. b Comparison of the tumour radiological presentation (diffuse, circumscribed, or mixed) of DMG according to their genotype (chi-square test for trend: ns, ****p value < 0.0001). c Comparison of presence of macro-calcifications in DMG CT-scans according to the presence of MAPK alteration (Fisher’s exact test: ****p value < 0.0001, *p = 0.0210, *p = 0.0348)

Fig. 4

Analysis of DNA methylation profiles of DMG H3.3-K27M BRAF^MUT/FGFR1^MUT. CNS tumour classification based on DNA methylation profiles. Unsupervised clustering by t-SNE analysis of tumours based on their DNA methylation profiles using 10,000 topmost differentially methylated probes across the reference sample set composed of samples from Capper et al. (n = 936) and Castel et al. (n = 41)

Fig. 5

Clinical specificities of patients with DMG H3-K27M BRAF^MUT/FGFR1^MUT and transcriptomic tumour profiling. a Comparison of OS estimated using Kaplan–Meier method according to the mutation status of Histone H3, BRAF, FGFR1 and TP53 (log-rank test, p value =  < 0.0001). b Distribution of age at diagnosis according to BRAF and FGFR1 mutation status (Mann–Whitney test; *p value = 0.0168, ***p value = 0.0001, ****p value =  < 0.0001). c Comparison of tumour location according to the mutation status of BRAF and FGFR1 (chi-square test for trend: ns, ***p value = 0.0004 ****p value =  < 0.0001). d GSEA plot showing common transcriptomic signature from DMG H3.3-K27M vs. DMG H3.3-K27M BRAF^V600E or FGFR1^MUT. The normalised enrichment score (NES) and the false discovery rate (FDR q) are indicated in each plot