K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas

Our findings confirm that the K27M mutation in histone H3.3 is a frequent event in pediatric DIPG. We further show that both the type of H3.3 mutation and their association with ATRX mutations are age and location dependent. The G34V/R-H3.3 mutation was not found in DIPGs whereas it represents 13 % of H3.3 mutations in supratentorial GBM. A recent report similarly did not find mutations at the G34 residue of H3.3 in DIPG [25]. Similarly, ATRX mutations were infrequent in DIPG but were present in 29 % of supratentorial GBM. This may be an age-related phenomenon as the mean age of our DIPG cohort was 7.1 versus 12.24 years for the supratentorial GBM patient cohort. In support of this, G34V/R-H3.3 was seen in older patients [mean age 19.66 years (±1.56)] and almost exclusively in hemispheric GBM [23]. Similarly, ATRX mutation-positive patients were significantly older than wild-type patients. We did not identify K27M-H3.1, which has been recently identified in 18 % (9 samples) of DIPGs [25]. This difference in frequency may be due to sampling bias; however, our findings support H3.3 as the major histone to be targeted in pediatric GBM and K27 the major residue affected in DIPG.

H3.3 is the major histone to be loaded on chromatin during brain development. This histone variant is known to modulate specific chromatin changes and gene expression profiles and to be associated with active chromatin and translation. Histone lysine methylation has emerged as an important player in regulating gene expression and chromatin function [14]. K27 is a critical residue in all seven histone 3 variants and the subject of post-translational histone modifications as it can be both methylated and acetylated [2, 14, 20]. Acetylation may induce active transcription, while mono, bi or tri-methylation of K27 is associated with a repressive mark on chromatin and gene expression. Abrogation of acetylation and/or potential mimicry of a methylated lysine through the methionine substitution are likely to interfere with chromatin function, inducing defects in chromatin remodelling and tumorigenesis. This is supported by our observation of specific copy number changes associated with mutant K27M-H3.3. Further studies aiming to model this mutation are required to precisely determine the effect of this mutation in chromatin remodelling in pediatric GBM.

ATRX mutations were only identified in 9 % of DIPGs compared to 29 % of supratentorial pediatric GBM. Notably, the presence of ATRX mutation significantly overlapped with TP53 mutations in GBM (p = 0.01) regardless of the location within the brain and with G34V/R mutants in supratentorial GBM (p < 0.0001), and were age-dependent as they mainly occurred in older children (p < 0.0001). The requirement for ATRX mutations in GBM may thus be due to tumor location and/or the age of the patient. This is potentially indicative of a different cell of origin or age-related plasticity of the tumor, similar to differences in genetic alterations seen based on age in infant MLL-positive leukemia [5]. TP53 mutations are associated with the vast majority of both K27M and G34V/R H3.3 mutations identified in pediatric and young adult GBM. In both DIPG and supratentorial GBM, TP53 alterations were commonly identified (77 and 54 %, respectively). Interestingly, in DIPGs, the K27M-H3.3 and wild-type H3.3 subsets had similarly high TP53 mutation and allelic loss rates. In K27M-H3.3-mutated tumors, this may thus represent an important second hit; however, our data are also indicative of an important role of TP53 mutations in the pathogenesis of GBM independent of H3.3 mutational status.

One of the most common copy number gains reported in multiple genomic studies of DIPG and pediatric GBM is that of PDGFRA [17, 18, 26]. Here, we report PDGFRA gain or amplification to be seen exclusively in the patients carrying K27M-H3.3 mutations, where it is present in 40 % of cases. We also identify gains and amplifications in a gene locus containing MYC/PVT1, also exclusively in K27M-H3.3 mutants. PVT-1 is an oncogene and a Myc protein target known to be over-expressed in transformed cells [6]. Amplification of MYC/PVT1 has been shown to contribute to the pathogenesis of ovarian and breast cancer, and is part of the chromosome 8q24 prostate cancer risk locus [16]. The finding of these copy number changes in a subset of K27M-H3.3 mutants suggests that PDGFRA and MYC/PVT1 locus gains/amplifications are subsequent to K27M-H3.3 mutations. The addition of histone modifying agents to RTK inhibitors may thus be of therapeutic benefit in this group of patients.

A clinically significant finding of this study is the fact that patients who harbor the K27M-H3.3 mutation have worse overall survival when compared to patients who are wild-type for H3.3. This association with survival was independent of patient age and histologic diagnosis. The only attributable histologic feature exclusive to the K27M mutant group is glial differentiation. However, not all of the samples mutated for K27M-H3.3 met criteria for GBM. The hypothesis that mutation status identifies distinct subtypes is further supported by the differences in copy number profiles and age distribution between these patient groups. Importantly, long-term survivors were only identified in the group of patients who are wild-type for this gene. Some of these patients had an atypical clinical presentation (longer duration of symptoms or atypical radiology) and thus were biopsied, demonstrating high grade histologic features. Despite high grade histology, this group of H3.3 wild-type patients did not follow the expected clinical course of what can be considered classic for children with DIPG, suggesting a potentially different clinical and molecular entity which should be added to the group of “atypical” DIPG when considering clinical trial design. These H3.3 wild-type tumors may be more heterogeneous in terms of histology and biological features than K27M-H3.3 tumors, and may perhaps, with larger numbers, be further divided into different sub-groups. One uncommon subgroup of H3.3 wild-type brainstem tumors which is hinted at by our series is the PNET. Interestingly, the two PNET patients in our cohort presented with “classical” DIPG features were treated as DIPG patients and both had a poor outcome despite the H3.3 wild-type status. This raises the hypothesis that H3.3 wild-type status may suggest better outcome only in glial neoplasms.

In contrast, the H3.3-mutated group contained patients who, at autopsy, had tumors which, if classified by WHO guidelines, would be considered diffuse astrocytoma, grade II. Nevertheless, these patients had the short survival expected of classic DIPG. Conversely, some patients with high-grade histology such as those with features of GBM were in the wild-type group. Thus, mutational status of H3.3 may be more helpful than histologic appearance alone in identifying patients expected to have a poor clinical outcome at presentation.

Our findings support performing a stereotactic biopsy, particularly for atypical clinical cases of DIPG. The finding of the K27M-H3.3 mutation can be considered as diagnostic of classic DIPG with its expected poor outcome. These patients may benefit from agents targeted at chromatin remodelling and/or histone post-translational modifications with an additional RTK inhibitor. Although not universally indicative of better clinical behavior, wild-type patients should perhaps be considered as atypical DIPG, and if coupled with atypical radiology and/or clinical presentation may warrant a different therapeutic approach.