Ependymoma Research Articles

Cytological features of ependymal and choroid plexus tumours.

Ependymal and choroid plexus tumours arise in anatomically related regions. Their intraoperative differential diagnosis is large and depends on factors such as age, tumour site and clinical presentation. Squash cytology can provide valuable information in this context. Cytological features of conventional ependymomas, subependymomas and myxopapillary ependymomas as well as choroid plexus tumours are reviewed and illustrated. Differential diagnostic considerations integrating morphological and clinical information are discussed.

Cortically-based Brain Tumors in Children: A Decision-tree Approach in the Radiology Reading Room.

Cortically-based brain tumors in children constitute a unique set of tumors with variably aggressive biological behavior. As radiologists play an integral role on the multidisciplinary medical team, a clinically useful and easy-to-follow flowchart for the differential diagnoses of these complex brain tumors is essential.This proposed algorithm tree provides the latest insights into the typical imaging characteristics and epidemiologic data that differentiate the tumor entities, taking into perspective the 2021 World Health Organization's classification and highlighting classic as well as newly identified pathologic subtypes using current molecular understanding.ABBREVIATIONS: Astroblastoma=AB) Angiocentric glioma (AG) Atypical teratoid rhabdoid tumor (ATRT) Central Nervous System tumor (CNS) CNS neuroblastoma FOXR2-activated (NB-FOXR2) Desmoplastic infantile glioma/astrocytoma (DIG/DIA) Diffuse hemispheric glioma, H3 G34-mutant (DHG) Diffuse glioneuronal tumor with oligodendroglioma-like features and nuclear clusters (DGONC) Dysembryoplastic neuroepithelial tumor (DNET) Embryonal Tumors with Multilayered Rosettes (ETMR) Ependymoma (EP) Focal cortical dysplasia (FCD) Ganglioglioma/gangliocytoma (GG) Infant-type hemispheric glioma (IHG) Intracranial pressure (ICP) Long-term epilepsy-associated tumors (LEATs) Pediatric diffuse low-grade gliomas (pLGG) MR spectroscopy (MRS) Multinodular and vacuolating neuronal tumor (MVNT) Overall survival (OS) Pediatric diffuse high-grade gliomas (pHGG).

Dominant Malignant Clones Leverage Lineage Restricted Epigenomic Programs to Drive Ependymoma Development.

1. Specific chromatin modules accessible during brain development render distinct cell lineage programs at risk of transformation by pediatric fusion oncoproteins.2. Cross-species single cell ATAC and RNA (scMultiome) of mouse and human ependymoma (EPN) reveals diverse patterns of lineage differentiation programs that restrain oncogenic transformation.3. Early intermediate progenitor-like EPN cells establish a tumor cell hierarchy that mirrors neural differentiation programs.4. ZFTA-RELA transformation is compatible with distinct developmental epigenetic states requiring precise 'goldilocks' levels of fusion oncoprotein expression.5. Dominant tumor clones establish the entire EPN cellular hierarchy that reflects normal gliogenic and neurogenic differentiation programs.

Coccygeal tumours unveiled: a retrospective cohort analysis from a tertiary referral centre.

Isolated tumours affecting the coccyx are infrequent, with only a handful of documented cases in the literature. Herein, we highlight the most extensive consecutive case series involving various isolated coccyx tumours with varied clinical presentations and imaging features. A retrospective search of our tertiary Orthopaedic oncology institute's oncology and Radiology database [Radiology Information System, Picture Archiving and Communication System, and Computerised Radiology Information System] for the keyword "Coccyx" and "Tumour" was performed over 15 years (between December 2007 and August 2022). Data collected was correlated with local histopathology and laboratory records. Patient demographics, clinical characteristics, and complementary imaging findings were recorded for analysis. One hundred and three lesions originating in the coccyx with a mean age of 62 years (range 25-90 years) were identified. There was a male preponderance with 59 male and 44 female patients (1.3:1.0). The most typical tumour noted was chordoma. Other lesions included a dermoid cyst, a myxopapillary ependymoma, a notochordal remnant, an osteochondroma, an Ewing sarcoma, and a teratoma. Our analysis suggests that most of the tumours involving coccyx are chordomas with a few rarely encountered benign and malignant tumours. Radiological imaging plays a vital role in characterising isolated tumours affecting the coccyx and guiding appropriate patient management. This is the largest reported series of coccygeal tumours. Chordoma is the commonest coccygeal tumour. Patients with unexplained coccydynia should undergo detailed investigations, preferably with cross-sectional imaging.

Histone serotonylation regulates ependymoma tumorigenesis.

Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment3. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.

Machine Learning Analysis in Diffusion Kurtosis Imaging for Discriminating Pediatric Posterior Fossa Tumors: A Repeatability and Accuracy Pilot Study.

Background and purpose: Differentiating pediatric posterior fossa (PF) tumors such as medulloblastoma (MB), ependymoma (EP), and pilocytic astrocytoma (PA) remains relevant, because of important treatment and prognostic implications. Diffusion kurtosis imaging (DKI) has not yet been investigated for discrimination of pediatric PF tumors. Estimating diffusion values from whole-tumor-based (VOI) segmentations may improve diffusion measurement repeatability compared to conventional region-of-interest (ROI) approaches. Our purpose was to compare repeatability between ROI and VOI DKI-derived diffusion measurements and assess DKI accuracy in discriminating among pediatric PF tumors. Materials and methods: We retrospectively analyzed 34 children (M, F, mean age 7.48 years) with PF tumors who underwent preoperative examination on a 3 Tesla magnet, including DKI. For each patient, two neuroradiologists independently segmented the whole solid tumor, the ROI of the area of maximum tumor diameter, and a small 5 mm ROI. The automated analysis pipeline included inter-observer variability, statistical, and machine learning (ML) analyses. We evaluated inter-observer variability with coefficient of variation (COV) and Bland-Altman plots. We estimated DKI metrics accuracy in discriminating among tumor histology with MANOVA analysis. In order to account for class imbalances, we applied SMOTE to balance the dataset. Finally, we performed a Random Forest (RF) machine learning classification analysis based on all DKI metrics from the SMOTE dataset by partitioning 70/30 the training and testing cohort. Results: Tumor histology included medulloblastoma (15), pilocytic astrocytoma (14), and ependymoma (5). VOI-based measurements presented lower variability than ROI-based measurements across all DKI metrics and were used for the analysis. DKI-derived metrics could accurately discriminate between tumor subtypes (Pillai's trace: p < 0.001). SMOTE generated 11 synthetic observations (10 EP and 1 PA), resulting in a balanced dataset with 45 instances (34 original and 11 synthetic). ML analysis yielded an accuracy of 0.928, which correctly predicted all but one lesion in the testing set. Conclusions: VOI-based measurements presented improved repeatability compared to ROI-based measurements across all diffusion metrics. An ML classification algorithm resulted accurate in discriminating PF tumors on a SMOTE-generated dataset. ML techniques based on DKI-derived metrics are useful for the discrimination of pediatric PF tumors.

Myxopapillary ependymoma of the spinal cord: A case with literature review

Myxopapillary ependymoma is a rare spinal cord tumors. The intramedullary myxopapillary ependymomas are. Herewith case of a 41-year-old male complaint of a nonspecific low back pain, progressive inability to bend, right lower limb pain with numbness of six month duration. On clinical evaluation right lower limb weakness was noted while the sensations were normal. There was no any significant past history. MRI lumbo-sacral spine with whole spine screening was performed. It showed lobulated heterogenous intensity enhancing intra medullary mass lesion in thecal sac extending from L4-L5 to S1-S2 level. It measured about 52 x 28 x 21 mm. MRI features suggestive of neoplasm – ependymoma was made. The gross total surgical resection was performed. On histopathological findings reported as myxopapillary ependymoma grade 2. The tumor on immunohistochemistry showed positive for EMA, GFAP. While negative for CMYC. The Ki-67 proliferation index (MIB1) was 3%. On follow up there was no tumor recurrence. Rehabilitation therapy was initiated and follow up is advised.

EPEN-02. CAR T-CELLS TARGETED TO B7-H3 OR HER2 FUNCTION INDEPENDENTLY OF ANTIGEN DENSITY IN XENOGRAFT MODELS OF EPENDYMOMA

Abstract BACKGROUND Targeted treatment options are desperately needed for ependymomas (EPN). Chimeric antigen receptor (CAR) T-cells have immense potential to positively transform treatment outcomes; however, little is known regarding antitumor efficacy of CAR T-cells against EPNs. Specifically, antigen density has emerged as a major influencer of CAR T-cell potency. However, the specific high and low antigen density thresholds governing CAR T-cell efficacy for EPN are unknown. We hypothesize that antigen density heterogeneity governs CAR T-cell potency against EPN. METHODS Using quantitative flow cytometry, we determined antigen density of B7-H3 and HER2 (the two CAR targets currently in pediatric EPN clinical trials) on EPN patient derived cell lines (1425, ST1, CPITT, ST2, EPI, L46SJ). We generated second generation B7-H3 and HER2-CAR T-cells with CD28z signaling domain and compared long-term cytolytic activity, expansion, and cytokine production against EPNs. RESULTS We found that all EPN cell lines express remarkably high yet variable density of B7-H3 (6.3e4 – 3.4e5 molecules/ cell). In contrast, surface density of HER2 was consistently and significantly lower (860 – 2.5e4 molecules/ cell). In repeated-stimulation assays, B7-H3-CAR T-cells killed 7-10 times and expanded up to 325,360-fold when cultured against EPN. However, we observed no significant difference in total expansion or correlation with antigen density against EPNs. Surprisingly, expansion of HER2-CAR T-cells against EPNs was drastically higher (up to over 100-billion-fold) and greatly exceeded that of B7-H3-CAR-T cells against the same EPN cells despite much lower antigen density. CONCLUSIONS Our studies reveal significant antigen density heterogeneity of B7-H3 and HER2 in EPN, which may lead to target-specific impacts on CAR T-cell persistence. Indeed, results suggest that lower antigen density of HER2 may be superior for sustained CAR T-cell activity. Future work will validate whether HER-CAR T-cells have superior antitumor efficacy in vivo to further delineate role of antigen density thresholds in CAR T-cell potency.

EPEN-09. TARGETING EPHB2/ABL1 RESTORES ANTI-TUMOR IMMUNITY IN A PRECLINICAL MODEL OF EPENDYMOMA

Abstract BACKGROUND Ependymoma (EPN) is a childhood brain cancer that is often resistant to cytotoxic therapies. Molecular profiling has led to a better understanding of unique EPN subtypes and revealed a critical role of EPHB2 in driving disease. With this in mind, new treatments for EPN are desperately needed and should be developed in a molecular subtype-specific fashion. METHODS We developed a syngeneic mouse model of EPHB2-driven genetically engineered EPN tumor cells. We performed transcriptomic profiling and analysis of EPHB2-driven murine EPN tumors and human EPN datasets to reveal multiple protein kinases as potential druggable targets. After identifying the tyrosine kinase inhibitor, Dasatinib, as a potentially effective FDA-approved agent, we measured changes in the murine microenvironment during EPN growth and after Dasatinib treatment. RESULTS We determined that Dasatinib inhibited the growth of EPN both in vitro and in vivo, through blocking EPHB2 and ABL1 signaling. Furthermore, we identified an increased frequency of immunosuppressive M2-like tumor- associated macrophages (TAMs), which proportionally increased with tumor size during tumor progression. However, treatment with Dasatinib reprogrammed the EPN immune microenvironment by polarizing TAMs toward an anti-tumor M1-like phenotype and increasing CD8 T cell activation. In addition, Dasatinib treatment induced complete regression of established EPN tumors in 78% of the animals and protected survivors against tumor recurrence. Depletion of CD8 T cells compromised the durability of response and reduced overall survival. CONCLUSION In conclusion, these data indicate that Dasatinib may be an effective therapy for EPHB2-driven molecular subgroup of EPN by activating the anti-tumor immune response and support further investigation of Dasatinib in clinical trials.

EPEN-03. THE MANAGEMENT OF PEDIATRIC AND ADOLESCENT SPINAL EPENDYMOMA; A 20-YEAR NATIONAL EVALUATION INVOLVING UNITED KINGDOM PEDIATRIC ONCOLOGY PRIMARY TREATMENT CENTERS ON BEHALF OF THE UK CHILDREN’S CANCER AND LEUKAEMIA GROUP (CCLG)

Abstract INTRODUCTION Pediatric spinal ependymomas account for approximately 1% of childhood CNS tumors. Most are myxopapillary ependymomas (MPEs). For symptomatic cases, maximal safe surgical resection is the accepted therapeutic standard. However, a role for adjuvant therapy is unclear, underpinned by low incidence rates, inadequate trial data and recent WHO upgrading of the MPE variant. This retrospective national service evaluation aimed to provide safety and outcome data, from which future management guidance could be developed. METHOD Children, teenagers and young adults diagnosed with a spinal ependymoma of any WHO grade since 2000 across 17 UK CCLG centres were evaluated. Patient demographics, therapies, adverse events, and outcomes were captured. RESULTS 102 patients were evaluated. Ten children had NF2 (median age 11 (7.6 – 14) years) with nine located in the cervicomedullary region. All ten patients were alive following surveillance (n=3) or surgical intervention (n=7) with an estimated median PFS of 5.4 (1.7 – 9.1) years. For the remaining 92 patients (median age 12 (0.7 – 19) years), 63% of tumors were lumbosacral, 73% demonstrated MPE morphology and 16% were metastatic. The median symptom interval was 4 (0 - 36) months. Progression or relapse was reported in 30/92 (33%), with a 5-year PFS of 62(+ 6)%, yet only 4 disease-related deaths occurred (mean follow-up 6.8 years; no MPEs). Across this non-NF2 cohort, gross/near total resection and upfront adjuvant radiotherapy independently reduced disease progression risk (p &amp;lt; 0.05). However, improved local control from early irradiation was not statistically proven when localized disease or MPEs were exclusively scrutinized. No radiotherapy-related high-grade toxicity was reported. DISCUSSION Pediatric spinal ependymomas are rare tumors demonstrating excellent survival, particularly MPEs. Maximal safe surgery and early adjuvant radiotherapy can improve local control rates, but European collaboration will be required to evaluate efficacy for certain tumor and clinical subgroups. Conclusions could support future national management guidelines.

CNSC-04. EXPLORATION OF INTERCELLULAR NETWORKS IN EPENDYMOMA REVEALS POTENTIAL THERAPEUTIC VULNERABILITIES

Abstract BACKGROUND Aggressive ependymoma (EPN) exhibit primary and secondary radio- and chemotherapy resistance with the poorest prognosis in the pediatric groups PF-EPN-A and ST-EPN-ZFTA. Recently, tumor cell networks formed by membrane protrusions, such as tunneling nanotubes (TNT) or tumor microtubes (TM), have been identified in other glial brain tumors. These structures facilitate intercellular hierarchical Ca2+-communication and contribute to therapy resistance. Previous data from our group suggested enhancer-regulated Ca2+-communication to be essential in EPN. This study aims to further characterize intratumoral interactions and to understand potential correlations between tumor cell connections and treatment resistance in EPN. METHODS Intercellular connections in EPN patients and model systems were characterized by scanning electron microscopy, immunofluorescence and ultra-high content imaging. Transcriptomic and proteomic data were analyzed to identify potential targetable vulnerabilities within network dynamics. Candidates were functionally validated in cell culture models applying Ca2+ live cell imaging. RESULTS Connectivity-related terms such as microtube-based movement or microtube bundle formation showed increased expression in EPN transcriptome data compared to healthy controls. EPN networks were found to be build of TM- and TNT-like structures with a higher abundance of TNT-like structures in PF-EPN-A compared to ST-EPN-ZFTA. All networks showed strong Nestin-positivity in cells, mouse models and human tumor tissue of ST-EPN-ZFTA and PF-EPN-A. Thrombospondin-1, a matricellular protein enhancing TMs in glioma cells, was strongly enriched in ST-EPN-ZFTA patients. The gap junction channel connexin 43, connecting glioma cells, was highly upregulated in PF-EPN-A. Active electrochemical communication was observed between ST-EPN-ZFTA cells performing Ca2+ imaging. Moreover, inhibition of T-type Ca2+ channels impaired intercellular signaling, leading to network perturbation in vitro. CONCLUSIONS Our study revealed the importance of gap junction-coupled networks for communication of aggressive EPN cells. Targeted disconnection of the EPN-specific network as demonstrated for Ca2+ channel inhibition suggests a new avenue for therapeutic strategies in EPN to overcome resistance.

EPEN-19. IMPACT OF MOLECULAR SUBTYPING ON PROGNOSIS IN CHILDREN AND ADOLESCENTS WITH SPINAL EPENDYMOMA

Abstract PURPOSE Ependymomas of the spinal cord are rare among children and adolescents, and the individual risk of disease progression is difficult to predict. Recent advances in subtyping the main molecular groups, myxopapillary ependymoma (MPE) and spinal ependymoma (SP-EPN), have successfully identified distinct risk groups among adult patients. This study aims at evaluating the prognostic impact of MPE subtypes A and B in a pediatric cohort. METHODS Eighty-three patients with spinal ependymoma ≤22 years registered in the HIT-MED database between 1992 and 2022 were included. Forty-seven tumors were analyzed regarding global DNA methylation. In six cases, antibodies against HOXB13 and MYCN were used as surrogate markers for the methylation group. Twenty-eight MPE were further classified into subtypes by t-distributed stochastic neighbor embedding (t-SNE) analysis. RESULTS MPE (n=32, 63%) was the most common molecular type followed by SP-EPN (n=17, 33%) and MYCN-amplified ependymoma (n=2, 4%). One case remained molecularly unclear, and one was excluded due to reclassification as anaplastic pilocytic astrocytoma. MPE subtyping identified 18 MPE-A and 9 MPE-B (inconclusive: n=1). 5-year progression-free survival (5y-PFS) did not significantly differ between MPE and SP-EPN (65% vs. 78%, p=0.64). MYCN-amplification was associated with early relapses and with death in one patient. Patients with MPE-B may have a tendency towards superior 5y-PFS compared to MPE-A (86% vs. 56%, p=0.15), irrespective of the use of adjuvant treatment. MPE-A was more frequently disseminated at time of diagnosis (n=6/18) than MPE-B (n=1/8, not evaluable: n=1; p=0.28). CONCLUSION Molecular subtyping of spinal ependymoma in children does not allow to separate patients into distinct risk groups, yet. However, the potentially favorable PFS among patients with MPE-B prompts the consideration of treatment de-intensification for this group. Larger cohorts and further insights into the molecular heterogeneity of these tumors are needed to complete the basis for future clinical decision-making.

EPEN-06. IDENTIFICATION OF CSF MIRNA BIOMARKERS FOR THE EARLY DETECTION OF MEASURABLE RESIDUAL DISEASE IN PAEDIATRIC EPENDYMOMA: A BIOMECA STUDY

Abstract BACKGROUND Ependymoma (EPN) is the second most common malignant paediatric brain tumour. 50% of children relapse. Detection of Measurable Residual Disease (MRD) via MRI is difficult and lacks sensitivity for very early recurrence, necessitating an urgent need for identifying new, robust biomarkers for the early diagnosis, prognosis, and clinical management of disease progression in EPN. We hypothesise that microRNA (miRNA) quantification in the cerebrospinal fluid (CSF) will have clinical utility in the early detection of MRD in these patients. PATIENTS/METHODS A discovery cohort of EPN CSF samples (n=12) from patients enrolled on the SIOP Ependymoma II clinical trial (trials.gov identifier: NCT02265770) and control leukaemia CSF samples (n=8) underwent RNA-sequencing (QIAGEN) after total RNA extraction using QIAGEN miRNeasy Serum/Plasma kit. The EPN cohort consisted of infratentorial (IT; n=9), supratentorial (ST; n=1) and spinal (SP; n=2) cases taken prior to, or at, resection (primary/relapse). RESULTS Raw sequencing data entered a quality-control pipeline (Tally/Cutadapt), followed by alignment to miRBase-v22 (https://mirbase.org/) using Chimira (PMID:26093149). Two outlying (infratentorial) EPN samples were removed, leaving a 10 (IT+ST+SP) versus 8 comparison. Extracted raw counts were normalised across samples using DESeq2 (PMID:25516281) and 87 differentially-expressed miRNAs identified (adj-p&amp;lt;0.05). EPN samples completely segregated from controls on clustering/heatmap analysis. From these 87 miRNAs, a core set of 53 were overlapping in comparisons removing spinal (IT+ST remaining) and supratentorial (IT alone) samples. These miRNAs underwent combined ranking by adjusted-p and abundance (normalised count) values, and top-ranking candidates taken forward for confirmation using highly-sensitive Taqman PCR, prior to validation in additional samples. CONCLUSION Circulating miRNA quantification offers promise for EPN diagnosis and MRD detection. Further CSF study in larger patient cohorts, and serum/plasma analysis, are now warranted.

EPEN-20. LEVERAGING ABERRANT EXTRACELLULAR VESICLE SIGNALING FOR TARGETED DRUG REPURPOSING IN PEDIATRIC EPENDYMOMA

Abstract Increasing evidence highlights the diverse functions of extracellular vesicles (EVs) as intercellular messengers in all facets of cancer progression, pointing towards a potential new avenue in cancer treatment. Limited understanding of tumor-specific deregulations and lack of suitable drug compounds have hampered clinical translation of targeted interference with EV signaling. The most aggressive types of intracranial ependymoma (EPN) in children and adults are resistant to chemotherapy. Despite enormous diagnostic advancements in classification and stratification, EPN lack recurrent effective molecular targets precluding access of affected patients to precision-based oncology therapeutics. Here, we characterized the proteome of ninety-five EPN and five matching healthy control brain tissue samples. Employing a mulit-omic data integration approach, we identified EVs as an excessively augmented pathway on the functional level. Isolation and molecular characterization of EPN EVs combined with single cell sequencing analysis, and ultra-high content imaging unraveled perivascular mesenchymal cell populations as predominant EV originators. Targeting EV pathways through chemical inhibition of sphingomyelinases (SMPD1, SMPD2, SMPD3) including re-purposed FDA approved compounds revealed growth inhibition, reduced invasion capacity in vitro and significant survival probability in vivo. Transcriptomic analysis and scanning electron microscopy delineated transition of mesenchymal-to neuroepithelial subpopulations in cells and patient-derived xenograft (PDX) models of EPN upon reduction of EV release. This study showcases the inhibition of EV signaling as a promising therapeutic strategy against mesenchymal tumor persister cells, which are the suspected origins of repeated EPN relapses. Our study provides a framework to leverage cell state-specific vulnerabilities and drug repurposing to attack the most aggressive cell populations in cancers with unfavorable prognosis.

EPEN-01. DISTINCT RELAPSE PATTERN ACROSS MOLECULAR EPENDYMOMA TYPES

Abstract BACKGROUND Ependymoma (EPN) is not a uniform disease but represents different tumor types with biological and clinical heterogeneity. Some types have an excellent prognosis; others display frequent relapse. However, the pattern of when and where different types of EPN relapse is largely unknown. METHODS We assembled 552 intracranial EPN from pediatric (n=445) and adult (n=107) patients from European and Northern American cohorts and correlated DNA methylation patterns and copy-number alterations with clinical information. Median age at diagnosis was 13.4±18.8 (0.1 – 80) years. RESULTS In 271/552 (49.1%) of included EPN relapse occurred. First relapses occurred in the initial tumor bed in 70.9%, 22.4% were metastatic only, and 6.7% were combined local and metastatic. First relapses of PF-EPN-B (PF: posterior-fossa) and PF-EPN-SE (SE: subependymoma) occurred later than of PF-EPN-A, ST-EPN-YAP (ST: supratentorial), or ST-EPN-ZFTA (median time to relapse: 4.3 and 6.0 years vs. 1.9/1.0/2.5 years; p&amp;lt;0.01). Metastatic or combined recurrences in PF-EPN-B and -A occurred more often in the spinal cord than in ST-EPN-ZFTA (75.0% and 40.0% vs. 12.5%; p&amp;lt;0.01). In the latter, relapse occurred more frequently when harboring an alternative fusion or CDNK2A loss (p&amp;lt;0.01, respectively). No distant relapses were observed in ST-EPN-YAP (n=9) or PF-EPN-SE (n=60). Post-relapse survival (PRS) was poor for PF-EPN-A and ST-EPN-ZFTA (5-year PRS: 44.5±4.4/ 47.8±9.1%), whereas PF-EPN-B and PF-EPN-SE displayed a 5-year PRS of 89.5±7.1/ 90.0±9.5% (p=0.03). However, 10-year PRS for PF-EPN-B dropped to 45.8±17.3%. CONCLUSIONS Relapse patterns of specific EPN types are different. Duration of surveillance and diagnostics should be planned incorporating entity-specific relapse information. Follow-up should be performed longer than 5 years after diagnosis. Spinal MRI should be considered as standard procedure during surveillance, particularly for PF-EPN-A, PF-EPN-B and ST-EPN-ZFTA.

EPEN-10. INDUCING A SENESCENCE PHENOTYPE BY BROMODOMAIN INHIBITION IN HIGH-RISK EPENDYMOMA

Abstract BACKGROUND Ependymomas (EPN) constitute 10% of pediatric brain tumors. WHO classification identified 10 molecular subtypes, with ZFTA fusion-driven supratentorial tumors (ST-ZFTA) and posterior fossa group A (PFA) being amongst the most aggressive ones. Current therapeutic success relies on local treatments, emphasizing the urgent need for novel targets. Epigenetic dysregulation is a major contributor to EPN aggressiveness. Bromodomain and extra-terminal domain (BET) proteins are transcriptional mediators involved in proto-oncogene transcription, thereby representing promising targets in cancer therapy. As the BET protein BRD4 co-regulates ZFTA-RELA fusion-mediated aberrant transcription, BET inhibitors emerge as novel targeted therapy approach in ST-ZFTA. METHODS Initial assessments encompassed primary, patient-derived (n=9) cell models representing ST-ZFTA and PFA EPN. RNA sequencing of two ZFTA-RELA models was performed, using DGE and GSEA analyses to discern altered pathways following BET-inhibition. On functional level, we investigated sensitivity towards BET-inhibition using cell viability, clone- and sphere formation assays, as well as Western blot and qPCR after treatment. Additionally, anti-tumor effects in genetically engineered cerebral organoids were assessed. RESULTS First investigations showed elevated BRD2 and BRD4 levels in ST-ZFTA compared to PFA models. Accordingly, treatment with BET inhibitors resulted in reduced survival and cell cycle arrest, evidenced by cell viability and clone formation assays, along with increased p21 and cleaved PARP levels in Western blot analyses. RNA sequencing revealed altered pathways associated with cell-cycle regulation and senescence following BET inhibition, supported by downregulation of the telomerase subunit TERT and genes of the senescence-associated secretory phenotype. Additionally, BET inhibitors exhibited synergistic effects with EZH2- and PARP inhibitors primarily in ZFTA-RELA cell lines, as well as decreased tumor volumes in ZFTA-RELA-driven cerebral organoids. CONCLUSIONS Summarizing, we show drastic effects of BET-inhibition in ZFTA-RELA EPN. Subsequently, the influence of Bromodomain inhibition on stemness and senescence, particularly combined with radio- and chemotherapy will be investigated.

EPEN-18. MULTI-OMICS APPROACH REVEALED A TIGHT BLOOD-BRAIN BARRIER IN EPENDYMOMA

Abstract BACKGROUND A major challenge in the treatment of brain tumors is the limited penetration of many drugs through the blood-brain barrier (BBB). BBB characteristics include endothelial cells connected by tight junction proteins (e.g. Claudin5, ZO1, occludin) and the expression of efflux transporters (e.g. P-gp) with the physiological role to limit the accumulation of potentially toxic substances in brain tissue. Our study aims to enhance our understanding of the BBB composition across different molecular groups of ependymoma (EPN) with the goal of leveraging this knowledge to improve therapeutic strategies against these tumors. METHODS We applied a multi-omics approach integrating single-nuclear RNA sequencing and ultra-high content imaging to unravel BBB composition at both transcriptomic and proteomic levels. Patient-derived xenograft (PDX) models were utilized to explore differences in BBB penetration between tumor and healthy brain tissue following drug treatments. RESULTS Expression of tight junction and transporter proteins revealed strong dependencies specific to molecular groups, but were independent of the corresponding brain regions. Human tissue of ST-EPN-ZFTA exhibited highest expression of claudin5, while both ST-EPN-ZFTA and PF-EPN-A represented upregulation of occludin and ZO1 in comparison to healthy tissue. These findings on claudin5 and ZO1 were further confirmed in PDX models. Single-cell data analysis from patients localized the expression of relevant BBB factors to tumor-associated endothelial cells. Treatment of PDX models with drugs revealed disparities in drug penetration between EPN tumors and healthy brain regions. Notably, for most drugs BBB penetration was lower in ST-EPN-ZFTA tumors than healthy brain region. CONCLUSIONS Molecular BBB specifics may contribute to drug resistance of aggressive EPN particularly in ST-EPN-ZFTA tumors, which presented high tight junction expressions and lower drug penetration. This resource aims to improve BBB penetration prediction in EPN, to identify combination therapies targeting BBB components and to select innovative drug delivery approaches.

EPID-04. CAUSES AND LONG-TERM COURSE OF OTOTOXICITY IN PEDIATRIC MEDULLOBLASTOMA AND EPENDYMOMA PATIENTS – CUMULATIVE PLATINUM AND COCHLEAR IRRADIATION DOSE MATTER

Abstract BACKGROUND Ototoxicity is a well-known side effect of treatment with platinum-containing chemotherapy and radiotherapy (RT). Nevertheless, precise dose-effect relationships are still lacking. METHODS Patients from the HIT2000 trial receiving first-line treatment for medulloblastoma (MB) or ependymoma (EPN) who were alive at last follow-up and had at least one audiogram prior to and after administration of chemotherapy with cisplatinum and radiotherapy were included into this study. Treatment details (incl. cochlear irradiation dose [CID]) and audiological outcome were analyzed. The ototoxicity was graded using the HIT scoring. RESULTS Courses of 101 patients were analyzed. Median audiology follow-up was 6 years [range: 1.0–18.1 years]. Ototoxicity grade ≥1 was documented for n=66 patients (65.4%). For n=27 this resulted in treatment modification. Univariable analyses revealed different proportions of ototoxicity (grade ≥1) regarding histology (CMB: 50/65, LCAMB: 3/3, DMB/MBEN: 8/12, EPN: 5/21; p&amp;lt;0.001), tumor location (supratentorial: 3/9, infratentorial: 63/92; p&amp;lt;0.001), metastatic status at diagnosis (M0: 46/77, M+: 20/24; p=0.003), radiation field (CSI: 59/75, local RT: 6/21, no RT: 1/5; p&amp;lt;0.001), CID (continuous variable, p=0.001) and cumulative cisplatinum dose (continuous variable, p&amp;lt;0.001). Certainly, disease specific parameters (histology/staging) were related to treatment intensity (cisplatinum/irradiation dose). Multivariable analyses confirmed the impact of cumulative cisplatinum dose (p=0.01) and CID (p=0.04). ROC-analyses allowed the definition of cut-offs for high risk of ototoxicity grade ≥1 (cisplatinum: 70mg/m2 BSA, CID: 37.5 Gy) and ototoxicity risk estimation, when applying doses above the respective cut-offs (cisplatinum dose increase by 1mg/m2 BSA results in 4% higher risk for ototoxicity grade ≥1; CID dose increase by 1 Gy results in 4.6% higher risk for ototoxicity grade ≥1). CONCLUSIONS Ototoxicity affects a majority of children receiving adjuvant treatment for MB and EPN. To avoid disabilities and negative effect on quality of live, dosage of cisplatinum and radiotherapy should be scrutinized and reduced whenever possible.

LMIC-11. OUTCOMES OF INFANTS WITH CNS TUMORS: REPORT OF 85 CASES FROM 6 MEXICAN HOSPITALS

Abstract INTRODUCTION Reports on central nervous system (CNS) tumors in infants from low- and middle-income countries are scarce. This is a high-risk population, we sought to analyze their outcomes in Mexico. METHODS Patients &amp;lt;36 months diagnosed CNS tumors between January 2014 to December 2023 from 6 hospitals in Mexico were included. Demographic, treatment, and outcomes were collected. RESULTS In total, 85 patients with a median age of 21 months were included. Of these, 51 (60%) were male. Diagnoses included low-grade glioma (LGG) in 26 (33%), medulloblastoma (MB) in 19 (22%), other embryonal tumors in 12 (14%), ependymoma (EPN) in 11 (13%), high-grade glioma (HGG) in 6 (7%), and other tumors in 11 (13%). Regarding surgery, a near or gross total resection was achieved in 22 patients (26%). For LGG it was 23%, while it was 37% for for MB, 36% for for EPN, and 17% for HGG. Ten patients (12%) died from postoperative complications. Overall, 12 (14%) patients, including 4 patients with MB, had metastasis at diagnosis. In total, 19 (22%) patients received radiotherapy, including 10 patients who received CSI (average age 35 months) and 9 who received focal radiotherapy, including 6 with EPN, 2 with LGG, and 1 with ATRT. Of patients with MB, 18 (95%) received chemotherapy, including 1 with autologous stem cell transplant and 3 with intraventricular chemotherapy. The 5-year event-free survival was 38±11%, while the 5-year overall survival (OS) was 52±10%. The 5-year OS were: 84±10% for LGG, 43±21% for MB, 55±23% for EPN, 9±25% for other embryonal tumors, and 17±35% for HGG. CONCLUSIONS These data show low survival rates, associated with very poor surgical outcomes. A fraction of patients received CSI radiotherapy, showing deviation from international standards. Further analyses are needed to identify factors that lead to better prognosis.

IMG-18. DIFFERENTIAL DIAGNOSIS OF POSTERIOR FOSSA TUMORS USING RADIOMICS; A FIRST-LINE DIAGNOSTIC STEP REQUIRED FOR DOWNSTREAM ANALYSES

Abstract BACKGROUND Accurate radiographic diagnosis of pediatric brain tumors (PBTs) that originate in the brainstem and posterior fossa, including medulloblastoma (MB), pilocytic astrocytoma (PA), ependymoma (EPN), atypical teratoid/rhabdoid tumor (ATRT), and diffuse intrinsic pontine glioma (DIPG), is crucial for optimizing surgical approaches and enhancing neoadjuvant therapies. Existing research on the radiographic differential diagnosis of posterior fossa tumors has limitations, including small sample sizes, lack of inclusion of certain histologies especially rarer tumors such as ATRTs, and incomplete analysis of the whole tumor, including peritumoral edema. In this study, we aimed to perform a comprehensive analysis using radiomics and machine learning to differentiate among the common posterior fossa and brainstem tumors. METHODS We employed 927 radiomic features extracted from whole tumor regions within treatment-naïve, standard multiparametric MRI sequences (pre-/post-contrast T1-weighted, T2-weighted, FLAIR) of 264 patients (106 MBs, 78 PAs, 28 EPNs, 27 ATRTs, and 25 DIPGs), collected from the Children’s Brain Tumor Network (CBTN). We adopted a one-versus-rest classification strategy, employing Support Vector Machines combined with the Least Absolute Shrinkage and Selection Operator (LASSO) for feature selection, and implementing nested cross-validation for robustness. RESULTS The performances of the classifiers were evaluated using the Area Under the Receiver Operating Characteristic Curve, yielding values of 0.84 for MBs, 0.84 for PAs, 0.70 for EPNs, 0.75 for ATRTs, and 0.71 for DIPGs. CONCLUSIONS Our method effectively differentiates between various tumor types in the posterior fossa and brainstem, paving the path towards the development of comprehensive diagnostic and prognostic AI tools for pre-treatment histological diagnosis of these tumors. These AI tools can lead to more tailored, risk-adjusted treatments for PBTs, reducing morbidities and improving patient outcomes. Based on our promising initial results, we will expand our dataset to include more samples and incorporate rarer tumor types, as well as piloting in different molecular subclassifications.