A subset of pediatric-type thalamic gliomas share a distinct DNA methylation profile, H3K27me3 loss and frequent alteration of EGFR

Abstract

Background: Malignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the epidermal growth factor receptor gene (EGFR) have recently been identified in a distinct subset of pediatric-type bithalamic gliomas with a unique DNA methylation pattern.

Methods: Here, we investigated an epigenetically homogeneous cohort of malignant gliomas (n = 58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in comparison with this recently identified subtype of pediatric bithalamic gliomas.

Results: EGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; 5 of them co-occurring with EGFR amplification). Additionally, 8 of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (6 of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of overexpression of the EZH inhibitory protein (EZHIP) in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations.

Conclusions: Our findings present a distinct molecular class of pediatric-type malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.

Keywords: EGFR mutation; (bi)thalamic; H3 K27M mutation; K27me3; pediatric-type high-grade glioma.

Fig. 1

t-SNE analysis of DNA methylation profiles of the 58 gliomas investigated (DMG_EGFR; 10 of the cases included from the Mondal et al cohort (UCSF) are highlighted in brown) alongside selected reference samples. Reference DNA methylation classes: glioblastoma IDH-wildtype, subclass RTK 1 (GBM_RTK1); glioblastoma IDH-wildtype, subclass RTK 2 (GBM_RTK2); glioblastoma IDH-wildtype, subclass mesenchymal (GBM_MES); glioblastoma IDH-wildtype, pediatric RTK 1 (GBM_pedRTK1); glioblastoma IDH-wildtype, pediatric RTK 2 (GBM_pedRTK2); glioblastoma IDH-wildtype, pediatric MYCN (GBM_pedMYCN); diffuse midline glioma H3 K27M-mutant (DMG_K27M); diffuse midline glioma H3-wildtype overexpressing EZHIP (DMG_EZHIP); glioblastoma IDH-wildtype, H3.3 G34-mutant (GBM_G34); ependymoma, posterior fossa group A (EPN_PFA).

Fig. 2

(A) Clinicopathological characteristics and recurrent genetic alterations of the 58 gliomas. (B).Visualization of the EGFR mutation profile in the investigated glioma cohort created using the online tool ProteinPaint available at https://proteinpaint.stjude.org/

Fig. 3

(A) Hematoxylin and eosin (H&E) staining of one of the gliomas included in the investigated cohort (case #1) revealing a pleomorphic astrocytic neoplasm with mitotic figures. (B) Immunohistochemical staining for histone H3 lysine 27 trimethylation (H3K27me3) showing a loss of nuclear expression in the tumor cells.

Fig. 4

(A) EGFR expression in the DMG_EGFR group in comparison to different other glioma showing that tumors within the DMG_EGFR group exhibit the highest expression of EGFR. (B) Distribution of normalized EZHIP gene expression level in the investigated glioma cohort in comparison to reference cases of H3 K27M-mutant and wildtype diffuse midline gliomas showing that H3 K27M-wildtype tumors exhibit higher expression of EZHIP.

Fig. 5

MR images from 2 representative patients of the investigated cohort showing (A) a 21-year-old male patient (case #6) with a mass in the left thalamus, extending into mesencephalon and left cerebellar peduncle and (B) a 12-year-old boy (case #21) with a mass in the right thalamus with diffuse infiltration of the right hemisphere and T2 changes also in the contralateral hemisphere.

Fig. 6

Kaplan–Meier curves for OS of 14 patients from the investigated cohort (DMG_EGFR) for whom OS data were available. OS of 14 patients is compared with OS of 46 patients with diffuse midline glioma, H3 K27M-mutant, WHO grade IV (DMG H3 K27M) diagnosed at the University Hospital Heidelberg.