Pediatric Ependymoma Research Articles

MULTI-OMIC INTEGRATION REVEAL INTRA-TUMOUR HETEROGENEITY AND IDENTIFY DISULfiRAM AND COPPER AS A SYNERGISTIC THERAPY IN PAEDIATRIC EPENDYMOMA

Abstract AIMS Ependymoma (EPN) is the second most malignant paediatric brain tumour. The PF-A subgroup, associated with a hypoxic microenvironment, has a dismal survival rate of 50% with clinical trials failing to demonstrate a significant survival advantage from chemotherapy to date. As the lack of genomic alterations makes it difficult to uncover novel therapeutic targets, we present a multi-omic integration investigating whether spatially-distinct tumour microenvironments represent targetable metabolic niches in PF-A EPN. METHOD Surgical sampling of spatially-distinct regions was performed on eight PF-A patients. Metabolites and RNA were simultaneously extracted from the same cellular population and analysed using liquid chromatography-mass spectrometry (LC-MS) and RNAseq. Multi-omic integration was performed using Metscape3 and functional assays evaluating proliferation, invasion and siRNA-mediated knockdown were performed in 2D and 3D models of intra-tumour region patient-derived cell lines. Selected single and drug combination therapy was performed in normoxia and hypoxia, and synergy assessed using the Chou-Talalay method. RESULTS Multi-omic integration revealed 124 dysregulated metabolic pathways, encompassing 156 genes and 49 metabolites with the largest number of interactions occurring in the gluconeogenesis pathway. Each anatomical region also presented at least one unique gene-metabolite interaction demonstrating heterogeneity within and across PF-A EPN tumours. Four selected drug targets (Disulfiram, Vandetanib, Mildronate and FBP1 inhibitor) based on metabolically relevant genes (ALDH3A1, FMO3, BBOX1, FBP1) showed impairment of metabolic viability in vitro, with limited chemosensitivity observed in control human cerebellar astrocytes. The addition of Cu2+ significantly sensitized cells to Disulfiram, particularly under hypoxic conditions. This synergistic combination significantly impaired invasion in both normoxia and hypoxia. CONCLUSION This is the first instance where multi-omic data integration and intra-tumour heterogeneity has been investigated for paediatric EPN revealing novel therapeutic targets in the context of gene-metabolite correlations. Drug tolerability will be further assessed in vivo using the previously characterised PF-A EPN orthotopic mouse xenograft MAF-928.

2532: Comparison of proton arc therapy and intensity-modulated proton therapy for pediatric ependymoma

2532: Comparison of proton arc therapy and intensity-modulated proton therapy for pediatric ependymoma

The medical and functional burden of surviving childhood ependymoma: A population-based study in Ontario, Canada.

Few studies have characterized the burden of late effects among childhood ependymoma survivors. To address this gap, we examined these sequelae using real-world health services data in a population-based ependymoma survivor cohort. All individuals younger than 18years diagnosed with an ependymoma in Ontario, Canada between 1987 and 2015 who had survived at least 5years from their latest pediatric cancer event (index date) were matched 1:5 with population controls. Following linkage with provincial health services data, the cumulative incidences of multiple medical and functional outcomes between survivors and controls were compared. Among 96 survivors, 77.1% had been irradiated and 9.4% had received cisplatin. At 10years post-index, survivors were at significantly higher risk of all-cause mortality (7.1%, 95% confidence interval [CI]: 1.0-13.3 vs. 0.3%, 95% CI: 0.0-1.0; p=.0002), non-obstetric hospitalization (45.1%, 95% CI: 32.6-56.7 vs. 10.6%, 95% CI: 7.6-14.1; p<.0001), stroke (6.5%, 95% CI: 2.3-13.7 vs. 0%; p<.0001), severe hearing loss requiring an amplification device (7.5%, 95% CI: 2.7-15.7 vs. 0%; p<.0001), receiving homecare service (27.6%, 95% CI: 18.5-37.5 vs. 7.7%, 95% CI: 5.3-10.7; p<.0001), and submitting a disability support prescription claim (24.0%, 95% CI: 14.8-34.3 vs. 5.4%, 95% CI: 3.5-7.8; p<.0001) compared to controls. Pediatric ependymoma survivors are highly vulnerable to severe late sequelae, including death, stroke, severe hearing loss, and disability. Urgent efforts are needed to improve risk-stratification approaches that mitigate exposure to toxic therapies for children with lower risk disease. Interventions to prevent or decrease the risk of developing late sequelae are critical to optimizing survivor long-term health.

EPEN-13. PRE-IRRADIATION CHEMOTHERAPY EXPERIENCE FOR PEDIATRIC EPENDYMOMA POLISH PROTOCOL

Abstract BACKGROUND Pediatric ependymomas are aggressive central nervous system malignancies arising from the ependymal linings of ventricles and spinal canal. Despite better understanding of tumor biology ependymomas remain therapeutic challenge in pediatric neurooncology. The role of chemotherapy remains controversial. Herein, we describe the outcomes of patients treated at the largest center in Poland who underwent surgery, pre-irradiation chemotherapy and irradiation regardless of extent of surgical resection. METHODS This retrospective review includes pediatric patients with ependymoma treated according to national protocol at the Children’s Memorial Health Institute. We reviewed histology, disease extent, treatment, and report on overall survival (OS) and progression free survival rates (PFS). Descriptive statistics were analyzed using SPSS software. RESULTS Forty-nine patients were included (median age: 84 months). Most patients (96%) had cranial ependymomas (43% supratentorial and 57% posterior fossa lesions). Disseminated disease was found in 16% of cases. Complete resection was achieved in 43%. All patients received pre-irradiation chemotherapy; 6.1% had progressive disease and 41% achieved complete response (CR). All patients, except one, underwent irradiation (67% focal, 2% focal/ventricular, 29% craniospinal, and 2% whole brain). Thirty-five percent of patients relapsed. Five-year PFS and OS were 69% and 80% respectively. Comparing posterior fossa to supratentorial lesions, there was no difference in response after surgery and pre-irradiation chemotherapy (CR 37% versus 50% (p=0.51)) or irradiation (CR 42% versus 55% (p=0.37)). However, relapse was more likely in posterior fossa ependymomas (OR 0.30 IQR (0.70, 1.29), p=0.03) with lower 5-year PFS (56% versus 85%, p=0.03) and 10-year PFS (52% versus 85%, p=0.02). The benefit of chemotherapy was marked in patients with Grade 3 (5-year OS 82% versus 69%) CONCLUSIONS We describe a large cohort of pediatric ependymoma patients who received pre-irradiation chemotherapy. This strategy did not negatively impact the incidence of progression before irradiation.

QOL-11. LATE MORBIDITY AND MORTALITY IN SURVIVORS OF CHILDHOOD EPENDYMOMA: A REPORT FROM THE CHILDHOOD CANCER SURVIVOR STUDY (CCSS)

Abstract BACKGROUND Between the 1970s and 1990s, therapy for childhood ependymoma evolved to include chemotherapy and focal rather than whole-brain radiation. The impact of these changes on health outcomes is unknown. METHODS 404 five-year survivors of ependymoma (52.5% male, median 6 [range 0-20] years old at diagnosis, 22 [5-49] years from diagnosis) were evaluated for cumulative incidence of late mortality, subsequent neoplasms, and chronic health conditions (CHC). Outcomes were analyzed by diagnosis decade and brain radiation and chemotherapy exposures. Fine-Gray’s test compared cumulative incidence curves. Relative Risks (RRs) with 95% CIs estimated outcomes using multivariable piecewise exponential models. RESULTS Throughout the three decades whole brain radiation exposure decreased (32.7% to 2.3%) while focal brain radiation increased (16.4% to 59.2%), and chemotherapy use increased (23.6% to 47.9%). Fifteen-year all-cause late mortality (incidence, 95% CI) estimates were similar across treatment eras: 1970s (9.3%, 3.4-18.8%), 1980s (14.7%, 9.4-21.2%), 1990s (10.3%, 6.7-14.9%), (p=0.9). Mortality was higher with whole brain radiation (22.5%, 11.2-36.5%) compared to focal radiation (11.4%, 7.5-16.1%) or no brain radiation (3.5%, 0.9-9.1%), (p &amp;lt;0.001). Mortality was also higher with chemotherapy (14.4%, 9.6-20.0%) compared to no chemotherapy (6.8%, 3.8-11.0%) (p=0.004). Differences in mortality were due to recurrence/progression of the primary cancer and health-related causes. Compared to no brain radiation, there were increased risks (RR, 95% CI) of any grade 3-4 CHCs for focal (2.6, 1.3-5.4) and whole brain radiation (3.5, 1.5-8.1) and &amp;gt;1 Grade 3-4 CHCs for whole brain radiation (6.5, 1.3-31.6). There were no differences in CHCs or subsequent neoplasms by chemotherapy exposure. CONCLUSIONS Despite the treatment changes over the 1970s to 1990s, late morbidity and mortality in pediatric ependymoma remained unchanged. Whole and focal brain radiation were associated with increased risk of grade 3-4 CHCs compared to no radiation. Future therapies for childhood ependymoma should target reductions in late morbidity and mortality.

EPEN-27. GAINING A CLUE FROM 1Q: A HIGH-RISK CHROMOSOMAL GAIN IN PEDIATRIC POSTERIOR FOSSA EPENDYMOMA

Abstract BACKGROUND Ependymomas are neuroepithelial tumours that can be classified into distinct molecular subgroups, each exhibiting a unique clinical outcome. Therapeutic efforts against the most aggressive and abundant subgroup of pediatric ependymoma (posterior fossa group A, PFA) have traditionally been hampered by a lack of known recurrent driver mutations. However, approximately 25% of PFA tumours exhibit gains of the chromosome arm 1q which is associated with an extremely poor prognosis despite aggressive treatment. By interrogating the multi-omic landscape of PFAs harbouring this copy number variation, we aimed to uncover genetic dependencies from which actionable therapeutics could be derived. METHODS Transcriptomic (bulk/single-cell RNA sequencing), proteomic (LC-MS/MS) and phosphoproteomic analyses were conducted on patient tissues and primary patient-derived cell lines to gain insight into the pathways that drive 1q gain pathogenesis. Chromatin immunoprecipitation sequencing of H3K27me3 and H3K27ac marked histones was carried out to profile the epigenetic landscape of these tumours, and whole genome CRISPR knockout screens were performed to reveal the contribution of essential genes unique to tumours harboring 1q gains. RESULTS Through the comparison of 1q gain and balanced PFA samples, our multi-omic analyses identified the up-regulation and essentiality of known glioma oncogenic drivers previously uncharacterized in PFA. The investigation of essential candidate genes showed a convergence on pathways related to ciliogenesis, with genetic vulnerabilities informing in vitro drug screening. Candidate genes were further validated by in vivo knockdown using established xenograft mouse models for pre-clinical testing. CONCLUSIONS This combined approach has allowed for the derivation of a functional cancer signature underlying 1q gains in PFA and presents novel therapeutic targets for this high-risk subgroup. PFA ependymoma is a deadly malignancy whose complex biology has so far eluded therapy, and new insights from this project will help to identify the first effective therapies for this vulnerable patient population.

EPEN-14. MOLECULAR MARKERS AND PREDICTORS OF OUTCOME FOR PAEDIATRIC INTRACRANIAL EPENDYMOMA IN THE PROSPECTIVE, MULTICENTRE E-HIT2000 TRIAL AND SUBSEQUENT HIT-REGISTRIES: A POOLED ANALYSIS OF 244 PATIENTS

Abstract BACKGROUND Throughout the past decade, molecular tools have revolutionised ependymoma classification, yet their application has been limited in clinical trial cohorts. METHODS We present the molecular analysis of paediatric ependymoma patients from the E-HIT2000 trial (n = 165), or subsequent HIT2000-interim and I-HIT-MED registries (n = 79) based on DNA-methylation profiling with multi-level classification using the Heidelberg Brain Tumor Classifier v12.5 and the analysis of chromosome 1q, 6q, and CDKN2A copy number status. Prior to further analysis, cases without ependymoma group prediction were excluded (n = 16/244). RESULTS 5-year PFS and OS rates varied by group: EPN-PFA (n=146/228) 45±4% and 76±4%; EPN-PFB (n=19/228) 90±7% and 100%; EPN-ZFTA (n=59/228) 63±7% and 87±5%; and EPN-YAP1 (n=4/228) 50±25% and 100%. EPN-PFA was subclassified into nine subtypes. Poor median PFS was observed for PFA1c (n=22, 5-yr-PFS 32±10%/5-yr-OS 77±9%), PFA1d (n=13, 35±14%/72±14%), PFA1e (n=15, 47±13%/59±13%), and PFA2a (n=16, 27±12%/50±14%). Presence of 1q gain was associated with inferior survival (n = 39/146, 29±8%/65±8%). 6q loss impacted PFS, but not OS (n=8, 25±14%/100%). Among 59 EPN-ZFTA cases, 46 clustered with EPN ZFTA-RELA fused, and 12 clustered within recently described subgroups of EPN-ZFTA (EPN-ZFTA alt., Cluster 1: 1/12, Cluster 2: 11/12). Strikingly, 6/11 EPN ZFTA-alt. (Cluster 2) cases showed infratentorial (4/6) or supratentorial midline location (2/6). Homozygous deletion of CDKN2A (CDKN2A-/-; n=8) was only observed in EPN ZFTA-RELA. Presence of EPN ZFTA-alt. (cluster 2) (5-yr-PFS: 36±15%; p&amp;lt;0.001), and CDKN2A-/- (19±16%; p&amp;lt;0.0001) was associated with inferior PFS compared to EPN ZFTA-RELA CDKN2A-wt (81±6%). OS was only impacted for patients with EPN ZFTA-RELA CDKN2A-/- (5-yr-OS: 38±20%, p&amp;gt;0.0001). CONCLUSIONS We present strong evidence supporting the inclusion of molecular criteria into the next generation of clinical trials for children with ependymoma.

EPEN-04. OVERWHELMING ZFTA-RELA NUCLEAR LOCALIZATION AS A THERAPEUTIC STRATEGY AGAINST EPENDYMOMA

Abstract BACKGROUND Pediatric ependymoma is a malignant brain tumor without targeted therapies. The 10-year survival is close to 50%, as this relentless tumor often relapses years following initial diagnosis. Over two thirds of supratentorial ependymomas harbor a gene fusion between ZFTA and RELA (ZFTA-RELA). Despite evidence that ZFTA-RELA drives tumorigenicity, mechanistic details remain elusive, hindering efforts to uncover therapeutic vulnerabilities. Since direct targeting of ZFTA-RELA is not currently possible, we hypothesized that ZFTA-RELA fusion binding partners would represent vulnerabilities amenable to therapeutic targeting. METHODS We investigated the biochemical basis of ZFTA-RELA, with the goal of identifying key members of the protein interaction landscape. This was performed in our in utero electroporation mouse model of ZFTA-RELA ependymoma. A series of sucrose gradient and immuno-precipitation experiments were performed, coupled with mass spectrometry. ZFTA-RELA interaction proteins were functionalized using focused CRIPSR-Cas9 library screens in cell cultures. Critically, these revealed novel dependencies and druggable targets, with XPO1 emerging as a lead candidate. In vitro and in vivo studies were conducted to evaluate targeting XPO1 in ZFTA-RELA ependymoma. RESULTS ZFTA-RELA regulates XPO1 gene transcription, and elevated gene expression is associated with poor survival in these patients. While expression of ZFTA-RELA is necessary for oncogenic transcription, extremely elevated nuclear localization is associated with reduced growth. This suggests a ‘goldilocks’ model in which XPO1 titrates precise levels of nuclear ZFTA-RELA protein. We reveal that a potential therapeutic vulnerability emerges through modulation of ZFTA-RELA nuclear localization. Supporting this, chemical inhibition of XPO1 using Selinexor, has a direct effect on ZFTA-RELA cellular localization patterns. Critically, in patient derived orthotopic xenograft models, XPO1 inhibition exerts anti-tumor effects both in vitro and in vivo. CONCLUSION Our findings reveal a novel therapeutic strategy in ependymoma, and provide the mechanistic and pre-clinical data needed to develop a clinical trial for patients with ZFTA-RELA ependymoma.

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.

Abstract 5467: Identification of possible new treatment options for rare pediatric CNS tumors using a panel of validated and characterized PDX models

Abstract Central nervous system (CNS) malignancies are the most frequent group of childhood solid tumors. While pediatric mortality due to CNS tumors has decreased over the past 40 years, however, there has been no significant change in brain and other CNS tumor mortality in children and adolescents since 2007*. Advances in research and innovative therapies are crucial to improving outcomes and reducing mortality. Collaborative efforts between researchers, healthcare professionals, and the pharmaceutical industry play a pivotal role in developing more effective treatments for these indications. One important piece in this endeavor is the availability of well characterized preclinical models for rigorous preclinical testing. In the framework of the ITCCP4 consortium (www.ITCCP4.eu) our group determined the pharmacological profile of ten orthotopically implanted pediatric brain tumor PDX models. The panel comprises amongst others four CNS-BCOR models, one ZFTA::RELA ependymoma model and two models of PFA ependymoma. We tested the models in a single mouse trial format with 16 arms covering standard of care therapy including radiation as well as targeted agents. All tumors were intracranially implanted into immune-compromised NSG mice. Tumor load was determined using a fluorescence based in vivo imaging technology based on the lentiviral transient transduction of PDX cells with iRFP713 prior to implantation into the brain. In addition, body weight and neurological scoring were monitored to determine the overall condition of the animals. At the end of the study brain tissue was harvested and tumor load confirmed by immunohistochemistry. Vismodegib, a first in class inhibitor of the hedgehog signaling pathway, was the most efficacious treatment with a significantly extended overall survival (Log Rank, p&amp;lt; 0.001) and a reduced tumor load compared to untreated control arms. The effect was most prominent in the subclass of CNS-BCOR models leading to growth delay or stable disease. Several targeted agents such as Ipatasertib (AKT inhibitor), Idasanutlin (MDM2-antagonist) and standard of care such as Carboplatin and Radiotherapy induced stable disease as well but had no significant effect on overall survival. The positive results for Vismodegib, Ipatasertib as well as Idasanutlin will be validated in a smaller subset of models in a conventional set-up to confirm the current dataset. In summary, the single mouse trial format using intracranially implanted PDX models proved to be a feasible tool to identify most promising drug candidates for pediatric ependymoma. *Ostrom QT, Price M, Ryan K, Edelson J, Neff C, Cioffi G, et al.. CBTRUS statistical report: pediatric brain tumor foundation childhood and adolescent primary brain and other central nervous system tumors diagnosed in the United States in 2014-2018. Neuro-Oncology (2022) 24(Supplement_3):iii1-38. Citation Format: Eva Oswald, Dorothee Lenhard, Kanstantsin Lashuk, Olivier Delattre, Didier Surdez, Johannes Gojo, Daniela Lötsch-Gojo, Walter Berger, Stefan Pfister, Julia B. Schueler. Identification of possible new treatment options for rare pediatric CNS tumors using a panel of validated and characterized PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5467.

Prognostic factors of pediatric ependymomas at a National Cancer Reference Center in Peru.

Ependymomas are central nervous system tumors that significantly impact the quality of life and carry a high mortality rate. Both the disease itself and its treatment cause significant morbidity. At a national level in Peru, there are no reports on clinical characteristics of the disease. This retrospective study captured patient aged less than 19 years with a diagnosis of ependymoma from 2012 to 2022 at a tertiary center in Lima. 85 patients were included with a median follow-up time was 51.6 months. The 5-year overall survival and progression-free survival were 55.89% (95% CI: 44.28 - 65.99) and 37.71% (95% CI: 26,21-49,16) respectively. The main prognostic factors identified were completed treatment (p=0.019), adjuvant chemotherapy (p=0.048), presence of metastasis (p=0.012), and disease recurrence (p=0.02). The survival of patients with ependymoma is below that reported in high-income countries. Incomplete treatment and treatment abandonment are factors that negatively impact the prognosis. Further studies are needed to identify barriers in the referral and treatment process for patients with ependymoma.

10154-PEDT-6 RETROSPECTIVE STUDY OF 12 PEDIATRIC EPENDYMOMAS TREATED AT OUR HOSPITAL

Abstract Ependymoma is an important disease in pediatric brain tumors, but reports with a large number of cases are scarce. In this study, we investigated the clinical course of 12 cases of ependymoma in children treated at our hospital.The median age was 2.5 years (0-15 years), and 10 had posterior fossa and 2 had supratentorial onset. Surgery was performed in all cases, and total resection was achieved in 9 cases (75%). Five cases were diagnosed as ependymoma, Grade II, and seven cases were diagnosed as anaplastic ependymoma, Grade III, according to the WHO revised 4th edition. Adjuvant therapy was administered in 8 patients, 6 with radiotherapy and 2 with combination chemotherapy and radiotherapy. Recurrence was seen in 6 cases, 4 of which were local recurrences and 2 of which were dissemination. The 2-year and 5-year progression-free survival rates were both 60%, and the 2-year and 5-year survival rates were 100% and 66.6%. Progression-free survival/overall survival analysis was performed, and the above results were considered to be equivalent to the published data of 27 cases (Peralia et al. Cell 2022). Progression-free survival tended to be favorable in patients with complete resection (HR = 0.18, 95% CI: 0.025-1.28) and patients with supratentorial disease (HR = 0.18, 95% CI: 0.030-1.11). No difference was seen by WHO grade (HR = 1.022, 95% CI: 0.20-5.24). Our cohort also yielded the same results regarding prognostic factors as previously reported, and WHO grade did not correlate with prognosis. The WHO 5th edition classification adopts molecular diagnosis and further subclassifies ependymoma. Since molecular diagnostics require advanced analytical techniques, there is a problem that it is difficult to put them into practical use, but surrogate markers that can be applied clinically have also been reported. This time, we will reclassify pediatric ependymoma using surrogate markers and compare it with the old classification.

Incorporating variable RBE in IMPT optimization for ependymoma.

To study the dosimetric impact of incorporating variable relative biological effectiveness (RBE) of protons in optimizing intensity-modulated proton therapy (IMPT) treatment plans and to compare it with conventional constant RBE optimization and linear energy transfer (LET)-based optimization. This study included 10 pediatric ependymoma patients with challenging anatomical features for treatment planning. Four plans were generated for each patient according to different optimization strategies: (1) constant RBE optimization (ConstRBEopt) considering standard-of-care dose requirements; (2) LET optimization (LETopt) using a composite cost function simultaneously optimizing dose-averaged LET (LETd ) and dose; (3) variable RBE optimization (VarRBEopt) using a recent phenomenological RBE model developed by McNamara etal.; and (4) hybrid RBE optimization (hRBEopt) assuming constant RBE for the target and variable RBE for organs at risk. By normalizing each plan to obtain the same target coverage in either constant or variable RBE, we compared dose, LETd , LET-weighted dose, and equivalent uniform dose between the different optimization approaches. We found that the LETopt plans consistently achieved increased LET in tumor targets and similar or decreased LET in critical organs compared to other plans. On average, the VarRBEopt plans achieved lower mean and maximum doses with both constant and variable RBE in the brainstem and spinal cord for all 10 patients. To compensate for the underdosing of targets with 1.1 RBE for the VarRBEopt plans, the hRBEopt plans achieved higher physical dose in targets and reduced mean and especially maximum variable RBE doses compared to the ConstRBEopt and LETopt plans. We demonstrated the feasibility of directly incorporating variable RBE models in IMPT optimization. A hybrid RBE optimization strategy showed potential for clinical implementation by maintaining all current dose limits and reducing the incidence of high RBE in critical normal tissues in ependymoma patients.

PATH-46. A NOVEL POT1-TPD PRESENTATION: A GERMLINE POT1 MUTATION DISCOVERED IN A PATIENT WITH NEWLY DIAGNOSED POSTERIOR FOSSA EPENDYMOMA

Abstract INTRODUCTION POT1 tumor predisposition (POT1-TPD) is an autosomal dominant disorder characterized by increased lifetime malignancy risk. Melanoma, angiosarcoma and chronic lymphocytic leukemia are the most frequently reported malignancies. Other related cancers include gliomas. Protection of telomeres protein 1 is part of the shelterin protein complex to maintain/protect telomeres. Proposed mechanisms for oncogenesis with POT1 loss of function include telomere elongation and DNA damage response causing genomic instability. Ependymomas are a heterogeneous group comprising 12% of pediatric brain tumors and are locally aggressive with frequent recurrence. CASE PRESENTATION A healthy 3-year-old male presented with worsening vertigo, headaches, and emesis. Radiographic studies demonstrated a midline posterior fossa mass in the fourth ventricle. Following a gross total resection, pathology demonstrated a posterior fossa ependymoma, group A. Next generation sequencing (NGS) using our institution’s clinically validated panel, “CinCSeq”, demonstrated a POT1 splice site variant (c.1164-1G &amp;gt;A; variant allele frequency 46%). Paired germline testing via the molecular characterization initiative confirmed this variant as a heterozygote in the patient. Genetic testing confirmed a germline mutation in his mother who has a history of multiple nevi. He completed treatment with focal proton radiotherapy with no evidence of disease recurrence to date. DISCUSSION To our knowledge, this represents the first documented pediatric ependymoma with a familial, germline POT1 mutation. Somatic POT1 mutational frequency as determined by NGS in 60,000 solid tumors occurs at a rate of 2.94%. 45/60,000 were ependymomas with 1 non-benign POT1 mutation. Alterations of telomere maintenance have been reported in infratentorial ependymomas through increased human telomerase reverse transcriptase (hTERT) expression. This case sheds light on potential new mechanisms/predispositions for ependymoma development and the expanding phenotype of POT1-TPD. We recognize the POT1 mutation may have been discovered incidentally in this case. Further research into an association between POT1 and ependymomas is needed to advance our understanding.

THE LINK BETWEEN M6A RNA METHYLATION AND R LOOPS IN PAEDIATRIC HIGH-GRADE GLIOMAS AND EPENDYMOMAS

Abstract AIMS Paediatric high-grade glioma (HGG) and ependymoma (EPN) are two devastating malignant tumours of the central nervous system that unfortunately carry a poor prognosis. As R loops are a known source of genomic instability in eukaryotic cells and have previously been revealed to undergo m6A RNA methylation, we sought to investigate the potential link between m6A methylation and R loops in relation to the pathogenesis of paediatric gliomas. METHOD Paediatric HGG cell lines including KNS42, GCE62 and SF188, and paediatric EPN cell lines including BXD- 1425EPN and EPN9, were utilised for experimental analysis, in addition to normal human astrocytes. Immuno- cytochemistry was performed to assess m6A and R loop levels in tumour cell lines compared to normal human astrocytes. Moreover, DNA damage levels and the effects of anti-cancer drugs on R loop levels were also examined. RESULTS Immunocytochemistry analysis revealed that overall m6A and R loop levels were higher in tumour cell lines compared to normal human astrocytes. Furthermore, differences in R loop levels were observed as a result of pharmacological and biological manipulation, including reduction in R loop abundance with PARP inhibitors. CONCLUSIONS The link between m6A methylation and R loops in relation to the pathogenesis of paediatric HGG and EPN is being investigated. The results of these experiments will be presented.

Multi-omic approach identifies hypoxic tumor-associated myeloid cells that drive immunobiology of high-risk pediatric ependymoma

Ependymoma (EPN) is a devastating childhood brain tumor. Single-cell analyses have illustrated the cellular heterogeneity of EPN tumors, identifying multiple neoplastic cell states including a mesenchymal-differentiated subpopulation which characterizes the PFA1 subtype. Here, we characterize the EPN immune environment, in the context of both tumor subtypes and tumor cell subpopulations using single-cell sequencing (scRNAseq, n= 27), deconvolution of bulk tumor gene expression (n= 299), spatial proteomics (n= 54), and single-cell cytokine release assays (n= 12). We identify eight distinct myeloid-derived subpopulations from which a group of cells, termed hypoxia myeloid cells, demonstrate features of myeloid-derived suppressor cells, including IL6/STAT3 pathway activation and wound healing ontologies. In PFA tumors, hypoxia myeloid cells colocalize with mesenchymal-differentiated cells in necrotic and perivascular niches and secrete IL-8, which we hypothesize amplifies the EPN immunosuppressive microenvironment. This myeloid cell-driven immunosuppression will need to be targeted for immunotherapy to be effective in this difficult-to-cure childhood brain tumor.

Respective Roles of Surgery, Chemotherapy, and Radiation Therapy for Recurrent Pediatric and Adolescent Ependymoma: A National Multicentric Study

Half of the children and adolescents treated for intracranial ependymoma experience recurrences that are not managed in a standardized manner. This study aimed to retrospectively evaluate recurrence treatments. We assessed overall survival (OS) and progression-free survival (PFS) after a first relapse in a population of patients from the Pediatric Ependymoma Photons Protons and Imaging study (PEPPI study) who were treated with surgery and radiation therapy in French Society of Childhood Cancer reference centers between 2000 and 2013. Data were analyzed using the Cox model as well as a landmark analysis at 4 months that accounted for the guarantee-time bias. The median follow-up of the whole population of 202 patients was 105.1 months, with a 10-year OS of 68.2% and PFS of 45.5%. Among the 100 relapse cases, 68.0% were local relapses, 20.0% were metastatic, and 12.0% were combined (local and metastatic). Relapses were treated by surgery (n = 79) and/or reirradiation (n = 52) and/or chemotherapy (n = 22). The median follow-up after relapse was 77.8 months. The OS and PFS at 5 years were 43.1% and 16.2%, respectively. After surgery or radiation therapy of the first relapse, OS and PFS were more favorable, whereas treatments that included chemotherapy with or without focal treatment were associated with worse OS and PFS. In the multivariate analysis, stereotactic hypofractionated reirradiation after surgery was associated with a significantly better outcome (OS, P = .030; PFS, P = .008) and chemotherapy with a worse outcome (OS, P = .028; PFS, P = .033). This analysis of relapse treatments within the PEPPI study determined that irrespective of whether the relapse was localized or metastatic, treatments that included surgery and/or reirradiation had better outcomes.

Linear energy transfer-inclusive models of brainstem necrosis following proton therapy of paediatric ependymoma.

Radiation-induced brainstem necrosis after proton therapy is a severe toxicity with potential association to uncertainties in the proton relative biological effectiveness (RBE). A constant RBE of 1.1 is assumed clinically, but the RBE is known to vary with linear energy transfer (LET). LET-inclusive predictive models of toxicity may therefore be beneficial during proton treatment planning. Hence, we aimed to construct models describing the association between brainstem necrosis and LET in the brainstem. A matched case-control cohort (n=28, 1:3 case-control ratio) of symptomatic brainstem necrosis was selected from 954 paediatric ependymoma brain tumour patients treated with passively scattered proton therapy. Dose-averaged LET (LETd) parameters in restricted volumes (L50%, L10% and L0.1cm3, the cumulative LETd) within high-dose thresholds were included in linear- and logistic regression normal tissue complication probability (NTCP) models. A 1keV/µm increase in L10% to the brainstem volume receiving dose over 54Gy(RBE) led to an increased brainstem necrosis risk [95% confidence interval] of 2.5 [0.0, 7.8] percentage points. The corresponding logistic regression model had area under the receiver operating characteristic curve (AUC) of 0.76, increasing to 0.84 with the anterior pons substructure as a second parameter. 19 [7, 350] patients with toxicity were required to associate the L10% (D>54Gy(RBE)) and brainstem necrosis with 80% statistical power. The established models of brainstem necrosis illustrate a potential impact of high LET regions in patients receiving high doses to the brainstem, and thereby support LET mitigation during clinical treatment planning.

WDR82-Mediated H3K4me3 Is Associated with Tumor Proliferation and Therapeutic Efficacy in Pediatric High-Grade Gliomas.

Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.

EPEN-05. TRANSCRIPTIONAL FACTOR-DIRECTED EPIGENETIC REGULATION IN CANCER NEUROSCIENCE OF ZFTA-RELA EPENDYMOMA

Abstract ZFTA-RELA (ZRFUS) ependymoma is a pediatric brain tumor with dismal 10-year progression free survival rates. Prior studies revealed low somatic mutation load in pediatric ependymoma compared to adult glioma, suggesting that developmental mechanisms play a key role during disease development. Using human tissues and mouse tumors generated from our autochthonous in utero electroporation (IUE) model, we identified a cohort of developmental transcription factors (TFs) linked to ZRFUS by investigating histone serotonylation changes with other epigenetic datasets. We further performed in vivo barcode-screening and identified the ETS variant transcription factor 5 (ETV5) is sufficient and required for disease progression. To further reveal the disease mechanism driven by ETV5, we applied transcriptomic, epigenetic, and proteomic analyses, and found ETV5 suppresses neuropeptide Y (NPY) expression in mouse ZRFUS tumors by binding with epigenetic modulator, chromobox 3 (CBX3). We further found NPY overexpression delays tumor progression and suppressed tumor-driven neuronal activity in mice. This study suggests developmental TF can shape tumor-promoting microenvironment during disease progression through epigenetic regulation.