An exceptionally rare case of a diffuse midline glioma with concomitant H3.1 K27M and G34R mutations in the HIST1H3C (H3C3) gene

Abstract

Histone mutations (H3 K27M, H3 G34R/V) are molecular features defining subtypes of paediatric-type diffuse high-grade gliomas (HGG) (diffuse midline glioma (DMG), H3 K27-altered, diffuse hemispheric glioma (DHG), H3 G34-mutant). The WHO classification recognises in exceptional cases, these mutations co-occur. We report one such case of a 2-year-old female presenting with neurological symptoms; MRI imaging identified a brainstem lesion which was biopsied. Histology showed diffusely infiltrating pleomorphic astrocytes, multinucleated cells, and conspicuous mitotic activity; the diagnosis was DMG, H3 K27-altered (immunohistochemistry: H3K27me3 loss, H3K27M positivity). DNA methylation profiling (Illumina EPIC BeadArrays, brain tumour classifier (MNP v12.5 R package)) classified the tumour as ‘DMG, H3 K27-altered’ (calibrated score = 0.99). Further molecular studies (whole exome, whole genome sequencing) revealed concurrent H3.1 K27M and G34R mutations (clonal, in the same reads) of H3C3, FGF11 and PIK3CA somatic variants, and a pathogenic germline NBN variant. The RNAseq profile clustered with H3K27M-mutant tumours. A patient-derived cell culture was established enabling unbiased in vitro drug screening; no selective sensitivities were identified. Chromatin immunoprecipitation assays with sequencing (ChIP-seq; H3K27ac, H3K27me3, H3K36me3, RNApol2 marks) showed features in keeping with DMG H3 K27M-mutant tumours (H3K27ac loci including OLIG2, IRX1/2, PKDCC). The patient was treated with adjuvant radiotherapy, but progressed and passed away 13 months post-diagnosis. This case is an exceptionally rare, complex variant of histone-mutant paediatric HGG, illustrating that the H3.1 K27M mutation demonstrates a dominance over the molecular and clinical profiles compared to G34R, and highlights the importance of broad molecular profiling to identify such examples for further study.

Introduction

The fifth edition of the WHO Classification of the Central Nervous System Tumours recognises two clinicopathologically distinct paediatric-type high-grade gliomas (HGG) defined by mutations of histone H3 genes [1]. Diffuse midline glioma (DMG), defined by H3 K27 alterations, are located in midline structures (including pons, thalamus, spinal cord). They are characterised by a global decrease of H3 tri-methylation (H3 p.K28me3 (p.K27me3)), associated with either H3 p.K28M (K27M) or rarely p.K28I (K27I) substitutions in histone H3.3 or H3.1/H3.2 isoforms, abnormal overexpression of EZHIP, or EGFR alterations [1,2,3,4]. The second subtype is the H3 G34-mutant diffuse hemispheric glioma (DHG), typically occurring in adolescents and young adults [5,6,7]. They contain a mutation in the H3F3A gene that results in an amino-acid substitution on the histone H3.3 tail at codon 35, most commonly glycine to arginine (c.103G > A or c.103G > C p.G35R - G34R) or rarely valine (c.104G > T p.G35V - G34V) [8]; these mutations have not been reported to occur in histone H3.1, unlike the K27M mutations. Each histone-mutant subgroup has a distinct methylation profile [9]. According to the WHO classification, it is exceptional for H3 K27M mutations to co-occur with H3 G34R/V mutations [1]. We report the clinicopathological and molecular characteristics of one such rare case with dual K27M and G34R mutations in the HIST1H3C (H3C3) gene, which has not been reported before.

Case presentation

A 2-year-old girl was admitted as an emergency case with ataxia, left-sided weakness, new-onset strabismus, and increasing lethargy. MRI head revealed an expansile ventral brainstem lesion centred on the pons, with extension into the superior aspect of the medulla and right side of midbrain (Fig. 1A). There was mass effect in the surrounding parenchyma with partial effacement of the prepontine cistern and distortion of the fourth ventricle, but no hydrocephalus. Elective stereotactic needle biopsy was undertaken (Fig. 1B).

Fig. 1

Characterising the tumour. (A) T1-weighted axial MRI image (left) and T2-weighted axial MRI image (right). (B) Clinical timeline showing the key timepoints of the patient’s diagnosis, treatment and progress. The timeline corresponds to the months post-diagnosis. (C) H&E sections of the tumour. (D) Panel of immunohistochemistry used to diagnose the tumour. The name of each stain is labelled in the bottom left corner of the image. (E) t-SNE plot of the Jones Lab glioma reference set (n = 3357). The primary tumour and the cell cultures are indicated by a black circle. Different glioma subgroups are represented by different colours and labelled with the group name. (F) IGV plot of DNA panel sequencing data showing the dual histone H3.1 HIST1H3C (H3C3) K27M and G34R mutations, occurring in the same reads, across the different cell cultures and the primary tumour. Oncoprint showing the variants identified in the primary tumour and cell cultures

Histology showed an infiltrative highly cellular glial tumour comprising pleomorphic and angulated nuclei, coarse chromatin, scanty eosinophilic cytoplasm and rare multinucleated cells, embedded in a fibrillary stroma (Fig. 1C). Mitotic activity was prominent, but there was no definite microvascular proliferation. There were focal areas of incipient necrosis (Fig. 1C). Immunohistochemistry showed strong immunoreactivity for OLIG2, GFAP, and H3K27M, H3K27me3 expression was lost from tumour cells and ATRX expression was retained by the tumour cells (Fig. 1D). Immunohistochemistry for the H3G34R mutation (specific to histone H3.3), EZHIP and mutant p53 was negative (Fig. 1D), with moderate-high Ki67 labelling (Fig. 1D). The immunohistochemistry profile therefore confirmed the diagnosis of diffuse midline glioma, H3 K27-altered, CNS WHO grade 4 and did not show a profile reflective of G34R/V-mutated tumours. The DKFZ Brain tumour classifiers (v12.5, v12.8) assigned the tumour to the DMG class with calibrated scores of 0.92 and 0.99 respectively.

DNA and RNA panel sequencing identified concurrent p.K28M (c.83 A > T) and p.G35R (c.103G > C, AF 0.44) mutations in the HIST1H3C (H3C3) gene in cis, and a pathogenic PIK3CA variant (c.1031T > G, AF 0.46) (Fig. 1F). Whole genome sequencing (WGS) identified an FGF11 variant and a pathogenic germline NBN variant (c.698_701delAACA p.Lys233SerfsTer5). Copy number profiles showed gains of chr1q and chr2 (Supp. Figure 1A). The final integrated diagnosis was diffuse midline glioma, H3 K27-altered, with concomitant H3.1 K27M and H3.1 G34R mutations, CNS WHO grade 4.

At biopsy, a tumour sample was collected for in vitro model establishment. 2D and 3D models (ICR-CXJ074) were established (Fig. 2A). DNA methylation profiling (passage 5), classified all of the cultures as diffuse midline glioma with high calibrated scores (0.74 and 0.72), clustering with the DMG group in our glioma reference cohort t-SNE (n = 3357) (Fig. 1E). Whole exome sequencing (WES) identified the same mutations as the primary tumour including the PIK3CA mutation (Fig. 1F). Copy number profiling, derived from the Illumina array data, showed a gain of chromosome 1q and 2, similar to that of the primary tumour (Supp. Figure 1A). Immunofluorescence showed expression of glial markers GFAP, OLIG2, stem-cell markers SOX2 and Nestin (Fig. 2B). RNA sequencing, for both the patient-derived models and the primary tumour, confirmed an expression profile consistent with other K27M-mutant DMGs (Supp. Figure 1B, C). Having established in vitro patient-derived models representative of the primary tumour, we undertook unbiased drug screening using the ICR drugs & tools library of over 800 compounds on cells cultured in 2D; no selective sensitivities were identified (Fig. 2C).

Fig. 2

In vitro patient-derived modelling. (A) Photo of ICR-CXJ074 2D culture, grown on laminin, passage 5 (top). Photo of ICR-CXJ074 3D neurosphere culture, passage 5 (below). (B) Immunofluorescence panel exploring the profiles of ICR-CXJ074 (3D). GFAP (red), Nestin (green), OLIG2 (green), SOX2 (red). (C) Heatmap showing screen results from the ICR drugs & tools library, compared with other screens using histone-mutant and wild-type models. ICR-CXJ74 results are shown in the heatmap on the left. (D) Heatmaps showing profiles of the H3K27me3 (left) and H3K27ac (right) peaks for ICR-CXJ074 compared to other histone-mutant tumours (the different histone subtypes are highlighted by a coloured outline according to the key provided). (E) Oncoprints showing ChIP-Seq gene level comparisons for H3K27ac (left) and H3K27me3 (right) compared to other histone-mutant models

To further characterise this tumour, chromatin immunoprecipitation assays with sequencing (ChIP-Seq) were undertaken looking at loci associated with the H3K27ac, H3K27me3, H3K36me3, RNApol2 marks, comparing with published data for H3.3 K27M- and H3.3 G34R-mutant patient-derived cell lines. The ICR-CXJ074 H3K27me3 peaks were in keeping with that of H3.3 K27M-mutant lines with a similar pattern observed for H3K27ac peaks (Fig. 2D). At the gene level, H3K27ac loci highlighted OLIG2, PKDCC and CBLN1 genes and H3K27me3 loci were associated with DLX1, DLX2, DLX5 and OTX1 genes (Fig. 2E, Supp. Figure 2A). The H3K27ac super-enhancer profile of ICR-CXJ074 clustered with other K27M-mutant tumours (Supp. Figure 2B). Overall, ICR-CXJ074 showed genetic, epigenetic and transcriptomic features in keeping with DMG H3 K27M-mutant tumours.

The patient received radiotherapy (39 Gy, 13 fractions) [10] and subsequently the mTOR inhibitor everolimus (5-month duration) due to the identification of a somatic PIK3CA alteration, a relevant molecular target within the PI3-kinase/AKT/mTOR signalling pathway. Initial follow-up scans after radiotherapy showed shrinkage of the tumour on treatment. However, areas of expansion and foci of enhancement in the pons were identified on repeat scans. The tumour extended into the medulla, and posterior limb of the internal capsule, with increased enhancement in keeping with tumour progression 6 months post-diagnosis. These matched evolving clinical symptoms of disease progression – increasing ataxia, weakness, deteriorating speech and eventually unsafe swallow. The patient was re-irradiated (20 Gy, 10 fractions) in keeping with national and international guidelines [11], but the disease continued to progress and the patient sadly passed away 13 months post-diagnosis (Fig. 1B).

Discussion and conclusions

Two cases of dual-histone mutant tumours have been previously reported in the literature, and both displayed low-grade histological features and a global decrease of H3K27me3. The first, a 7-year-old male with a tumour in the spinal cord, harboured an H3F3A K27M mutation co-occurring with a novel G34W variant on the same allele [12]. Although novel in HGG, this G34W variant is known to characterise giant cell tumours of bone which are a rare group of bone tumours occurring more frequently in long bones at the meta-epiphyseal region [13]. The second, a thalamic tumour in a 38-year-old male, showed low-grade gemistocytic histological features with concomitant K27M and G34R mutations in the HISTH3B gene [14]. Accompanying mutations in the FGFR1, NF1, PTPN11, PPM1D and ATRX genes were also seen [14]. Our case represents the first reported DMG with concomitant H3 K27M and G34R mutations involving the HIST1H3C (H3C3) gene and high-grade histology; the molecular profile reflects that of a K27M-mutant DMG rather than a G34R-mutant DHG and suggests that the K27M mutation is the dominant driver over G34R on histone H3.1 and when in cis. It also should be noted that we are comparing the data with H3.3 G34 tumours and we do not know the role and function of the H3.1 G34 mutation at this stage. The germline pathogenic NBN variant is associated with autosomal recessive Nijmegen breakage syndrome (NBS), but there were no suggestive clinical features in this patient upon review by a clinical geneticist; variants in this gene have been reported in other astrocytic tumours [15, 16], but the possible role and implications are yet be determined.

This case highlights that there are rare but complex genetic aberrations of histone mutant tumours which are important to identify and research in order to understand more about their underlying biology.