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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.

BACKGROUND: Gliomatosis cerebri (GC), a radiologically defined diffusely infiltrating glioma, is no longer considered a distinct entity since the 2016 WHO classification for tumors of the CNS. Due to its rarity and dismal prognosis treatment recommendations in children remain ambiguous. Using central neuroradiological review, we performed a multi-institutional, retrospective study of GC providing comprehensive radiological, clinical, and (epi)genetic characterization. RESULTS: We included 104 patients between 1-19 years. Within a median follow-up of 15.5 months (range, 2.3-138.8), 93 patients (89.4 %) had died, 4 (3.8 %) were lost to follow-up and 7 (6.8 %) were alive with stable/progressive disease. Median progression-free- (PFS) and overall survival (OS) were 8.6 months (interquartile range, 4.3-14.0) and 15.5 months (10.9-27.7), respectively. Former WHO grading correlated significantly with median OS: WHO °II: 47.8 months (25.2-55.7); WHO °III: 15.9 months (11.4-26.3); WHO °IV: 10.4 months (8.8-14.4) (p

PURPOSE Constitutional Mismatch Repair Deficiency (CMMRD) is a cancer predisposition due to bi-allelic mutations in one of the four main mismatch repair (MMR) genes (PMS2, MSH2, MSH6 or MLH1) associated with early onset of cancers, especially glioblastomas (GBM). Our aim was to decipher the molecular specificities of gliomas occurring in this context. METHODS A comprehensive analysis of clinical, histopathological and genomic data (whole exome sequencing) was performed for 12 children with a CMMRD for which we had available frozen brain tumor material (10 GBM and 2 anaplastic astrocytomas). RESULTS Eight patients harbored an ultra-mutated phenotype with more than 100 somatic non synonymous (NS) SNV/Mb. No correlation was observed between the number of mutation and sex, age, overall survival or mutated MMR gene. POLE and POLD1 exonuclease domain driver somatic mutations were described for eight and one patients respectively. The 4/12 tumors without POLE somatic mutation did not show the classical ultra-hypermutation pattern. All patients with POLE mutation had already more than 20 NS SNV/Mb (median 40NS SNV/Mb, [range 23-114]) suggesting that the hypermutation phenomenon started before the appearance of the somatic POLE mutation. The mutational signatures of the tumors, dominated by the MMR signatures, were not modified after the onset of the POLE mutation when analyzing the different mutation bursts. Specific recurrent somatic mutations were observed in SETD2 (9/12), TP53 (9/12), NF1 (9/12), EPHB2 (8/12), and DICER1 (7/12). Only half of the tumors overexpressed PDL1 by immunohistochemistry and this overexpression was not associated with a higher tumor mutation burden. CONCLUSION CMMRD-associated gliomas have a specific oncogenesis that does not trigger usual pathways and mutations seen in sporadic pediatric or adult GBM. Frequent alterations in other pathways (e.g. MAPK or DNA-PK pathway) may suggests the use of other targeted therapies aside from PD1 inhibitors.

PURPOSE: Constitutional Mismatch Repair Deficiency (CMMRD) is a cancer predisposition due to bi-allelic mutations in one of the four main mismatch repair (MMR) genes (PMS2, MSH2, MSH6 or MLH1) associated with early onset of cancers, especially glioblastomas (GBM). Our aim was to decipher the molecular specificities of gliomas occurring in this context. METHODS: A comprehensive analysis of clinical, histopathological and genomic data (whole exome sequencing) was performed for 12 children with a CMMRD for which we had available frozen brain tumor material (10 GBM and 2 anaplastic astrocytomas). RESULTS: Eight patients harbored an ultra-mutated phenotype with more than 100 somatic non synonymous (NS) SNV/Mb. No correlation was observed between the number of mutation and sex, age, overall survival or mutated MMR gene. POLE and POLD1 exonuclease domain driver somatic mutations were described for eight and one patients respectively. The 4/12 tumors without POLE somatic mutation did not show the classical ultra-hypermutation pattern. All patients with POLE mutation had already more than 20 NS SNV/Mb (median 40, [range 23-114]) suggesting that the hypermutation phenomenon started before the appearance of the somatic POLE mutation. The mutational signatures of the tumors, dominated by the MMR signatures, were not modified after the onset of the POLE mutation when analyzing the different mutation bursts. Specific recurrent somatic mutations were observed in SETD2 (9/12), TP53 (9/12), NF1 (9/12), EPHB2 (8/12), and DICER1 (7/12). Only half of the tumors overexpressed PDL1 by immunohistochemistry and this overexpression was not associated with a higher tumor mutation burden. CONCLUSION: CMMRD-associated gliomas have a specific oncogenesis that does not trigger usual pathways and mutations seen in sporadic pediatric or adult GBM. Frequent alterations in other pathways (e.g. MAPK or DNA-PK pathway) may suggests the use of other targeted therapies aside from PD1 inhibitors.

Diffuse midline gliomas (DMG) H3 K27M-mutant were introduced in the 2016 WHO Classification unifying diffuse intrinsic pontine gliomas (DIPG) and gliomas from the thalamus and spinal cord harboring a histone H3-K27M mutation leading to Polycomb Repressor Complex 2 (PRC2) inhibition. However, few cases of DMG tumors presenting a H3K27 trimethylation loss, but lacking an H3-K27M mutation were reported. To address this question, we combined a retrospective cohort of 10 patients biopsied for a DIPG at the Necker Hospital or included in the BIOMEDE trial (NCT02233049) and extended our analysis to H3-wildtype (WT) diffuse gliomas from other midline locations presenting either H3K27 trimethylation loss or ACVR1 mutation from Necker, ICR, the HERBY trial, the INFORM registry study and the St. Jude PCGP representing 9 additional cases. Genomic profiling identified alterations frequently found in DMG, but none could explain the observed loss of H3K27 trimethylation. Similar observations were previously made in the PF-A subgroup of ependymoma, where the H3K27me3 loss resulted from EZHIP/CXorf67 overexpression rather than H3-K27M mutations. We thus analyzed EZHIP expression and observed its overexpression in all but one H3-WT DMGs compared to H3-K27M mutated tumors (EZHIP negative). Strikingly, based on their DNA methylation profiles, all H3-WT DMG samples analyzed clustered close to H3-K27M DIPG, rather than EZHIP overexpressing PF-A ependymomas. To conclude, we described a new subgroup of DMG lacking H3-K27M mutation, defined by H3K27 trimethylation loss and EZHIP overexpression that can be detected by IHC. We propose that these EZHIP/H3-WT DMGs extend the spectrum of DMG with PRC2 inhibition beyond H3-K27M mutation.

Diffuse Intrinsic Pontine Glioma (DIPG) and more largely Diffuse Midline Gliomas H3 K27M-mutant (DMG) harbor a unique property of infiltration. Our objective is to elucidate/describe the cellular and molecular determinants of micro-environmental modifications resulting from the tumour/stroma dialogue as it might provide pro-invasive conditions that favour the development of the disease. To this end, we performed RNA-seq analyses to characterize exhaustively the bidirectional molecular modifications of the stroma/tumour in DIPG xenograft models. Gene expression changes in murine microenvironment compartment were investigated as continuous or semi-continuous traits of tumor load by measuring transcriptome in zone with high vs. low infiltration. We observed substantial modulations in gene expression in the microenvironment associated with increasing tumor cell content, pointing to a modification of the macrophage/microglial infiltrate. The expression or overexpression of several modulated genes was validated by IHC in the stroma of DMG primary tumors. Among them, overexpression of the cytokine CCL3 was confirmed, reflecting the activation status of microglial cells. Moreover, we observed in patients that the density of IBA-1 positive microglial cells increases according to the extent of tumor infiltration and that a significant part of them harbor a mitotic status, supporting their interaction with DMG cells. The involvement of this interaction in DMG development needs further evaluation and might represent opportunity to slow down DIPG extension.

Diffuse Intrinsic Pontine Glioma (DIPG) remains the most fatal form of pediatric brain tumor. The specific somatic mutation (H3K27M) in the H3 histone gene was found in around 95% of patients making this alteration as the initial oncogenic event. However, until now this alteration cannot be targeted. Consequently, we conducted a RNAi-based loss-of-function screen targeting the human kinome to decipher vulnerabilities in DIPG, unravel their biology and provide therapeutic opportunities in this devastating disease. An integrative lentiviral pooled library of 7450 shRNAs against 770 kinases was used to transduce four GSC (glioma stem cells) cultures harboring either H3.1 (n=2) or H3.3-mutation (n=2) in order to cover the diversity observed in patients, and 2 control NSC (neural stem cell) cultures. The entire set of shRNAs was identified by sequencing 40 hours and 22 days after transduction. Genes required for cell expansion over 22 days of outgrowth in GSC but with limited deleterious effect on NSC proliferation were identified. Among those, we selected the target genes with at least 3 distinct shRNAs presenting a significant decrease in their abundance and identified in particular VRK3 (Vaccinia-Related Kinase 3) as one of the top scoring kinases whose extinction was lethal to DIPG. This serine-threonine kinase was recently linked to cell cycle progression, DNA repair and neuronal differentiation. The role of this protein in DIPG biology is currently assessed especially to identify actionable downstream targets. Drugs mimicking the effect of VRK3 extinction will be evaluated in our vitro and vivo models of DIPG.

INTRODUCTION: BIOMEDE is a multicentre, phase-II trial comparing efficacy of erlotinib, everolimus, and dasatinib in combination with radiotherapy, in newly diagnosed diffuse intrinsic pontine glioma (DIPG) (NCT02233049). Randomisation is based on biomarkers determined in upfront stereotactic biopsies: mTOR pathway activation detected by the loss of PTEN expression and EGFR overexpression. METHODS: A central pathology review is mandatory in BIOMEDE for DIPG diagnosis confirmation, assessment of H3K27M immunostatus, PDGFRA amplification by FISH and biomarkers evaluation for treatment allocation (no erlotinib if no EGFR overexpression; no everolimus if no PTEN loss, no specific marker for dasatinib allocation). We evaluated the process feasibility and the agreement between H3.3 immunoprofil and genotyping on the first 120 patients. RESULTS: DIPG was centrally confirmed in 94% of cases (median time=1 day; range 0–6). The differential diagnoses were HGNET-MYCN (n=1) and angiocentric glioma MYB-QKI (n=1). No tumoral infiltration was found in 3 cases and extensive necrosis in 1 case. Overall, 38/118 evaluable cases showed EGFR overexpression (32%) and 90/118 had PTEN loss (76%). 15/105 evaluable cases had PDGFRA amplification (14%). Trimethylation loss at H3K27 was detected in all samples but 4 corresponding to HGNET-MYCN, angiocentric glioma and to 2 H3 wild-type gliomas. Agreement between genotyping (H3.3 and/or H3.1) and H3K27M immunostatus (loss of trimethylation and/or H3K27M) is substantial (Kappa coefficient=0.79, 95%-confidence interval, 0.41 to 1). CONCLUSIONS: These data document the feasibility of real-time molecular pathology and biomarkers screening in international multicentre trials.

Diffuse intrinsic pontine gliomas (DIPG) are the most severe and common form of malignant brain tumours in children and adolescent. Radiotherapy is the only validated treatment, albeit only bringing a transient symptom relief followed by a constant subsequent progression within a variable delay. We previously identified a heterogeneous response to radiotherapy at diagnosis in DIPG patients depending on the type of mutated histone H3 (Castel et al., 2015 & 2016). We thus hypothesized that there are possible molecular determinants of this initial response, and evaluated the radiosensitivity of a compendium of 13 treatment-naïve cellular models of DIPG deriving from stereotactic biopsies at diagnosis. Our results show a heterogeneous response of DIPG cells to RT in vitro correlating with the duration of the clinical response in the corresponding patients. This phenomenon thus implies a cell-autonomous response, and we decided to confront radioresistance with the molecular profiles of the corresponding cells. It appears that the main driver of resistance to irradiation in DIPG is the inactivation of TP53, and not the type of mutated histone per se. We are currently validating this result in isogenic DIPG cells with or without functional TP53, and studying the underlying molecular mechanisms that could explain the differences in radiosensitivity of these tumour cells. By confronting tumour molecular data with clinical responses to RT in a monocentric retrospective cohort, we are able to consolidate our in vitro prediction and propose new possible stratifications in DIPG with respect to response to therapy.

BACKGROUND: Genomic analyses of DIPG have been so far mostly performed on autopsy samples or in retrospective studies. In the frame of the BIOMEDE randomized trial of three targeted therapies (erlotinib, everolimus, dasatinib) based on three biomarkers (EGFR, PTEN and PDGFRA respectively), a systematic whole exome and RNA sequencing of stereotactic biopsies was performed at diagnosis. These analyses were implemented to inform the results of the trial and define additional targets that could be used to guide therapies at relapse. PATIENTS AND METHODS: The results of the first 100 patients from France, Denmark and Belgium are presented. Seventy-five percent of the samples could be profiled for DNA &/or RNA, no frozen material was available for 6% of the tumors and only one sample was not contributive. RESULTS: Tumor samples from all but one patients included in the study on the basis of histology showed a loss of H3-K27 trimethylation. With respect to histone H3-K27M mutation, 75% of the samples were mutated in the H3F3A gene, 17% in the HIST1H3B gene and 8% were wild-type for all histone H3 genes. TP53 or PPM1D mutations were identified in 75% of H3F3A-K27M DIPG while never found in HIST1H3B-K27M tumours. ACVR1 mutations were almost exclusively found in HIST1H3B-K27M mutated samples, except for two histone H3 wild-type and one H3F3A-K27M mutated samples. Receptor Tyrosine-Kinases mutations or amplifications were restricted to H3F3A-K27M DIPG. Most tumors showed copy number variations. But gains of chromosome 1q and whole chromosome 2 were more frequent in the HIST1H3B-K27M mutated samples. On the opposite, chromothripsis-like structural copy number aberrations mostly affected TP53 or PPM1D mutated samples. Translocations were only rarely identified and none was recurrent. CONCLUSION: This study refines the description of the differences in the subtypes of DIPG and may support specific treatment adaptations with respect to potential biomarkers.