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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 Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. While genome and transcriptome landscapes are well studied, data of the complete methylome, tumor cell composition, and immune infiltration are scarce. Methods We generated whole genome bisulfite sequence (WGBS) data of 9 PAs and 16 control samples and integrated available 154 PA and 57 control methylation array data. RNA sequence data of 49 PAs and 11 control samples as well as gene expression arrays of 248 PAs and 28 controls were used to assess transcriptional activity. Results DNA-methylation patterns of partially methylated domains suggested high stability of the methylomes during tumorigenesis. Comparing tumor and control tissues of infra- and supratentorial location using methylation arrays revealed a site specific pattern. Analysis of WGBS data revealed 9381 significantly differentially methylated regions (DMRs) in PA versus control tissue. Enhancers and transcription factor (TF) motifs of five distinct TF families were found to be enriched in DMRs. Methylation together with gene expression data–based in silico tissue deconvolution analysis indicated a striking variation in the immune cell infiltration in PA. A TF network analysis showed a regulatory relation between basic leucine zipper (bZIP) transcription factors and genes involved in immune-related processes. Conclusion We provide evidence for a link of focal methylation differences and differential gene expression to immune infiltration.

Gliomas are the most common primary central nervous system tumors occurring in children and adults with neurofibromatosis type 1 (NF1). Over the past decade, discoveries of the molecular basis of low-grade gliomas (LGGs) have led to new approaches for diagnosis and treatments. However, these new understandings have not been fully applied to the management of NF1-associated gliomas. A consensus panel consisting of experts in NF1 and gliomas was convened to review the current molecular knowledge of NF1-associated low-grade “transformed” and high-grade gliomas; insights gained from mouse models of NF1-LGGs; challenges in diagnosing and treating older patients with NF1-associated gliomas; and advances in molecularly targeted treatment and potential immunologic treatment of these tumors. Next steps are recommended to advance the management and outcomes for NF1-associated gliomas.

Background Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive pediatric brain tumors that are characterized by a recurrent mutation (K27M) within the histone H3 encoding genes H3F3A and HIST1H3A/B/C. These mutations have been shown to induce a global reduction in the repressive histone modification H3K27me3, which together with widespread changes in DNA methylation patterns results in an extensive transcriptional reprogramming hampering the identification of single therapeutic targets based on a molecular rationale. Methods We applied a large-scale gene knockdown approach using a pooled short hairpin (sh)RNA library in combination with next-generation sequencing in order to identify DIPG-specific vulnerabilities. The therapeutic potential of specific inhibitors of candidate targets was validated in a secondary drug screen. Results We identified fibroblast growth factor receptor (FGFR) signaling and the serine/threonine protein phosphatase 2A (PP2A) as top depleted hits in patient-derived DIPG cell cultures and validated their lethal potential by FGF ligand depletion and genetic knockdown of the PP2A structural subunit PPP2R1A. Further, pharmacological inhibition of FGFR and PP2A signaling through ponatinib and LB-100 treatment, respectively, exhibited strong tumor-specific anti-proliferative and apoptotic activity in cultured DIPG cells. Conclusions Our findings suggest FGFR and PP2A signaling as potential new therapeutic targets for the treatment of DIPGs.

Background Patients with glioblastoma without O6-methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation are unlikely to benefit from alkylating chemotherapy with temozolomide (TMZ). Trials aiming at replacing TMZ with targeted agents in unselected patient populations have failed to demonstrate any improvement of survival. Advances in molecular understanding and diagnostic precision enable identification of key genetic alterations in a timely manner and in principle allow treatments with targeted compounds based on molecular markers. Methods The NCT Neuro Master Match (N2M2) trial is an open-label, multicenter, phase I/IIa umbrella trial for patients with newly diagnosed isocitrate dehydrogenase (IDH) wildtype glioblastoma without MGMT promoter hypermethylation to show safety, feasibility, and preliminary efficacy of treatment with targeted compounds in addition to standard radiotherapy based on molecular characterization. N2M2 is formally divided into a Discovery and a Treatment part. Discovery includes broad molecular neuropathological diagnostics to detect predefined biomarkers for targeted treatments. Molecular diagnostics and bioinformatic evaluation are performed within 4 weeks, allowing a timely initiation of postoperative treatment. Stratification for Treatment takes place in 5 subtrials, including alectinib, idasanutlin, palbociclib, vismodegib, and temsirolimus as targeted therapies, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asinercept (APG101) and the standard of care, TMZ. For the phase I parts, a Bayesian criterion is used for continuous monitoring of toxicity. In the phase II trials, progression-free survival at 6 months is used as endpoint for efficacy. Results Molecular diagnostics and bioinformatic evaluation are performed within 4 weeks, allowing a timely initiation of postoperative treatment. Stratification for Treatment takes place in 5 subtrials, including alectinib, idasanutlin, palbociclib, vismodegib, and temsirolimus as targeted therapies, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asinercept (APG101) and the standard of care, TMZ. For the phase I parts, a Bayesian criterion is used for continuous monitoring of toxicity. In the phase II trials, progression-free survival at 6 months is used as endpoint for efficacy. Discussion Molecularly informed trials may provide the basis for the development of predictive biomarkers and help to understand and select patient subgroups who will benefit.

Background Wingless-activated medulloblastoma (WNT MB) represents a well-characterized molecular variant accounting for 10-15% of all MB and is associated with a favorable clinical outcome. Patients with localized WNT MBs could benefit from de-intensification of combined treatment, which would require an accurate diagnosis of these tumors. However, despite the presence of molecular features related with a WNT MB signature (nuclear s-catenin immunoexpression, CTNNB1 mutation, and monosomy 6), a prompt and reliable diagnostic verification of these tumors is not yet feasible. Methods In the current study, we analyzed 78 samples of WNT MB treated in a single institute through genome-wide DNA methylation and targeted next generation sequencing to elaborate an optimal method for WNT MB molecular verification. Results We found that DNA methylation profiling discloses significant advantages for molecular diagnostic of WNT MB. All other "routine" methods applied, such as s-catenin immunohistochemistry, CTNNB1 mutation analysis, and detection of monosomy 6, failed to identify all WNT MB cases. Survival analysis revealed that application of a reduced radiotherapy protocol for WNT MB treatment had no influence on patients' survival. Only one patient died due to local relapse but recurrent tumor was pathologically and molecularly diagnosed as a secondary glioblastoma. Conclusions DNA methylation analysis should be considered as a method of choice for further clinically relevant stratification of WNT MB and for correct diagnosis of the recurrent tumors. WNT MB patients with localized disease could benefit from treatment de-intensification.

Background This study aims to evaluate the impact of tumor location on key molecular alterations on a single voxel level in patients with newly diagnosed glioma. Methods A consecutive series of n = 237 patients with newly diagnosed glioblastoma and n = 131 patients with lower-grade glioma was analyzed. Volumetric tumor segmentation was performed on preoperative MRI with a semi-automated approach and images were registered to the standard Montreal Neurological Institute 152 space. Using a voxel-based lesion symptom mapping (VLSM) analysis, we identified specific brain regions that were associated with tumor-specific molecular alterations. We assessed a predefined set of n = 17 molecular characteristics in the glioblastoma cohort and n = 2 molecular characteristics in the lower-grade glioma cohort. Permutation adjustment (n = 1000 iterations) was used to correct for multiple testing, and voxel t-values that were greater than the t-value in >95% of the permutations were retained in the VLSM results (α = 0.05, power > 0.8). Results Tumor location predilection for isocitrate dehydrogenase (IDH) mutant tumors was found in both glioblastoma and lower-grade glioma cohorts, each showing a concordant predominance in the frontal lobe adjacent to the rostral extension of the lateral ventricles (permutation-adjusted P = 0.021 for the glioblastoma and 0.013 for the lower-grade glioma cohort). Apart from that, the VLSM analysis did not reveal a significant association of the tumor location with any other key molecular alteration in both cohorts (permutation-adjusted P > 0.05 each). Conclusion Our study highlights the unique properties of IDH mutations and underpins the hypothesis that the rostral extension of the lateral ventricles is a potential location for the cell of origin in IDH-mutant gliomas.

Introduction Precise diagnoses and robust detection of actionable alterations is required for individualized treatments. By using extended molecular diagnostics, the Pediatric Targeted Therapy (PTT) 2.0 program aims at the improvement of diagnostic accuracy and detection of actionable alterations for pediatric high-risk patients. The impact of these analyses on clinical management is reported. Methods Pediatric patients with relapsed or progressive tumors after standard of care treatment were included, independent of histological diagnosis. Formalin fixed paraffin embedded material and a blood sample for germline correction were requested. DNA methylation array, targeted gene panel sequencing (130 genes), RNA and Sanger sequencing in selected cases, and immunohistochemistry (IHC) of selected markers (pERK, pAKT, pS6, PD-L1) were performed. A questionnaire-based follow-up was used to determine the clinical impact of the analysis. Results We enrolled n=263 patients from February 2017 to February 2019. Complete molecular analysis was possible for n=260 cases (99%). The most common entities were brain tumors (n=172/260, 65%). In brain tumors, DNA methylation array alone allowed robust diagnostic classification (score of >=0.9) in n=104/172 cases (60%). Actionable targets as detected by copy number calculation, gene panel sequencing, RNA sequencing and IHC were found in n=94/172 (55%) brain tumor cases. The most common actionable targets in brain tumors were MAPK (pERK, BRAF fusions, BRAF V600E), mTOR (pS6), PI3K (pAKT), CDK4/6 (CDKN2A/B loss), and immune checkpoints (PD-L1). Pathogenic germline alterations with clinical relevance were identified in n=12/172 brain tumor cases (6.9%) and were confirmed by Sanger sequencing, 5/12 (41%) of which were previously unknown. Clinical follow-up of subsequent treatment and outcome are ongoing. Conclusion The combination of next-generation diagnostics such as methylation arrays and targeted sequencing in addition to selected IHC markers added robust information with regard to diagnosis and actionable alterations. The impact on clinical decision-making and on outcome is currently being evaluated.

Introduction Pilocytic astrocytoma (PA) is the most common brain tumor in children. Activation of the mitogen-activated protein kinase (MAPK) pathway is a hallmark of PA. Complete remission in non-resectable tumors is infrequently observed with current therapeutic approaches. Most PA tumors cells are in oncogene-induced senescence (OIS), which may explain the benign growth behavior of PAs but also account for resistance to therapy. Therefore, treatment of PA with senolytic agents such as BH3-mimetics is a promising new approach. Methods Three patient-derived PA cell lines, DKFZ-BT66, DKFZ-BT308 (both KIAA1549:BRAF-fusion positive) and DKFZ-BT314 (BRAF V600E-mutation positive) were used. Depending on inducible expression or repression of SV40 large T antigen all models can reflect both states of PA, proliferation and OIS. Cells in both states were treated with different BH3-mimetics. Inhibition of metabolic activity was measured after 72 hours. Target expression was assessed by RT-qPCR and Western blot. On-target activity of BH3-mimetics was determined by immunoprecipitation (IP) of Bcl-xL/BAK. Results BH3-mimetics with strong binding affinity for Bcl-xL (Navitoclax, A-1131852, A-1155463) showed selectivity for senescent cells in 2/3 models (DKFZ-BT66 and DKFZ-BT314) and acted in nanomolar ranges. IC50s for Navitoclax (Cmax 6600nM in patients) were 40nM (OIS) vs. 200nM (proliferation) and 170nM (OIS) vs. 3700nM (proliferation) in DKFZ-BT66 and DKFZ-BT314, respectively. Target engagement was evident in the Bcl-xL/BAK-IP, and target expression of Bcl-xL was similar in all models studied. The relative resistance of senescent DKFZ-BT308 despite on-target activity is currently being investigated. Conclusion Senolytic treatment of PA with BH3-mimetics targeting Bcl-xL is a promising new strategy directly targeting the major senescent part of the tumor in clinically archivable concentrations. However, our data suggests that not all PAs may respond to treatment. The analysis of comparative gene expression analysis and BH3-profiling is ongoing to define predictive biomarkers.

Introduction Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by MAPK pathway alterations, leading to its constitutive activation and modulating the balance between cell proliferation and the oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP) factors. This makes PA suitable for MAPK inhibitor (MAPKi) therapies, showing encouraging results in phase 1/2 clinical trials. Little is known about the molecular implications of MAPK downregulation in the proliferating and senescent compartments. Methods DKFZ-BT66 PA cells derived from a primary KIAA:BRAF-fusion positive PA cell line, were used as model system. Gene expression and phospho-proteomic datasets were generated from DKFZ-BT66 cells, in both the proliferative and senescent states, and treated with the MEKi trametinib for different time-spans. A time course analysis based on differentially expressed genes was performed, followed by a single-sample gene set enrichment analysis (ssGSEA). Analysis of the phospho-proteomic data is ongoing. Results Differential gene expression analysis revealed that MEK inhibition leads to the inhibition of the OIS-SASP gene program in senescent DKFZ-BT66. ssGSEA showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including FGFR, NTRK and TGFB pathways) were upregulated, in both proliferating and senescent DKFZ-BT66. Genes regulated by the MAPK pathway and involved in OIS-SASP were identified by analyzing genes differentially regulated between proliferating and senescent DKFZ-BT66, and modulated upon MEKi treatment. Conclusion This data suggests that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators in proliferating and senescent PA cells, identifying putative co-targets that could help prevent growth rebound upon MAPKi withdrawal. Furthermore, the identification of the MAPK-related OIS-SASP genes provide insight about the regulation of OIS-SASP by the MAPK pathway. Validation of this data with the ongoing phospho-proteomic analysis and in primary samples is needed.

The majority of supratentorial ependymomas (ST-EPN) are driven by fusion genes between RELA and zinc finger translocation associated, ZFTA, previously named C11orf95. Apart from fusions with a portion of the Hippo effector YAP1, which affects a small group of infant patients, the oncogenic mechanism of remaining ST-EPNs remains unclear. Aiming at refining the molecular classification of ST-EPNs, we have analyzed methylation profiles, RNA and DNA sequencing results as well as clinical data in a cohort of 613 ST-EPNs. An unbiased approach revealed distinct methylation clusters composed of tumors with ependymal but also various other histological features containing alternative translocations that shared ZFTA as a partner gene. Tumors within these additional clusters were characterized by fusions of ZFTA to numerous fusion partners different from RELA, e.g. MAML2, MAML3, NCOA2 and SS18, implying a general role of ZFTA in tumorigenesis of ST-EPN. Indeed, the transforming capacity of newly identified fusion genes was validated using an electroporation-based in vivo gene transfer technology in mice. All fusion genes themselves were sufficient to drive malignant transformation in the developing cerebral cortex and resulting tumors faithfully recapitulated molecular characteristics of their human counterparts. We found that both, the partner gene and the zinc finger DNA binding domain of ZFTA, were essential to exert tumorigenesis. Together with two additional studies, we performed a comprehensive analysis across datasets to derive a 93 gene signature of ZFTA-RELA-driven tumors, in which the Sonic Hedgehog effector gene GLI2 was identified as a promising downstream target. Subsequent co-expression of ZFTA:RELA and a dominant negative form of Gli2 indeed hampered tumorigenesis. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating GLI2 as a critical regulator of ZFTA fusion-positive tumorigenesis as well as a potential therapeutic vulnerability in these tumors.

Background The purpose of this study was to analyze the potential of radiomics for disease stratification beyond key molecular, clinical, and standard imaging features in patients with glioblastoma. Methods Quantitative imaging features (n = 1043) were extracted from the multiparametric MRI of 181 patients with newly diagnosed glioblastoma prior to standard-of-care treatment (allocated to a discovery and a validation set, 2:1 ratio). A subset of 386/1043 features were identified as reproducible (in an independent MRI test-retest cohort) and selected for analysis. A penalized Cox model with 10-fold cross-validation (Coxnet) was fitted on the discovery set to construct a radiomic signature for predicting progression-free and overall survival (PFS and OS). The incremental value of a radiomic signature beyond molecular (O6-methylguanine-DNA methyltransferase [MGMT] promoter methylation, DNA methylation subgroups), clinical (patient's age, KPS, extent of resection, adjuvant treatment), and standard imaging parameters (tumor volumes) for stratifying PFS and OS was assessed with multivariate Cox models (performance quantified with prediction error curves). Results The radiomic signature (constructed from 8/386 features identified through Coxnet) increased the prediction accuracy for PFS and OS (in both discovery and validation sets) beyond the assessed molecular, clinical, and standard imaging parameters (P ≤ 0.01). Prediction errors decreased by 36% for PFS and 37% for OS when adding the radiomic signature (compared with 29% and 27%, respectively, with molecular + clinical features alone). The radiomic signature was-along with MGMT status-the only parameter with independent significance on multivariate analysis (P ≤ 0.01). Conclusions Our study stresses the role of integrating radiomics into a multilayer decision framework with key molecular and clinical features to improve disease stratification and to potentially advance personalized treatment of patients with glioblastoma.

Pediatric brain tumor entities harbor a variety of gene fusions. Whilst other molecular parameters like somatic mutations and copy number alterations have become pivotal for brain tumor diagnostics, gene fusions are only less well covered by routinely applied methylation arrays or targeted next-generation sequencing of DNA. In a routine diagnostic setting we established and optimized a workflow for investigation of gene fusions in formalin-fixed paraffin-embedded (FFPE) tumor tissues by using RNA sequencing. Assessing different tools for calling fusions from raw data, we found relevant fusions in 66 out of 101 (65%) analyzed cases in a prospective cohort collected over 26 months. In 43 (43%) cases the fusions were of decisive diagnostic relevance and in 40 (40%) cases the fusion genes rendered a druggable target. Besides the relevance of pathognomonic fusions for diagnostics, especially the detection of druggable gene fusions yields direct benefit to the patients. This approach allows for an unbiased search for fusion events in the tested samples. Besides rare variants of established fusions which were not detected by prior targeted analyses, we identified previously unreported fusion events. Exemplified on KIAA1549:BRAF fusion, we in addition provide an overview of the detection accuracy of different methods, including breakpoint detection in DNA methylation array data and fusion gene detection in DNA panel sequencing data. Our data show that RNA sequencing has great diagnostic as well as therapeutic value by clinically detecting relevant alterations.

Isomorphic diffuse glioma (IDG) was first described in 2004 as an epilepsy-associated supratentorial diffuse glioma with low cellularity, low proliferation and very monomorphic tumour cells. Most patients had seizures since childhood but were operated on as adults. To study the position of these lesions among brain tumours we histologically, molecularly and clinically analysed 26 histologically typical IDGs. Tumour cells were GFAP-positive, MAP2-, OLIG2- and CD34-negative and the nuclear ATRX-expression was retained. Proliferation was very low. Sequencing of 24 cases revealed an IDH-wildtype status. Cluster analyses of DNA methylation data showed that IDG has a DNA methylation profile distinct from those of different glial/glio-neuronal brain tumours and normal hemispheric tissue. About half of IDGs had copy number alterations of MYBL1 or MYB (13/25) and half of the cases analysed by RNA-sequencing had gene fusions of MYBL1 or MYB with various gene partners (11/22), often associated with an increased RNA-expression of the respective MYB-family gene. Integrating all data available, 77% of IDGs had either MYBL1 (54%) or MYB (23%) alterations. All patients had a good outcome and most were seizure-free after surgery. In summary, we show that isomorphic diffuse glioma is a distinct benign tumour in the family of MYB/MYBL1-altered gliomas. DNA methylation analysis is very helpful for their identification. More recent analyses of a large cohort of MYB/MYBL1-altered brain tumours suggest the presence of a third methylation group that primarily contains paediatric cases and seems to be distinct from IDG and angiocentric gliomas. Further histological, molecular and clinical analyses are ongoing.

Pineoblastoma (PB) is an aggressive embryonal brain tumor comprising 1% of pediatric CNS tumors. The clinico-molecular heterogeneity and developmental origins underlying PB are poorly understood; therefore, we have assembled a molecular cohort of histologically defined PBs (n=43) with corresponding outcome data. Methylation profiling revealed four molecularly and clinically distinct PB subgroups, including two novel entities. Mutational and transcriptional analysis identified characteristic molecular features of each subgroup, such as mutations in the miRNA processing pathway or FOXR2 proto-oncogene overexpression. Furthermore, subgroups exhibited differences in propensity for metastasis, cytogenetics, and clinical outcomes. To dissect PB developmental origins and resolve PB subgroup biology, we have employed a combination of single-cell genomics and genetically engineered mouse modeling. We created a single-cell transcriptional atlas of the developing murine pineal gland across 11 timepoints and are currently integrating these data with single nuclei RNA-seq data of human PB (n=25). Single-cell analysis of the developing pineal gland revealed three distinct populations of pinealocytes, referred to as early, mid and late pinealocytes, which segregate by developmental stage yet lie along a single developmental trajectory. Preliminary results implicate significant associations between PBs and the early pinealocyte population as well as subgroup-specific differences in intratumoral heterogeneity. Furthermore, this knowledge has informed the downstream generation of biologically faithful disease models, including a transgenic mouse model of the PB-RB subgroup. Remarkably, this model shows up-regulation of key markers of PB such as Crx, Asmt and Otx2 and substantiates early pinealocytes as the probable cell-of-origin for this PB subgroup.

BACKGROUND Our previous analysis of established cancer predisposition genes in medulloblastoma (MB) identified pathogenic germline variants in ~5% of all patients. Here, we extended our analysis to include all protein-coding genes. METHODS Case-control analysis performed on 795 MB patients against >118,000 cancer-free children and adults was performed to identify an association between rare germline variants and MB. RESULTS Germline loss-of-function variants of Elongator Complex Protein 1 (ELP1; 9q31.3) were strongly associated with SHH subgroup (MBSHH). ELP1-associated-MBs accounted for ~15% (29/202) of pediatric MBSHH cases and were restricted to the SHHα subtype. ELP1-associated-MBs demonstrated biallelic inactivation of ELP1 due to somatic chromosome 9q loss and most tumors exhibited co-occurring somatic PTCH1 (9q22.32) alterations. Inheritance was verified by parent-offspring sequencing (n=3) and pedigree analysis identified two families with a history of pediatric MB. ELP1-associated-MBSHH were characterized by desmoplastic/nodular histology (76%; 13/17) and demonstrated a favorable clinical outcome when compared to TP53-associated-MBSHH (5-yr OS 92% vs 20%; p-value=1.3e-6) despite both belonging to the SHHα subtype. ELP1 is a subunit of the Elongator complex, that promotes efficient translational elongation through tRNA modifications at the wobble (U34) position. Biochemical, transcriptional, and proteomic analyses revealed ELP1-associated-MBs exhibit destabilization of the core Elongator complex, loss of tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response. CONCLUSIONS We identified ELP1 as the most common MB predisposition gene, increasing the total genetic predisposition for pediatric MBSHH to 40%. These results mark MBSHH as an overwhelmingly genetically-predisposed disease and implicate disruption of protein homeostasis in MBSHH development.

Background Medulloblastoma (MB) is one of the most common malignant pediatric CNS tumors. Patients with Group 3 MBs harboring MYC amplification exhibit low survival rates. Surviving patients suffer from therapy-induced sequelae, which calls for new targeted therapy strategies. We and others have previously shown the sensitivity of MYC-amplified MB to class I histone deacetylase (HDAC) inhibition. After demonstrating that the MYC target gene PLK1 is significantly downregulated upon class I HDACi treatment, we hypothesized that inhibition of both HDACs and PLK1 could have synergistic effects. Methods Cell metabolic activity changes upon HDAC and PLK1 inhibitor treatment were measured in MYC-amplified and non-amplified MB cell lines, as well as in an additional MYC-inducible cell line. The interaction effect of both inhibitors was determined by computation of the combination index (CI) using the Chou-Talalay method. Results were validated assessing cell viability, cell cycle, and apoptosis induction. Transcription profile changes after combination treatment were evaluated. Results MYC-amplified MB cell lines were more sensitive than non-amplified cell lines to PLK1i treatment, showing IC50 in clinically achievable concentration ranges. Inhibition of class I HDACs and PLK1 synergistically reduced cell metabolic activity in lower concentrations in MYC-amplified compared to non-amplified MB cell lines. We also observed a significant loss of viability and cells in G1 phase, as well as induction of apoptosis after combination treatment in MYC-amplified cells. MYC target gene sets were significantly downregulated in the MYC-amplified cell line HD-MB03 after treatment with combination. We demonstrated reduction of MYC protein levels upon PLK1i treatment. In vivo evaluation of combination treatment using orthotopic Group 3 MYC-amplified MB PDX models is ongoing. Conclusion Our data suggest that MYC-amplification is a predictive marker for PLK1i treatment in MB. The combination of HDACi and PLKi could be a candidate therapy for future clinical trials for MYC-amplified group 3 MB.

Introduction In the last decade, checkpoint inhibitor-based immunotherapy has been a groundbreaking development in the treatment of cancer. However, only a subset of patients treated with immune checkpoint inhibitors show long-lasting clinical benefit. Studies showed the tumor immune microenvironment (TME) as a particularly important factor influencing treatment response, critical for the design of other or combinatorial immunotherapy treatment strategies. Extensive research has been performed in the adult cancer field to unravel its immunogenomic aspects. However, in pediatric cancer this insight into tumor-infiltrating immune components is still lacking. This study aims to provide insight into the landscape of the immune microenvironment in pediatric primary nervous system tumors. Methods Bulk RNA-seq data of 936 pediatric primary solid tumors acquired from multiple international initiatives including Therapeutically Applicable Research To Generate Effective Treatments (TARGET), the International Cancer Genome Consortium (ICGC) and the Children’s Brain Tumor Tissue Consortium (CBTTC) were included in this study. We applied computational tumor immune microenvironment deconvolution, repurposed RNA-seq data to recover infiltrating T- and B-cell clonotypes and studied checkpoint gene expression across pediatric neural tumors. Results Among pediatric neural tumors, embryonal tumors with multilayered rosettes (ETMR) and medulloblastomas (MB) were least immune infiltrated. Neuroblastomas (NBL) had the highest T-cell infiltration among pediatric cancers, while atypical teratoid/rhabdoid tumors (ATRT) had the highest levels of CD8 T cell infiltration among pediatric CNS tumors. While tumor mutational burden (TMB) was associated with immune cell infiltration in adult lung cancers and melanomas, we found no significant associations in pediatric cancers. The majority of NBL samples expressed LAG3, but ~10% of samples had elevated levels of TIM3 gene expression, suggesting a distinct mode of immunosuppression in this subset.

Tumors of the CNS represent one of the most complex groups of human cancer, with a vast number of different entities occurring across a spectrum of ages and anatomic locations. This heterogeneity makes accurate diagnosis challenging, with the current gold standard relying on multiple subjective elements. We recently proposed a classification algorithm based on tumor DNA methylation profiling as an objective way to assign samples to over 80 distinct molecular classes. Here we present a substantial update to our machine learning-based algorithm, with more than 170 molecular classes now being represented amongst the 5,915 samples in our reference cohort. These new classes include further subclassification of known groups such as medulloblastoma and ependymoma, as well as multiple new molecular entities described here for the first time. A further improvement is the introduction of a more rationally layered output, making use of ‘families’ of closely-related molecular classes to improve the compatibility with the current WHO classification of CNS tumors. This approach is designed to increase the clinical relevance of the primary output, while also retaining the full information content from the random forest-driven classification. Benchmarking our new algorithm by cross-validation and on an independent validation cohort indicates a retention of the excellent accuracy of diagnosis (error-rate < 4%), with a significant improvement in the proportion of confidently classifiable tumors compared with our previous tool. We believe that this approach, freely accessible through an online web portal, has the potential to enhance diagnostic precision and thereby support clinical care for brain tumor patients.

AIM Recurrent structural variants involving MYB and MYBL1 transcription factors were recently identified in pediatric low-grade gliomas (pLGGs), such as the MYB-QKI rearrangement in Angiocentric Gliomas and truncations of MYBL1 (MYBL1tr) in Diffuse Astrocytomas. However, therapeutic dependencies induced by these alterations remain unexplored. METHODOLOGY We have generated in vitro pLGG mouse neural stem cell (NSCs) models engineered to harbor distinct MYB/MYBL1 genomic alterations. We used single cell RNA sequencing approaches to determine the transcriptional profile and dissect the central regulatory networks of our in vitro pLGG models over time. To identify specific genetic dependencies associated with MYB/MYBL1 mutations, we employed the Brie genome-wide mouse CRISPR lentiviral knockout pooled library, consisting of 78,637 single guide RNAs (sgRNAs) targeting 19,674 mouse genes. RESULTS MYB/MYBL1 expression in neural stem cells induced activation of cell-cycle related, glioma-related and senescence-related pathways that are involved in normal development, including activation of MAPK and mTOR signaling which are also activated in human pLGG samples. Genome-scale CRISPR-cas9 screens in isogenic NSCs expressing MYB-QKI or MYBL1tr identified differential genetic dependencies relative to GFP controls. These included regulators of cell-cycle progression and several modulators of the ubiquitin-proteasome degradation pathway. Analysis of RNA-sequencing data from human tumors revealed several of these dependencies identified in the cell line model to be differentially expressed in MYB-altered pLGG tumors relative to normal brain. CONCLUSION Expression of MYB family alterations induces expression of key developmental and oncogenic pathways and genetic dependencies that represent potential therapeutic targets for MYB or MYBL1 rearranged pLGGs.

INTRODUCTION The next generation of clinical trials for relapsed medulloblastoma demands a thorough understanding of the clinical behavior of relapsed tumors as well as the molecular relationship to their diagnostic counterparts. METHODS A multi-institutional molecular cohort of patient-matched (n=126 patients) diagnostic MBs and relapses/subsequent malignancies was profiled by DNA methylation array. Entity, subgroup classification, and genome-wide copy-number aberrations were assigned while parallel next-generation (whole-exome or targeted panel) sequencing on the majority of the cohort facilitated inference of somatic driver mutations. RESULTS Comprised of WNT (2%), SHH (41%), Group 3 (18%), Group 4 (39%), primary tumors retained subgroup affiliation at relapse with the notable exception of 10% of cases. The majority (8/13) of discrepant classifications were determined to be secondary glioblastomas. Additionally, rare (n=3) subgroup-switching events of Group 4 primary tumors to Group 3 relapses were identified coincident with MYC/MYCN pathway alterations. Amongst truly relapsing MBs, copy-number analyses suggest somatic clonal divergence between primary MBs and their respective relapses with Group 3 (55% of alterations shared) and Group 4 tumors (63% alterations shared) sharing a larger proportion of cytogenetic alterations compared to SHH tumors (42% alterations shared; Chi-square p-value < 0.001). Subgroup- and gene-specific patterns of conservation and divergence amongst putative driver genes were also observed. CONCLUSION Integrated molecular analysis of relapsed MB discloses potential mechanisms underlying treatment failure and disease recurrence while motivating rational implementation of relapse-specific therapies. The degree of genetic divergence between primary and relapsed MBs varied by subgroup but suggested considerably higher conservation than prior estimates.

Pilocytic astrocytomas (PA) are low-grade gliomas (pLGG) and are the most frequent childhood brain tumors. They are characterized by oncogene-induced senescence (OIS) initiated and sustained by senescence-associated secretory phenotype (SASP) factors. OIS and SASP in PA are thought to be driven by aberrations of the mitogen-activated protein kinase (MAPK) pathway (e.g. KIAA1549:BRAF fusion, BRAFV600E mutation, for the most common MAPK alterations occuring in PA), leading to its sustained activation. The MAPK pathway cascade is activated in a sequential manner: 1) ERK activation, which phosphorylates downstream partners in both cytoplasm and nucleus. 2) ERK-mediated induction of immediate early genes encoding transcription factors. 3) Induction of MAPK target genes expression. 4) Activation of downstream pathways. Our aim is to unravel the molecular partners involved at each level of the sustained MAPK pathway activation in pLGG with different genetic backgrounds (KIAA1549:BRAF fusion and BRAFV600E mutation), and leading to the induction of OIS and SASP factors expression. pLGG cell lines DKFZ-BT66 (KIAA1549:BRAF) and BT-40 (BRAFV600E) were treated with the MEK inhibitor trametinib at key time points, and gene expression profile analysis was performed, allowing transcriptome analysis at each step of the MAPK cascade. This will be combined with a whole proteomic and phospho-proteomic analysis. Combination of the transcriptome and proteome data layers will allow the identification of a) downstream targetable partners activated by the MAPK pathway involved in PA senescence, b) new putative targets that might bring benefit in combination with MAPK inhibitors.

BACKGROUND Impact of isolated spread into the cerebrospinal fluid (CSF) is still not investigated comprehensively for childhood medulloblastoma and the best therapeutic strategy is currently unclear. MATERIAL AND METHODS Sixty-six patients with isolated M1-MB registered to the HIT-MED-database from 2000–2018 were identified. CSF and MRI were centrally reviewed for all patients. Patients were stratified by age and either treated with upfront craniospinal irradiation (CSI) followed by maintenance chemotherapy (CT) or with postoperative CT and delayed CSI. RESULTS Forty-nine patients were non-infants ≥4 years and seventeen were infants CONCLUSION Isolated M1-MB is rare. Patients without contraindication for CSI appear to benefit from treatment by upfront CSI followed by maintenance CT, while cumulative CT-doses would be reduced compared to sandwich strategies.

Medulloblastoma (MB) is a highly malignant pediatric brain tumor originating from the cerebellum and brainstem. Identification of molecular subgroups forming this heterogeneous tumor entity was initially achieved from transcriptome characterization and further strengthened using DNA methylation profiling. While subgroup classification improved clinical diagnosis and treatment options, the lack of knowledge of the cell-of-origin for some of the subgroups hinders further treatment improvements. In addition identification of the precise cells of origin for each subgroup could help to understand tumor cell biology. Single cell sequencing is the optimal way to solve this task; recently, there were attempts to uncover putative MB cell-of-origin by using such information obtained from mouse embryonic cerebellum. However, such a comparative strategy can miss important results due to the differences between mouse and human. To solve this issue, we performed global single nucleus sequencing on human cerebellum pre- and postnatal materials across several developmental time points and generated transcriptome profiles from ~200k single cells. We identified known cell types forming the human cerebellum and performed detailed comparison of normal cells to RNA-seq bulk data from MB brain tumors across all subgroups. By selecting an optimal analysis strategy, we verified granule neuron precursors as cells of origin for the SHH MB subgroup. Additionally, we also found other cell types in conjunction with the remaining MB subgroups, suggesting new potential targets for investigation. Notably, this strategy can be further applied to the examination of other brain tumors and has perspectives in medical application.

AIM Advances in molecular classification of paediatric ependymoma have been pivotal in improving risk stratification and understanding of this disease. C11orf95-RELA fused supratentorial ependymoma (ST-EPN) have been reported to have a poor outcome, with 10-year overall survival (OS) of 49% and progression free survival (PFS) of 19%. A cohort of patients from multiple international institutions with molecularly confirmed C11orf95-RELA fused ST-EPN were reviewed to assess their disease behaviour. METHOD: We reviewed patients with molecularly determined C11orf95-RELA supratentorial ependymoma diagnosed between 1999 – 2019. Demographic information, extent of surgical resection, use of radiotherapy and/or chemotherapy, disease recurrence, treatment at recurrence and clinical outcome data was collected. PFS and OS of all patients were estimated using Kaplan-Meier method. RESULTS A total of 76 ST-EPN patients with C11orf95-RELA fusion were identified (median age: 7 years3 months, range: 5 months – 18 years7 months). 58 patients (76.3%) had complete surgical resection. 70 patients(92.1%) received radiotherapy. 55 patients(72.3%) received chemotherapy. The 10-year OS of C11orf95-RELA fused ST-EPN was 72.4% and PFS was 63.8%. In contrast, ST-EPN at a single institution with unconfirmed molecular status had an OS of 61.1% and PFS of 34.9%. CONCLUSION Detailed molecular analysis identified distinct subgroups of patients with ST-EPN. Patients from this cohort with C11orf95-RELA methylation profiles had a significantly higher OS compared to previous reports and those with unconfirmed fusion status, emphasising the critical importance of complete molecular profiling to assist in treatment decision making. Complete molecular analysis in future prospective cohorts is essential for accurate risk stratification and treatment selection.

Atypical teratoid/rhabdoid tumors (ATRT) represents a genomically homogeneous disease characterized by loss of SMARCB1 protein in the vast majority of cases. In recent years, it has become clear that these tumors display a high degree of intertumoral heterogeneity with three molecularly distinct subgroups. However, the degree of intratumoral heterogeneity and the information on cellular subpopulations currently remains largely an unchartered territory. To explore the transcriptomic composition of ATRTs, we performed single nuclei RNA sequencing for 16 ATRTs representing all three molecular subgroups (5 ATRT-TYR, 7 ATRT-SHH, 4 ATRT-MYC). By performing tSNE cluster analyses of all the single cell data (~50.000 cells have been sequenced), we were able to gain unprecedented insights into the phenotypic composition of ATRTs and unravelled substantial differences between the three subgroups. Integrating transcriptomic information from non-neoplastic brain cells and the data derived from single nuclei sequencing, we found an OPC like gene signature in ATRT-SHH. In contrast, ATRT-TYR subpopulations overexpressed more markers of neuronal stem cells suggesting a larger fraction of undifferentiated cells in this subgroup. We also identified a subpopulation of cells with a clear overexpression of cell cycle associated genes (CDK4, CDKN3), predominantly present in ATRT-MYC samples, a finding which may harbour important consequences for a targeted therapy with e.g. CDK inhibitors. In summary, our analyses reveal different cellular compartments in ATRT and provide important insights into the cellular differentiation of the three ATRT-subgroups. Further analyses to achieve a specific mapping of ATRT to its physiological cell of origin are currently being pursued.

OBJECTIVES Reduced neuropsychological outcomes are a major concern in pediatric patients with malignant brain tumors. We aimed to estimate decline in cognitive function according to treatment regimens. METHODS Cross-sectional analysis of cognitive functions tested with the Neuropsychological Basic Diagnostic tool (NBD) in 279 patients >4 years at diagnosis (median: 8.66; range: 4.01–18.98) with medulloblastoma (n=110, 23.7–25.0Gy CSI; n=131, >30Gy CSI) or posterior fossa ependymoma (n=38 local radiotherapy) who participated in the HIT-2000 trial. Multivariable regression analysis was conducted to adjust for postoperative cerebellar mutism syndrome, preoperative hydrocephalus, postoperative shunt placement, the interval between diagnosis and assessment, sex and age. RESULTS Mean time from diagnosis to assessment was 5.1 years. Increasing CSI-dose was significantly associated with a deterioration in performance of most subtests, particularly in areas of fluid intelligence (mean z-values per test for no CSI/23.4Gy/>30Gy respectively: matrix reasoning:-0.40/-0.52/-0.98, p30Gy CSI, selective attention, but no other function was reduced in patients treated with pre-radiotherapy chemotherapy including intraventricular MTX (selective attention (with chemotherapy/without chemotherapy mean z-values: -0.66/0.00, p=.006)). Patients with SHH-activated medulloblastoma did significantly better than WNT or Group3/Group4 medulloblastoma patients in fluid intelligence and fine motor skills. CONCLUSION CSI dose among other highly relevant factors had significant effects on neuropsychological outcome. Pre-radiotherapy intraventricular MTX had only minor effects. Patients with SHH-activated medulloblastomas showed a more favorable outcome when compared to patients in the other subgroups.

Medulloblastoma (MB) with extensive nodularity (MBEN) represent a rare subtype of cerebellar tumors of infancy which comprise two histologically distinct components, nodular reticulin-free zones and inter-nodular reticulin-rich regions. We applied single nucleus RNA-sequencing (snRNA-seq) using the 10X Genomics and the SMARTseq V2 protocols, bulk RNA-sequencing, DNA-methylation profiling and DNA-panel sequencing to ten histologically confirmed MBEN specimens. All tumors were classified as sonic hedgehog (SHH) MB based on DNA methylation. Somatic mutations within the SHH-pathway were detected in seven samples (3x SUFU, 2x PTCH1, 2x SMO) by DNA panel sequencing. The combined snRNAseq approach resulted in data on ~30.000 single cells. Several non-malignant cell types were identified, e.g. endothelial cells, astrocytes, and microglia. Amongst malignant cell populations SHH-pathway activation and mitotic activity differed revealing actively cycling embryonic stem (ES) cell-like and more differentiated neuronal-like cell types. In addition, distinct histological components of these tumours were subjected to bulk RNA sequencing following microdissection. This approach was repeated for DNA methylation profiling in an independent paraffin embedded MBEN cohort. However, these analyses did not reveal significant transcriptomic differences or differential methylation patterns between the two histological components. In summary, snRNA-seq identified a strongly proliferating, ES-like subset of cells in MBEN, which might represent the driving cell population in these malignancies, while direct analyses of nodular and inter-nodular regions did not reveal any significant differences. These findings suggest that both components originate from the same cell of origin but represent different cellular developmental stages.

The majority of supratentorial ependymomas (ST-EPN) are driven by fusions between RELA and a zinc finger containing gene, C11orf95. Apart from fusions to the Hippo effector YAP1, which affects a small group of infant patients, the oncogenic mechanism of remaining ST-EPNs is unclear. Aiming at refining the molecular classification of ST-EPNs, we analyzed methylation profiles, RNA and DNA sequencing results as well as clinical data in a cohort of 617 ST-EPNs. Unsupervised clustering analysis of DNA methylation data revealed four distinct clusters that formed in addition to the known molecular groups ST-EPN-RELA and –YAP1. Tumors within these additional clusters were characterized by fusions of C11orf95 to numerous fusion partners different from RELA, e.g. MAML2, MAML3, NCOA2 and SS18, suggesting a general role of C11orf95 in tumorigenesis of ST-EPN. Transforming capacity of newly identified fusion genes was validated using an electroporation-based in vivo gene transfer technology. All fusion genes were sufficient to drive malignant transformation in the cerebral cortex of mice and resulting tumors faithfully recapitulated molecular characteristics of their human counterparts. We found that both, the partner gene and the zinc finger DNA binding domain of C11orf95, were essential to exert tumorigenesis. When exploring genes commonly upregulated in C11orf95 fusion-expressing tumors of human and murine origin, the Sonic Hedgehog effector gene Gli2 was identified as a promising downstream target. Subsequent co-expression of C11orf95:RELA and a dominant negative form of Gli2 indeed hampered tumorigenesis. We thus propose GLI2 as a potential therapeutic downstream target of C11orf95 fusion-dependent oncogenic signaling in ST-EPN.

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.

Pineoblastomas (PB) are rare, aggressive pineal gland tumours with poor global OS of 50–70% and only 15–49% OS for patients

The large variety of CNS tumor entities affecting children and adolescents, some of which are exceedingly rare, results in very diverging patient outcomes and renders accurate diagnosis challenging. To assess the diagnostic utility of routine DNA methylation-based CNS tumor classification and gene panel sequencing, the Molecular Neuropathology 2.0 study prospectively integrated these (epi-)genetic analyses with reference neuropathological diagnostics as an international trial for newly-diagnosed pediatric patients. In a four-year period, 1,215 patients with sufficient tissue were enrolled from 65 centers, receiving a reference neuropathological diagnosis according to the WHO classification in >97%. Using 10 FFPE sections as input, DNA methylation analysis was successfully performed in 95% of cases, of which 78% with sufficient tumor cell content were assigned to a distinct epigenetic tumor class. The remaining 22% did not match any of 82 represented classes, indicating novel rare tumor entities. Targeted gene panel sequencing of >130 genes performed for 96% of patients with matched blood samples detected diagnostically, prognostically, or therapeutically relevant somatic alterations in 48%. Germline DNA sequencing data indicated potential predisposition syndromes in ~10% of patients. Discrepant results by neuropathological and epigenetic classification (29%) were enriched in histological high-grade gliomas and implicated clinical relevance in 5% of all cases. Clinical follow-up suggests improved survival for some patients with high-grade glioma histology and lower-grade molecular profiles. Routine (epi-)genetic profiling at the time of primary diagnosis adds a valuable layer of information to neuropathological diagnostics and will improve clinical management of CNS tumors.

About half of all pediatric high-grade gliomas (HGG) harbor mutations in histone 3 or IDH genes. The remaining HGG are currently broadly classified as H3-/IDH-wild-type. Since the introduction of a uniform approach to DNA methylation-based classification of CNS tumors in 2018, DNA methylation data from over 45,000 CNS tumor samples have been generated. From this large cohort, a number of smaller yet distinct subgroups start to emerge within H3-/IDH-wild-type HGG. Three such subgroups are enriched for focal gene amplifications and have been provisionally termed pedGBM_MYCN, pedGBM_RTK1 and pedGBM_RTK2. Since a significant subset of samples in each subgroup is lacking characteristic alterations, we further investigated the molecular and transcriptional composition of H3-/IDH-wild-type HGG. We evaluated DNA methylation and copy-number profiles in >1000 tumors classified as H3-/IDH-wild-type HGG. Tumors classified pedGBM_MYCN showed a focal MYCN amplification in 25%, with a similar fraction showing amplification of EGFR (8% of samples harbored both alterations) compared to 4% and 4% in pedGBM_RTK1 and 14% and 22% in pedGBM_RTK2. Deletion of CDKN2A/B was much more prevalent in the pedGBM_RTK2 subgroup (~50% compared to 27% in pedGBM_RTK1 and

OBJECTIVE Molecular subtypes of Group 3/4 medulloblastoma have been identified by unsupervised clustering methods in different studies. We hypothesized that risk stratification using these subtypes I-VIII improves outcome prediction. PATIENTS AND METHODS n=340 patients with Group 3 or Group 4 medulloblastoma defined by DNA methylation array profiling enrolled into the HIT2000 study and HIT-MED registries were subtyped by the Heidelberg Medulloblastoma Classifier. The discovery cohort consisted of n=162 previously published samples, the validation cohort of n=178 newly analyzed samples. RESULTS AND DISCUSSION: n=300/340 (88%) MBs could be assigned to one of the subtypes with confidence (score >0.8; Heidelberg Medulloblastoma classifier). Subtype II,III and V showed a poor PFS and OS and were classified as HR (discovery:5y-PFS 45%[95%-CI:33–62], 5y-OS 50%[37–67]; validation:5y-PFS 32%[20–50], 5y-OS 40%[27–61]). Subtypes I, IV, VI-VIII fared better (discovery:5y-PFS 67%[58–77], 5y_OS 84%[77–91]; Validation:5y-PFS 70%[58–83], 5y-OS 89%[81–99]). Survival prediction by subtype-based risk assessment was improved compared to Group 3 versus 4 differentiation in both cohorts in univariate and multivariable Cox regression models (PFS:Hazard ratio HR versus LR 2.474, p CONCLUSION We showed that molecular subtypes I-VIII improved risk stratification of Group 3/4 medulloblastomas. Group 3 subtype IV MB treated with RT had very high cure rates.

Pediatric low-grade gliomas (pLGGs) are the most common brain tumors in children. Despite recent advances in the molecular characterization of this heterogeneous set of tumors, the separation of specific tumor types is still not fully established. Pilocytic astrocytoma (PA; WHO grade I) and pleomorphic xanthoastrocytoma (PXA; WHO grade II) are two pLGG types that can be difficult to distinguish based on histology alone. Even though their clinical course is different, they are often grouped as ‘pLGG’ in clinical trials (and therefore treated similarly). Based on a cohort of 89 human pediatric tumor samples, we show that PAs and PXAs have clearly distinct methylation and transcriptome profiles. The difference in gene expression is mainly caused by cell cycle- and development-associated genes, suggesting a key difference in the regulatory circuits involved in tumor growth. In addition to BRAF V600E, we found NTRK fusions and a previously unknown EGFR:BRAF fusion as mutually exclusive driving events in PXAs. Both tumor types show marked signs of immune cell infiltration, but with significant qualitative differences, which might represent therapeutic vulnerabilities. To pave the way for further research on PA and PXA, we developed corresponding mouse models using the virus-based RCAS system, which allows introduction of an oncogenic driver into immunocompetent mice for molecular and preclinical research. The murine tumors do not only histologically resemble their human counterparts but also show a similar growth behavior. Expression analysis revealed that the murine PXAs have a stronger gene signature of proliferation and immune cell infiltration compared to PAs.

Pilocytic astrocytomas (PAs) and other pediatric low-grade gliomas (pLGGs) exhibit aberrant activation of the MAPK signaling pathway caused by genetic alterations, most commonly KIAA1549:BRAF fusions, BRAF V600E and NF1 mutations. In such a single-pathway disease, novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for treatment. We developed an assay suitable for pre-clinical testing of MAPKi in pLGGs, aiming at the identification of novel MAPK pathway suppressing synergistic drug combinations. We generated a reporter plasmid (pDIPZ) expressing destabilized firefly luciferase driven by a MAPK-responsive ELK-1-binding element, packaged in a lentiviral vector system. We stably transfected pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively. Measurement of MAPK pathway activity was performed using the luciferase reporter. pERK protein levels were detected for validation. We performed a screen of a MAPKi library and calculated Combination Indices of selected combinations. The MAPKi library screen revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and second generation RAF inhibitors. Synergistic effects in both BRAF-fusion and BRAFV600E mutation backgrounds were observed following combination treatments with different MAPKi classes (RAFi/MEKi, > RAFi/ERKi > MEKi/ERKi). We have generated a novel reporter assay for medium- to high-throughput pre-clinical drug testing of MAPKi in pLGG cell lines. MEK, ERK and next-generation RAF inhibitors were confirmed as potential treatment approaches for KIAA1549:BRAF and BRAFV600E mutated pLGGs. Synergistic suppression of MAPK pathway activity upon combination treatments was revealed using our assay in addition.

In the last few years, the extent of heterogeneity between tumors of a given class has become increasingly evident. Thus, tumors should optimally be treated differently according to their individual genetic alterations. Within the ICGC PedBrain Tumor Project, we have analyzed >150 low-grade and high-grade pediatric gliomas with whole-genome DNA and RNA sequencing, and have identified a wide variety of genetic alterations responsible for tumor growth. This includes novel point mutations, fusions and duplications in oncogenes such as NTRK2, FGFR1 and ALK. In some cases these alterations also define distinct molecular subgroups of glioma as judged by DNA methylation profiling. In order to identify new targeted therapy options for pediatric gliomas, mouse models recapitulating the human disease are required for testing of more specific inhibitors. We have therefore generated a series of novel models which are now being characterized and pre-clinically treated. Initial results with ALK or NTRK fusions show that our somatic gene transfer methods (RCAS- or in utero electroporation-based) can be used to rapidly generate new orthotopic brain tumor models, and that specific inhibitors against chosen oncogenes have a greater effect on tumor growth compared to standard therapy. The murine tumor cells can also be cultivated as neurospheres for functional testing and e.g. high-throughput screens. In addition to the basic biological understanding of tumorigenic mechanisms that can now be elucidated in more detail, we aim to rapidly translate our pre-clinical results in a manner that we hope will directly benefit patients in the clinic.

Ependymoma (EPN) is one of the most aggressive pediatric brain tumors, often arising in the supratentorial (ST) region. Our previous DNA methylation profiling study has identified ST-EPN subgroups that are characterized by YAP1-related fusions through genomic structural rearrangements (hereafter called as ST-EPN-YAP1). No reports of amplification and hyper-activation of the YAP1 gene in ST-EPN-YAP1s postulate that YAP1-related fusions could have unique oncogenic function(s) in this type of cancer. Nevertheless, the lack of adequate models for ST-EPN-YAP1 had hindered the discovery of YAP1-fusion-specific molecular mechanisms in EPN tumorigenesis and the development of effective targeted therapies for these tumors. Most recently, we developed a murine ST-EPN-YAP1 model by forced expression of YAP1-MAMLD1, the most dominant type of the fusion proteins in ST-EPN-YAP1 into cortical progenitors using in utero electroporation. Detailed histological analysis suggest that tumors arose from Pax6-positive neural stem cells, which is highly consistent with the highest expression of PAX6 in human ST-EPN-YAP1s across all EPN subgroups. MAMLD1-driven nuclear localization of the fusion protein is strongly associated with tumorigenesis. To investigate oncogenic functions of YAP1-MAMLD1, we performed a ChIP-seq analysis on human ST-EPN-YAP1 primary tumors with an anti-YAP1 antibody. Intriguingly, ST-EPN-YAP1-specific enhancers enriched for YAP1 peaks exhibited abundant transcriptional factor binding motifs of nuclear factor I and TEADs. Co-immunoprecipitation revealed that both TEAD and NFIA/B interact with YAP1-MAMLD1. Both the mutation of the TEAD binding site in the YAP1 fusion and dominant-negative inhibition of NFI proteins prevented tumor induction in mice. Collectively, we demonstrated that YAP1-MAMLD1 function as an oncogenic driver in ST-EPN-YAP1s through recruitment of TEAD and NFI transcriptional factors.

There is a broad variety of CNS tumor entities that can affect children and adolescents, associated with divergent clinical outcomes. We have recently proposed a DNA methylation-based classification to distinguish biologically distinct CNS tumor classes within and across neuropathological entities. The Molecular Neuropathology 2.0 study integrates genome-wide (epi-)genetic diagnostics with reference neuropathological assessment for newly-diagnosed pediatric CNS tumors in Germany. From 04/2015 to 09/2017, 607 patients with sufficient tissue were enrolled from 51 German centers. A reference neuropathological diagnosis according to the WHO classification was established for 95% of tumors and reflected the distribution of known histological CNS tumor entities in a pediatric population. Using 10 FFPE sections for DNA extraction, a DNA methylation-based molecular diagnosis was established in 95% of cases, of which 83% were assigned to a distinct CNS tumor methylation class. The remaining 17% did not match any of 82 currently established classes, but revealed evidence for novel rare molecular entities. Gene panel sequencing of >130 genes performed for 88% of patients with matched blood samples indicated diagnostically, prognostically, or therapeutically relevant somatic alterations in 47%. Germline DNA sequencing indicated potential predisposition syndromes in ~10% of patients, leading to human genetic counselling of affected families. Discrepant results by neuropathological and (epi-)genetic classification (~20%) were discussed in a weekly multi-disciplinary tumor board including reference neuroradiological evaluation. The majority of clinically-relevant discrepancies (67%) involved tumors neuropathologically diagnosed as high-grade gliomas, of which 25% were showed discrepant results. Clinical follow-up of enrolled patients through the German HIT study centers is ongoing. The MNP 2.0 study adds a valuable layer of information to clinical neuropathological diagnostics and has expanded to an international registry for molecular data analysis for pediatric CNS tumors. The results provide insight into tumors with divergent neuropathological and molecular classification with potential implications for patient management.

A greater understanding of the Ras-MAP kinase-signaling pathway in pediatric low-grade glioma (LGG) has led to the ability to target this pathway with therapeutic intent. The PBTC conducted a multi-institutional phase II study (NCT01089101) evaluating selumetinib (AZD6244, ARRY-142886), a MEK I/II inhibitor, in children with recurrent/refractory LGG assigned to 6 strata and treated at 25 mg/m2/dose PO BID for up to two years. Here we present the data from three of these strata. Stratum I included children with non-NF-1 and non-optic pathway recurrent/refractory pilocytic astrocytoma (PA) harboring BRAF aberrations (BRAF V600e mutation or the BRAF-KIAA 1549 fusion). Eight of 25 (32%) patients achieved a partial response (PR) with 2-year PFS of 66+11%. Two of 7 (29%) patient tumors with a BRAF V600e mutation and 6/18 (33%) with a BRAF KIAA-1549 fusion had a PR. Stratum 3 enrolled NF-1-associated LGG. Tissue for tumor BRAF evaluation was not required for eligibility. Ten of 25 (40%) achieved PR with a 2-year PFS of 96+4%. Only one patient progressed while on treatment. Stratum 4 included children with non-NF-1 optic pathway/hypothalamic LGG. Tissue for tumor BRAF evaluation was not required for eligibility. Two of 16 (12.5%) had a PR with a 2-year PFS of 65+13%. The BRAF aberration status of the responders in strata 3 and 4 is mostly unknown. All responses were confirmed centrally and seven patients remain on treatment. The most common toxicities were grade 1/2 CPK elevation, diarrhea, hypoalbuminemia, elevated AST and rash. Rare grade 3/4 toxicities included elevated CPK, rash, neutropenia, emesis and paronychia. Selumetinib was effective in treating children with recurrent/refractory LGG, including those with NF-1 associated LGG and PA harboring BRAF V600e mutation or BRAF-KIAA 1549 fusion. Larger prospective studies are necessary to determine the future, specific role of this agent in treating children with various LGG molecular subtypes.

In a previous study we identified two molecular groups of supratentorial (ST) ependymoma in pediatric patients driven by distinct gene fusions involving either the NF-κB effector RELA or the HIPPO signaling regulator YAP1, designated ST-EPN-RELA and ST-EPN-YAP1, respectively. The lack of adequate models for ST-EPN-YAP1, which predominantly occurs in very young children, has so far hindered the development of effective targeted therapies for these tumors. In an attempt to model this group, the most frequent fusion type, YAP1-MAMLD1, was cloned into a Luciferase-carrying transposable vector. Oncogenicity was subsequently tested using an electroporation-based in vivo gene transfer approach following injection of the vector into the lateral ventricle of E13.5 wildtype mouse embryos. After birth, YAP1-MAMLD1-expressing tumors, monitored using luciferase-based in vivo bioluminescence imaging, developed rapidly with 100% penetrance, indicating that the fusion alone is sufficient to initiate tumors. To further investigate the role of this fusion in human EPNs, we performed YAP1 ChIP-seq analyses on human ST-EPN-YAP1 and ST-EPN-RELA primary tumors. Despite similar gene expression levels of YAP1 in both molecular groups, our comparative analyses found that putative binding sites of TEADs, transcriptional factors interacting with YAP1, were significantly enriched in ST-EPN-YAP1-specific YAP1 target sites. In vivo validation further confirmed that interaction between YAP1-MAMLD1 and TEADs is crucial for oncogenicity of the fusion, since prevention of YAP1-TEAD binding did not result in tumor formation. Thus, targeting the YAP1-TEAD interaction might represent a promising therapeutic approach for this devastating infant disease.

Children can be affected by a large variety of CNS tumor entities with very divergent outcomes, some of which are exceedingly rare. DNA methylation analysis is a powerful tool to distinguish biologically distinct CNS tumor classes within and across histological entities. The Molecular Neuropathology 2.0 study aims to integrate genome wide (epi-)genetic diagnostics with reference neuropathological assessment for all newly-diagnosed pediatric CNS tumors in Germany. In the first 2 ½ years, 675 patients with sufficient tissue were enrolled from 55 centers. For >95% of patients, a diagnosis according to the WHO classification was assigned by reference neuropathology. Using 10 FFPE sections as input, a DNA methylation-based molecular diagnosis was established in 95% of cases, of which 84% were assigned to a distinct CNS tumor methylation class. The remaining 16% did not match any of 82 currently established classes, with evidence for novel rare entities. Targeted gene panel sequencing of >130 genes performed for 88% of patients with matched blood samples indicated diagnostically, prognostically, or therapeutically relevant somatic alterations in 47%. Germline DNA sequencing data indicated potential predisposition syndromes in ~10% of patients. Discrepant results by neuropathological and molecular classification (~20%) were discussed in a weekly multi-disciplinary tumor board including reference neuroradiological evaluation. Clinical follow-up data for all patients is being collected by the HIT study centers. This ongoing study adds a valuable layer of information to clinical neuropathological diagnostics in Germany and will provide insight into CNS tumors with divergent neuropathological and molecular classification with potential implications for future patient management.

We screened exon 4 of the gene isocitrate dehydrogenase 1 (NADP+), soluble (IDH1) for mutations in 596 primary intracranial tumors of all major types. Codon 132 mutation was seen in 54% of astrocytomas and 65% of oligodendroglial tumors but in only 6% of glioblastomas (3% of primary and 50% of secondary glioblastomas). There were no mutations in any other type of tumor studied. While mutations in the tumor protein p53 gene (TP53) and total 1p/19q deletions were mutually exclusive, IDH1 mutations were strongly correlated with these genetic abnormalities. All four types of mutant IDH1 proteins showed decreased enzymatic activity. The data indicate that IDH1 mutation combined with either TP53 mutation or total 1p/19q loss is a frequent and early change in the majority of oligodendroglial tumors, diffuse astrocytomas, anaplastic astrocytomas, and secondary glioblastomas but not in primary glioblastomas.

Previously, we have identified nine distinct molecular groups of ependymoma across all age groups, three in each major anatomical compartment of the CNS: spinal, posterior fossa, and supratentorial. These nine molecular groups are genetically, epigenetically, transcriptionally, demographically, and clinically distinct. Pediatric intracranial ependymomas are either affiliated with the RELA and YAP1 supratentorial groups or with the posterior fossa PFA group. RELA and YAP1 ependymomas harbor recurrent and distinct fusion genes that drive the disease, but PFA ependymomas lack significant recurrent mutations, and the specific oncogenic events underlying these tumors remain undefined. Observing distinct outcomes among children with PFA ependymomas, we tested the hypothesis that further molecular diversity with clinical utility, including possible novel genetic alterations, may exist among these tumors. Genome-wide DNA methylation profiles of 681 pediatric PFA ependymomas were analyzed by unsupervised consensus hierarchical clustering and t-distributed stochastic neighbor embedding (tsne), which identified not only three major distinct molecular subgroups of PFA ependymoma: PFA-1, PFA-2, and PFA-3, but additional small subgroups that segregate within PFA-1 and PFA-2. Subgroups were characterized by distinct clinical and genetic characteristics; most PFA-3 tumors harbored 1q gain and occurred in children significantly older than those with PFA-1 and PFA-2 tumors. Some of the small subgroups demonstrated distinctive copy number alterations or gene mutations, e.g. H3 K27M. Outcome data revealed considerable heterogeneity among the different subgroups, overall survival at 5 years was >90% for one subgroup, but

The benign but unpredictable growth behaviour of pilocytic astrocytoma (PA) is thought to originate from variations in proliferation caused by oncogene-induced senescence (OIS). An increase in MAPK pathway activation, present in virtually all PAs, serves a stimulus driving the tumor into OIS. Upregulation of p16, p21 and several other OIS-related genes has been observed in primary PAs. The secretion of a set of cytokines, growth factors and proteases, referred to as the senescence-associated secretory phenotype (SASP), is a characteristic feature of senescent cells. The SASP is thought to be involved in the induction and maintenance of the OIS state. The key SASP factors in PA and their functional significance are unknown. To study OIS in PA, the genetically engineered primary PA cell culture model DKFZ-BT66 was used. The model allows switching between senescence and proliferation via inducible expression of the SV40 Large T Antigen suppressing p53/RB signaling. Readouts used were gene-expression profiling (GEP), Western Blot, real-time qPCR, ELISA, cell counts and viability by automated trypan blue exclusion staining. Comparison of the GEP of senescent versus proliferating cells showed a significant increase in the SASP during OIS. The upregulation of the representative SASP factors IL-6 and IL-1B was confirmed on mRNA and protein level. The respective receptor proteins were both expressed, and activation of both pathways was detected in the OIS state. Stimulation of selected SASP pathways led to reduced cell growth of proliferating cells. In summary, the first representative patient-derived PA tumor model DKFZ-BT66 confirms the presence of OIS in PA, and additionally reveals activity of the OIS-characteristic SASP. Ongoing studies will show whether the OIS-induced growth arrest is reversible and if it may explain the unpredictable regrowth of PAs observed in patients. These findings will be relevant for treatment decisions concerning anti-inflammatory or immunosuppressive drugs in PA patients.