Castel et al. Acta Neuropathologica Communications (2018) 6:117 https://doi.org/10.1186/s40478-018-0614-1 RESEARCH Open Access Transcriptomic and epigenetic profiling of ‘diffuse midline gliomas, H3 K27M-mutant’ discriminate two subgroups based on the type of histone H3 mutated and not supratentorial or infratentorial location David Castel1,2*, Cathy Philippe1,12, Thomas Kergrohen1,2, Martin Sill3,4, Jane Merlevede1, Emilie Barret1, Stéphanie Puget5, Christian Sainte-Rose5, Christof M. Kramm6, Chris Jones7, Pascale Varlet8, Stefan M. Pfister3,4,9, Jacques Grill1,2, David T. W. Jones3,10 and Marie-Anne Debily1,11,13* Abstract Diffuse midline glioma (DMG), H3 K27M-mutant, is a new entity in the updated WHO classification grouping together diffuse intrinsic pontine gliomas and infiltrating glial neoplasms of the midline harboring the same canonical mutation at the Lysine 27 of the histones H3 tail. Two hundred and fifteen patients younger than 18 years old with centrally-reviewed pediatric high-grade gliomas (pHGG) were included in this study. Comprehensive transcriptomic (n = 140) and methylation (n = 80) profiling was performed depending on the material available, in order to assess the biological uniqueness of this new entity compared to other midline and hemispheric pHGG. Tumor classification based on gene expression (GE) data highlighted the similarity of K27M DMG independently of their location along the midline. T-distributed Stochastic Neighbor Embedding (tSNE) analysis of methylation profiling confirms the discrimination of DMG from other well defined supratentorial tumor subgroups. Patients with diffuse intrinsic pontine gliomas (DIPG) and thalamic DMG exhibited a similarly poor prognosis (11.1 and 10.8 months median overall survival, respectively). Interestingly, H3.1-K27M and H3.3-K27M primary tumor samples could be distinguished based both on their GE and DNA methylation profiles, suggesting that they might arise from a different precursor or from a different epigenetic reorganization. These differences in DNA methylation profiles were conserved in glioma stem-like cell culture models of DIPG which mimicked their corresponding primary tumor. ChIP-seq profiling of H3K27me3 in these models indicate that H3.3-K27M mutated DIPG stem cells exhibit higher levels of H3K27 trimethylation which are correlated with fewer genes expressed by RNAseq. When considering the global distribution of the H3K27me3 mark, we observed that intergenic regions were more trimethylated in the H3.3-K27M mutated cells compared to the H3.1-K27M mutated ones. H3 K27M-mutant DMG represent a homogenous group of neoplasms compared to other pediatric gliomas that could be further separated based on the type of histone H3 variant mutated and their respective (Continued on next page) * Correspondence: [email protected]; marie- [email protected] 1UMR8203,Vectorologie et Nouvelles Thérapies Anticancéreuses, CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, 94805 Villejuif, France Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Castel et al. Acta Neuropathologica Communications (2018) 6:117 Page 2 of 13 (Continued from previous page) epigenetic landscapes. As these characteristics drive different phenotypes, these findings may have important implicationforthedesignoffuturetrialsinthesespecifictypesofneoplasms. Keywords: Pediatric high-grade glioma, Diffuse midline glioma, H3 K27M-mutant, Diffuse intrinsic pontine glioma, Epigenetics, DNA methylation profiling, Gene expression profiling, H3K27me3 landscape, Glioma stem cell, Introduction these parameters on their biology described by their gene ex- Diffuse intrinsic pontine glioma and malignant midline pression, methylome, and clinical behaviour. gliomas have the worst prognosis of all types of malig- Moreover, we compared the H3-K27me3 landscape nant tumors in children and adolescents [3, 4, 10]. The between the two main subgroups of DIPG, H3.1-K27M nosological shift in the 2016 WHO classification now and H3.3-K27M, in patient deriving cellular models. based on both phenotype and genotype has redefined the family tree of diffuse gliomas [17]. Glial tumors are Materials & methods now grouped according to their driver mutation, e.g. Central pathology review IDH1 mutation, and their astrocytic or oligodendroglial High-grade glioma cases were reviewed centrally to con- phenotypes which are often associated with additional firm the diagnosis according to the 2007 WHO classifica- ATRX specific genetic alterations such as mutations or tion and its 2016 update as previously described [10, 20]. 1p/19q co-deletion, respectively. The discovery of recur- Specific immunostainings were performed to detect rent mutations in the histone H3 genes in pediatric nuclear expression of the trimethylation mark at position high-grade glioma has definitively separated these K27 of the histone 3 tail (1:1000, polyclonal rabbit anti- gliomas from the ones seen in adults [21, 26]. While body, Diagenode, Belgium) as well as nuclear expression H3F3A G34R/V mutations in the gene are exclusively of the K27M form of histone H3 (1:1000, polyclonal found in the hemispheres, K27M/I mutations in several rabbit antibody, Millipore, CA). histone H3 variants genes are specific to midline tumors [23]. The 2016 release of the WHO classification has therefore created a new entity to describe these latter Derivation and culture of glioma stem-like cells (GSCs) tumors as diffuse midline glioma, H3K27M mutant, irre- GSCs were derived from DIPG tumors at diagnosis as spective of their specific location along the midline. previously described [19]. Briefly, tumor cells were In pediatric brain tumors, location has however long mechanically dissociated from biopsies within 24 h of been seen as a master driver of oncogenesis that could surgery, and further cultured as an adherent monolayer reflect their different cells of origin [8, 9]. Whether the in laminin-coated flask (Sigma) in neural stem cells oncogenic driver mutation is overriding location as a medium consisting of NeuroCult NS-A proliferation crucial determinant of oncogenesis is therefore to be medium (Stemcell technologies) supplemented with hep- μ examined since biologic identity of all these tumors arin (2 g/mL, Stemcell technologies), human-basic FGF would call for a common therapeutic framework. There (20 ng/ml, Peprotech), human-EGF (20 ng/ml, Pepro- is however no reported data showing at once a similar tech), PDGF-AA (10 ng/ml, Peprotech), and PDGF-BB biology and outcome of diffuse midline gliomas (DMG) (10 ng/ml, Perprotech). Medium was renewed every irrespective of their location in the presence of a histone other day, and passaging performed when cells reached H3-K27M mutation. 80% confluence using Accutase (Thermo). Moreover, we have shown two distinct forms of diffuse intrinsic pontine gliomas according to the type of his- Case selection for overall survival analysis and gene tone H3 gene mutated, H3F3A versus HIST1H3B, with expression profiling by microarray respect to differentiation markers, oncogenic programs, Frozen tissue samples were obtained from 119 pediatric response to therapy and evolution [1, 2]. These muta- patients with brain tumors of WHO grade III and IV (all tions are mutually exclusive either because their effect is locations, below 18 years old). The samples were col- redundant [16] leading to a global loss of H3K27me3 lected at Necker Hospital (Paris, France). Complete repressive mark, or because they cannot transform the follow-up information was available for 82.5% of patients same cell, suggesting the idea of distinct cells of origin. (n = 99). Histone H3 gene mutational status was deter- The purpose of this work was therefore to better mined by Sanger sequencing for H3F3A, HIST1H3B/C characterize a large series of pediatric midline high grade and HIST2H3C [2]. The distribution of samples in the gliomas from the (epi)genomic, transcriptomic and anatomic distinct genotype subgroups and location are detailed point of view in order to identify the respective influences of in Table 1. Castel et al. Acta Neuropathologica Communications (2018) 6:117 Page 3 of 13 Table 1 Contingency table of samples used for microarray gene correlation, weighted by variance. Clustering analyses were expression profiling and overall survival analysis performed using the beta values of the top 10,000 most Tumor Histone H3 mutational status Total variably methylated probes by standard deviation. Methy- location number H3.3- H3.1- H3.3- H3.1 & lation probes in the heatmap representation were of samples G34R K27M K27M H3.3-WT reordered by unsupervised hierarchical clustering using Cortex 6 0 0 35 41 Pearson correlation distance and median linkage. Pons 0 13 26 6 45 Non-thalamic 0021214 Microarray gene expression profiling midline Gene expression analysis was conducted on an Agilent Thalamic 0 0 12 7 19 platform as previously