Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.

As the progression of low-grade diffuse astrocytomas into grade 4 tumors significantly impacts patient prognosis, a better understanding of this process is of paramount importance for improved patient care. In this project, we analyzed matched IDH-mutant astrocytomas before and after progression to grade 4 from six patients (discovery cohort) with genome-wide sequencing, 21 additional patients with targeted sequencing, and 33 patients from Glioma Longitudinal AnalySiS cohort for validation. The Cancer Genome Atlas data from 595 diffuse gliomas provided supportive information. All patients in our discovery cohort received radiation, all but one underwent chemotherapy, and no patient received temozolomide (TMZ) before progression to grade 4 disease. One case in the discovery cohort exhibited a hypermutation signature associated with the inactivation of the MSH2 and DNMT3A genes. In other patients, the number of chromosomal rearrangements and deletions increased in grade 4 tumors. The cell cycle checkpoint gene CDKN2A, or less frequently RB1, was most commonly inactivated after receiving both chemo- and radiotherapy when compared to other treatment groups. Concomitant activating PDGFRA/MET alterations were detected in tumors that acquired a homozygous CDKN2A deletion. NRG3 gene was significantly downregulated and recurrently altered in progressed tumors. Its decreased expression was associated with poorer overall survival in both univariate and multivariate analysis. We also detected progression-related alterations in RAD51B and other DNA repair pathway genes associated with the promotion of error-prone DNA repair, potentially facilitating tumor progression. In our retrospective analysis of patient treatment and survival timelines (n = 75), the combination of postoperative radiation and chemotherapy (mainly TMZ) outperformed radiation, especially in the grade 3 tumor cohort, in which it was typically given after primary surgery. Our results provide further insight into the contribution of treatment and genetic alterations in cell cycle, growth factor signaling, and DNA repair-related genes to tumor evolution and progression.

The large diversity of central nervous system (CNS) tumor types in children and adolescents results in disparate patient outcomes and renders accurate diagnosis challenging. In this study, we prospectively integrated DNA methylation profiling and targeted gene panel sequencing with blinded neuropathological reference diagnostics for a population-based cohort of more than 1,200 newly diagnosed pediatric patients with CNS tumors, to assess their utility in routine neuropathology. We show that the multi-omic integration increased diagnostic accuracy in a substantial proportion of patients through annotation to a refining DNA methylation class (50%), detection of diagnostic or therapeutically relevant genetic alterations (47%) or identification of cancer predisposition syndromes (10%). Discrepant results by neuropathological WHO-based and DNA methylation-based classification (30%) were enriched in histological high-grade gliomas, implicating relevance for current clinical patient management in 5% of all patients. Follow-up (median 2.5 years) suggests improved survival for patients with histological high-grade gliomas displaying lower-grade molecular profiles. These results provide preliminary evidence of the utility of integrating multi-omics in neuropathology for pediatric neuro-oncology.

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors manifesting in infancy. They split into four molecular types. The major three (AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC) all carry mutations in SMARCB1, the fourth quantitatively smaller type is characterized by SMARCA4 mutations (AT/RT-SMARCA4). Molecular characteristics of disease recurrence or metastatic spread, which go along with a particularly dismal outcome, are currently unclear. Here, we investigated tumor tissue from 26 patients affected by AT/RT to identify signatures of recurrences in comparison with matched primary tumor samples. Microscopically, AT/RT recurrences demonstrated a loss of architecture and significantly enhanced mitotic activity as compared to their related primary tumors. Based on DNA methylation profiling, primary tumor and related recurrence were grossly similar, but three out of 26 tumors belonged to a different molecular type or subtype after second surgery compared to related primary lesions. Copy number variations (CNVs) differed in six cases, showing novel gains on chromosome 1q or losses of chromosome 10 in recurrences as the most frequent alterations. To consolidate these observations, our cohort was combined with a data set of unmatched primary and recurrent AT/RT, which demonstrated chromosome 1q gain and 10 loss in 18% (n = 7) and 11% (n = 4) of the recurrences (n = 38) as compared to 7% (n = 3) and 0% (n = 0) in the primary tumors (n = 44), respectively. Similar to the observations made by DNA methylation profiling, RNA sequencing of our cohort revealed AT/RT primary tumors and matched recurrences clustering closely together. However, a number of genes showed significantly altered expression in AT/RT-SHH recurrences. Many of them are known tumor driving growth factors, involved in embryonal development and tumorigenesis, or are cell-cycle-associated. Overall, our work identifies subtle molecular changes that occur in the course of the disease and that may help define novel therapeutic targets for AT/RT recurrences.

Posterior fossa type A (PF-EPN-A, PFA) ependymoma are aggressive tumors that mainly affect children and have a poor prognosis. Histopathology shows significant intratumoral heterogeneity, ranging from loose tissue to often sharply demarcated, extremely cell-dense tumor areas. To determine molecular differences in morphologically different areas and to understand their clinical significance, we analyzed 113 PF-EPN-A samples, including 40 corresponding relapse samples. Cell-dense areas ranged from 0 to 100% of the tumor area and displayed a higher proportion of proliferating tumor cells (p < 0.01). Clinically, cell density was associated with poor progression-free and overall survival (pPFS = 0.0026, pOS < 0.01). Molecularly, tumor areas with low and high cell density showed diverging DNA methylation profiles regarding their similarity to distinct previously discovered PF-EPN-A subtypes in 9/21 cases. Prognostically relevant chromosomal changes at 1q and 6q showed spatial heterogeneity within single tumors and were significantly enriched in cell-dense tumor areas as shown by single-cell RNA (scRNA)-sequencing as well as copy number profiling and fluorescence in situ hybridization (FISH) analyses of different tumor areas. Finally, spatial transcriptomics revealed cell-dense areas of different tumors to be more similar than various different areas of the same tumor. High-density areas distinctly overexpressed genes encoding histone proteins, WNT5A, TGFB1, or IGF2. Relapsing tumors displayed a higher proportion of cell-dense areas (p = 0.036), a change in PF-EPN-A methylation subtypes (13/32 patients), and novel chromosome 1q gains and 6q losses (12/32 cases) compared to corresponding primary tumors. Our data suggest that PF-EPN-A ependymomas habor a previously unrecognized intratumoral heterogeneity with clinical implications, which has to be accounted for when selecting diagnostic material, inter alia, by histological evaluation of the proportion of cell-dense areas.

Copy-number variations (CNVs) are common genetic alterations in cancer and their detection may impact tumor classification and therapeutic decisions. However, detection of clinically relevant large and focal CNVs remains challenging when sample material or resources are limited. This has motivated us to create a software tool to infer CNVs from DNA methylation arrays which are often generated as part of clinical routines and in research settings.

Medulloblastoma, an embryonal tumor of the cerebellum/fourth ventricle, is one of the most frequent malignant brain tumors in children. Although genetic variants are increasingly used in treatment stratification, survival of high-risk patients, characterized by leptomeningeal dissemination, TP53 mutation or MYC amplification, is still poor. FOXM1, a proliferation-specific oncogenic transcription factor, is deregulated in various solid tumors, including medulloblastoma, and triggers cellular proliferation, migration and genomic instability. In tissue samples obtained from medulloblastoma patients, the significant upregulation of FOXM1 was associated with a loss of its putative regulating microRNA, miR-4521. To understand the underlying mechanism, we investigated the effect of miR-4521 on the expression of the transcription factor FOXM1 in medulloblastoma cell lines. Transfection of this microRNA reduced proliferation and invasion of several medulloblastoma cell lines and induced programmed cell death through activation of caspase 3/7. Further, downstream targets of FOXM1 such as PLK1 and cyclin B1 were significantly reduced thus affecting the cell cycle progression in medulloblastoma cell lines. In conclusion, a restoration of miRNA-4521 may selectively suppress the pathophysiological effect of aberrant FOXM1 expression and serve as a targeted approach for medulloblastoma therapy.

The HERBY trial was a phase II open-label, randomized, multicenter trial evaluating bevacizumab (BEV) in addition to temozolomide/radiotherapy in patients with newly diagnosed non-brainstem high-grade glioma (HGG) between the ages of 3 and 18 years. We carried out comprehensive molecular analysis integrated with pathology, radiology, and immune profiling. In post-hoc subgroup analysis, hypermutator tumors (mismatch repair deficiency and somatic POLE/POLD1 mutations) and those biologically resembling pleomorphic xanthoastrocytoma ([PXA]-like, driven by BRAF_V600E or NF1 mutation) had significantly more CD8+ tumor-infiltrating lymphocytes, and longer survival with the addition of BEV. Histone H3 subgroups (hemispheric G34R/V and midline K27M) had a worse outcome and were immune cold. Future clinical trials will need to take into account the diversity represented by the term "HGG" in the pediatric population.

Diffuse gliomas with K27M histone mutations (H3K27M glioma) are generally characterized by a fatal prognosis, particularly affecting the pediatric population. Based on the molecular heterogeneity observed in this tumor type, personalized treatment is considered to substantially improve therapeutic options. Therefore, clinical evidence for therapy, guided by comprehensive molecular profiling, is urgently required. In this study, we analyzed feasibility and clinical outcomes in a cohort of 12 H3K27M glioma cases treated at two centers. Patients were subjected to personalized treatment either at primary diagnosis or disease progression and received backbone therapy including focal irradiation. Molecular analyses included whole-exome sequencing of tumor and germline DNA, RNA-sequencing, and transcriptomic profiling. Patients were monitored with regular clinical as well as radiological follow-up. In one case, liquid biopsy of cerebrospinal fluid (CSF) was used. Analyses could be completed in 83% (10/12) and subsequent personalized treatment for one or more additional pharmacological therapies could be recommended in 90% (9/10). Personalized treatment included inhibition of the PI3K/AKT/mTOR pathway (3/9), MAPK signaling (2/9), immunotherapy (2/9), receptor tyrosine kinase inhibition (2/9), and retinoic receptor agonist (1/9). The overall response rate within the cohort was 78% (7/9) including one complete remission, three partial responses, and three stable diseases. Sustained responses lasting for 28 to 150 weeks were observed for cases with PIK3CA mutations treated with either miltefosine or everolimus and additional treatment with trametinib/dabrafenib in a case with BRAFV600E mutation. Immune checkpoint inhibitor treatment of a case with increased tumor mutational burden (TMB) resulted in complete remission lasting 40 weeks. Median time to progression was 29 weeks. Median overall survival (OS) in the personalized treatment cohort was 16.5 months. Last, we compared OS to a control cohort (n = 9) showing a median OS of 17.5 months. No significant difference between the cohorts could be detected, but long-term survivors (>2 years) were only present in the personalized treatment cohort. Taken together, we present the first evidence of clinical efficacy and an improved patient outcome through a personalized approach at least in selected cases of H3K27M glioma.

In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL-10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins' capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches.

Ependymomas in children can arise throughout all compartments of the central nervous system (CNS). Highly malignant paediatric ependymoma subtypes are Group A tumours of the posterior fossa (PF-EPN-A) and RELA-fusion positive (ST-EPN-RELA) tumours in the supratentorial compartment. It was repeatedly reported in smaller series that accumulation of p53 is frequently observed in ependymomas and that immunohistochemical staining correlates with poor clinical outcome, while TP53 mutations are rare. Our TP53 mutation analysis of 130 primary ependymomas identified a mutation rate of only 3%. Immunohistochemical analysis of 398 ependymomas confirmed previous results correlating the accumulation of p53 with inferior outcome. Among the p53-positive ependymomas, the vast majority exhibited a RELA fusion leading to the hypothesis that p53 inactivation might be linked to RELA positivity.In order to assess the potential of p53 reactivation through MDM2 inhibition in ependymoma, we evaluated the effects of Actinomycin-D and Nutlin-3 treatment in two preclinical ependymoma models representing the high-risk subtypes PF-EPN-A and ST-EPN-RELA. The IC-50 of the agent as determined by metabolic activity assays was in the lower nano-molar range (0.2-0.7 nM). Transcriptome analyses of high-dose (100 nM), low-dose (5 nM) and non-treated cells revealed re-expression of p53 dependent genes including p53 upregulated modulator of apoptosis (PUMA) after low-dose treatment. At the protein level, we validated the Actinomycin-D induced upregulation of PUMA, and of p53 interaction partners MDM2 and p21. Proapoptotic effects of low-dose application of the agent were confirmed by flow cytometry. Thus, Actinomycin-D could constitute a promising therapeutic option for ST-EPN-RELA ependymoma patients, whose tumours frequently exhibit p53 inactivation.

Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g., via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here we show that both static 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D-grown cells are substantially different from those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models more closely mimic tumor (patho-)physiology, including the upregulation of tumor relevant pathways, such as autophagy.

Pleomorphic xanthoastrocytoma (PXA) in its classic manifestation exhibits distinct morphological features and is assigned to CNS WHO grade 2 or grade 3. Distinction from glioblastoma variants and lower grade glial and glioneuronal tumors is a common diagnostic challenge. We compared a morphologically defined set of PXA (histPXA) with an independent set, defined by DNA methylation analysis (mcPXA). HistPXA encompassed 144 tumors all subjected to DNA methylation array analysis. Sixty-two histPXA matched to the methylation class mcPXA. These were combined with the cases that showed the mcPXA signature but had received a histopathological diagnosis other than PXA. This cohort constituted a set of 220 mcPXA. Molecular and clinical parameters were analyzed in these groups. Morphological parameters were analyzed in a subset of tumors with FFPE tissue available. HistPXA revealed considerable heterogeneity in regard to methylation classes, with methylation classes glioblastoma and ganglioglioma being the most frequent mismatches. Similarly, the mcPXA cohort contained tumors of diverse histological diagnoses, with glioblastoma constituting the most frequent mismatch. Subsequent analyses demonstrated the presence of canonical pTERT mutations to be associated with unfavorable prognosis among mcPXA. Based on these data, we consider the tumor type PXA to be histologically more varied than previously assumed. Histological approach to diagnosis will predominantly identify cases with the established archetypical morphology. DNA methylation analysis includes additional tumors in the tumor class PXA that share similar DNA methylation profile but lack the typical morphology of a PXA. DNA methylation analysis also assist in separating other tumor types with morphologic overlap to PXA. Our data suggest the presence of canonical pTERT mutations as a robust indicator for poor prognosis in methylation class PXA.

In the 5th edition of the WHO CNS tumor classification (CNS5, 2021), multiple molecular characteristics became essential diagnostic criteria for many additional CNS tumor types. For those tumors, an integrated, "histomolecular" diagnosis is required. A variety of approaches exists for determining the status of the underlying molecular markers. The present guideline focuses on the methods that can be used for assessment of the currently most informative diagnostic and prognostic molecular markers for the diagnosis of gliomas, glioneuronal and neuronal tumors. The main characteristics of the molecular methods are systematically discussed, followed by recommendations and information on available evidence levels for diagnostic measures. The recommendations cover DNA and RNA next-generation-sequencing, methylome profiling, and select assays for single/limited target analyses, including immunohistochemistry. Additionally, because of its importance as a predictive marker in IDH-wildtype glioblastomas, tools for the analysis of MGMT promoter methylation status are covered. A structured overview of the different assays with their characteristics, especially their advantages and limitations, is provided, and requirements for input material and reporting of results are clarified. General aspects of molecular diagnostic testing regarding clinical relevance, accessibility, cost, implementation, regulatory, and ethical aspects are discussed as well. Finally, we provide an outlook on new developments in the landscape of molecular testing technologies in neuro-oncology.

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

Accurate identification of brain tumor molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European "Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)" study.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood-brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model in freely moving mice and investigated its CNS disposition by quantifying actinomycin D in cerebral microdialysate, brain tissue homogenate, and plasma. For this purpose, we developed and validated an ultraperformance liquid chromatography-tandem mass spectrometry assay suitable for ultra-sensitive quantification of actinomycin D in the pertinent biological matrices in micro-samples of only 20 µL, with a lower limit of quantification of 0.05 ng/mL. In parallel, we confirmed actinomycin D as a substrate of P-glycoprotein (P-gp) in in vitro experiments. Two hours after intravenous administration of 0.5 mg/kg, actinomycin D reached total brain tissue concentrations of 4.1 ± 0.7 ng/g corresponding to a brain-to-plasma ratio of 0.18 ± 0.03, while it was not detectable in intracerebral microdialysate. This tissue concentration exceeds the concentrations of actinomycin D that have been shown to be effective in in vitro experiments. Elimination of the drug from brain tissue was substantially slower than from plasma, as shown in a brain-to-plasma ratio of approximately 0.53 after 22 h. Because actinomycin D reached potentially effective concentrations in brain tissue in our experiments, the drug should be further investigated as a therapeutic agent in potentially susceptible CNS malignancies, such as ependymoma.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant tumor that may not only contain rhabdoid tumor cells but also poorly differentiated small-round-blue cells as well as areas with mesenchymal or epithelial differentiation. Little is known on factors associated with histopathological diversity. Recent studies demonstrated three molecular subgroups of AT/RT, namely ATRT-TYR, ATRT-SHH, and ATRT-MYC. We thus aimed to investigate if morphological patterns might be related to molecular subgroup status. Hematoxylin-eosin stained sections of 114 AT/RT with known molecular subgroup status were digitalized and independently categorized by nine blinded observers into four morphological categories, that is, "rhabdoid," "small-round-blue," "epithelial," and "mesenchymal." The series comprised 48 ATRT-SHH, 40 ATRT-TYR, and 26 ATRT-MYC tumors. Inter-observer agreement was moderate but significant (Fleiss' kappa = 0.47; 95% C.I. 0.41-0.53; p < 0.001) and there was a highly significant overall association between morphological categories and molecular subgroups for each of the nine observers (p < 0.0001). Specifically, the category "epithelial" was found to be over-represented in ATRT-TYR (p < 0.000001) and the category "small-round-blue" to be over-represented in ATRT-SHH (p < 0.01). The majority of ATRT-MYC was categorized as "mesenchymal" or "rhabdoid," but this association was less compelling. The specificity of the category "epithelial" for ATRT-TYR was highest and accounted for 97% (range: 88-99%) whereas sensitivity was low [49% (range: 35%-63%)]. In line with these findings, cytokeratin-positivity was highly overrepresented in ATRT-TYR. In conclusion, morphological features of AT/RT might reflect molecular alterations and may also provide a first hint on molecular subgroup status, which will need to be confirmed by DNA methylation profiling.

More than 60% of supratentorial ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA-RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.This article is highlighted in the In This Issue feature, p. 2113.