bluuebunny/crossref_metadata_2025 huggingface dataset:
DIPG-23. Artificial intelligence for detecting ACVR1 mutations in patients with DIPG using MRI and clinical data
Authors: Khalid Fahad , Escobar Thibault , Goya-Outi Jessica , Frouin Vincent , Boddaert Nathalie , Grill Jacques , Frouin Frederique
Published: June 2022

INTRODUCTION: ACVR1 mutations are found in about 25% of patients with diffuse intrinsic pontine glioma (DIPG). Recent work has identified the combination of vandetanib and everolimus as a promising therapeutic approach for these patients. We investigate the predictive power of an AI model integrating clinical and radiomic information to predict ACVR1 mutation. METHODS: This retrospective monocentric study includes 65 patients with known ACVR1 status. Patients were scanned at the diagnosis time with at least one of the four structural MRI modalities (pre- and post-contrast T1, T2, FLAIR) and basic clinical information (age and sex) was collected. Radiomic features were extracted within the tumor region from each modality. For each modality, a recursive feature elimination method was used to select the most relevant features. Inside a leave-one-out framework, up to five logistic regression models were built: one per MRI modality and one for the clinical information. The final prediction for each patient was computed as the mean of the probabilities of ACVR1 mutation for the up to 5 different models. Assigning a different weight to clinical data according to age, (more or less than 10 years old) was also tested. RESULTS: Out of the 65 patients (mean age 7.9±3.7, 15 patients older then 10 years), ACVR1 mutations were identified with a 78% accuracy (sensitivity = 92% and specificity = 75%) in the leave-out-out process. Accounting for the clinical data in the model increase the accuracy to 82% (resp. sensitivity = 86% and specificity = 80%). CONCLUSION: The proposed multi model approach compensates for missing MR modalities while taking advantage of all the available information. Our first results suggest that a dedicated model could be developed for younger patients to improve the prediction. The different models will now be tested using additional data coming from the ongoing multi-centric BIOMEDE trials.
DIPG-29. Highly multiplexed digital spatial profiling of the tumor immune environment of pediatric and adultH3 K27M-mutant diffuse midline gliomas
Authors: Damodharan Sudarshawn , Helgager Jeffrey , Dey Mahua
Published: June 2022

BACKGROUND: H3 K27M-mutant diffuse midline gliomas (DMGs) are highly aggressive malignancies of the central nervous system that affect both pediatric and adult populations. The immune layout and genetic changes within the tumor microenvironment associated with these high-grade malignancies are thought to play an integral role in the phenotypic differences in tumor presentation and clinical course between both populations. Comparative immune landscape between pediatric and adult DMGs is not known. METHODS: The NanoString GeoMxTM Digital Spatial Profiler platform was used to determine the immune marker and genetic layout in a cohort of both pediatric and adult H3 K27M-mutant DMG tissue samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to identify and separate out the various components within the tumor tissues from selected regions of interest. The resultant information was then pooled into libraries that were run through the Illumina sequencing system to assess gene expression and proteomics for both cohorts of samples. RESULTS: Our data revealed that there were immune and genetic marker changes that were seen within both populations across multiple regions of interest within the tumor tissues. A number of immune cell types and protein markers exhibited significant difference between the pediatric and adult tissue cohorts. CONCLUSION: The digital spatial analysis of pediatric and adult H3 K27M-mutant DMGs show different immune and genetic profiles which are proposed to contribute to the clinical differences between both populations. These observed differences may play a role in future treatments for H3 K27M-mutant DMGs and further research is needed to explore this.
DIPG-30. A Phase 1, Safety and Dose Escalation Study of BXQ-350 in Combination with Radiation in Pediatric Patients with Diffuse Midline Glioma or Diffuse Intrinsic Pontine Glioma
Authors: O'Toole Dorris Kathleen McCarthy , Fouladi Maryam , Hummel Trent , Takigiku Ray , Tapolsky Gilles , Curry III Richard
Published: June 2022

The significance of sphingolipid metabolism in brain cancers has been demonstrated and enzymes involved in sphingolipid metabolism are being investigated as novel therapeutic targets for adult and pediatric brain cancers. Saposin C is a human protein encoded by the Psap gene and is an allosteric activator of enzymes involved in sphingolipid/ceramide metabolism. BXQ-350 is a nanovesicle of Saposin C that has broad anticancer activity, inducing apoptosis of cancer cells and an anti-tumoral immune response by lowering Sphingosine-1-Phosphate and increasing ceramides concentrations. Also, BXQ-350 is synergistic with radiation therapy. BXQ-350 was investigated in a Phase 1 dose-escalation safety study in pediatric brain cancer patients (NCT03967093); results showed that it was well-tolerated, a MTD was not established, and the safety profile warranted the investigation of combination studies at the highest dose tested (3.4 mg/kg). Additionally, results from a Phase 1 safety and dose escalation study in adult patients with advanced solid malignancies (NCT02859857) indicated that BXQ-350 was well tolerated and showed single agent activity with evidence of modulating sphingolipid metabolism and impacting the immune system positively. The primary objective of this dose-escalation study is to describe the safety profile and to determine the maximum tolerated dose of BXQ-350 in combination with radiation. BXQ-350 is administered intravenously in a modified Fibonacci cohort-based dose escalation design starting at 2.4mg/kg. Multiple secondary parameters are included to characterize BXQ-350’s pharmacokinetic parameters, efficacy profile and investigate potential biomarkers. This trial is currently enrolling at 3 US sites; as of January 2022, enrollment of cohort 1 (2.4 mg/kg and 54 Gy) has been completed while enrollment in cohort 2 (3.2 mg/kg and 54 Gy) is on-going. To this date, no dose-limiting toxicity has been observed and the combination BXQ-350 plus radiation has been well tolerated. Current results will be presented.
DIPG-42. Diffuse midline gliomas, H3K27-altered as an interdisciplinary challenge
Authors: Kammler Gertrud , Fritzsche Friederike , Kordes Uwe , Schüller Ulrich , Westphal Manfred
Published: June 2022

INTRODUCTION: Diffuse midline gliomas represent a particular challenge in treatment. DIPG behave like highly malignant glioma with extremely poor prognosis due to location and inoperability. Molecular genetic studies, complementary to classical histopathology, have found an entity for midline gliomas, newly described ten years ago and entered the WHO classification in 2016. This group of childhood tumor with DMG’s, H3K27-altered will be demonstrated in the following with 14 case reports from our clinic. METHODS: Clinical data of four patients with tectum/thalamic gliomas, six with diffuse intrinsic brainstem glioma, two with cerebellar, one with suprasellar, one with spinal glioma were retrospectively studied. MRI data, volume increase, contrast behavior was also analyzed. Tumor tissue was obtained by various surgical procedures and diagnostic workup included histopathology as well as genetics and epigenetics. RESULTS: 14 pediatric patients were treated from 2012 to 2021, median age 7,5 years. Leading symptoms were hydrocephalus, movement disorders, cranial nerve disorders. Four patients (29%) were partially resected, two (14%) received extended biopsy, seven (50%) were (stereo tactically) biopsied, one diagnosed by liquid biopsy (7%). Histological results revealed the presence of GBM in four cases (29 %). Subsequent methylome analyses confirmed that the tumors belonged to the group of diffuse midline gliomas, H3K27-altered. The other ten tumors (71%) were primarily assigned to this H3K27 group. CONCLUSION: The pediatric tumors of the brainstem, the further midline structures, including intraspinal manifestation show different MRI findings, histology, and clinical course. Complementary molecular genetic diagnosis is essential and a meaningful addition to the histological assignment. It is considered proven that the exclusivity of H3K27 – altered tumors of children and adolescents differs from that of IDH mutated gliomas and glioblastomas by their localization of hemispheric processes. Possible therapeutic approaches using targeted therapy require understanding of these oncological mechanisms.
DIPG-45. Radiation induces a robust interferon response in Diffuse Midline Glioma (DMG), improving the potential for combination immunotherapy
Authors: Minns Hanna E , Padilla Oscar , Wei Hong-Jian , Webster-Carrion Andrea , Tazhibi Masih , McQuillan Nicholas , Zhang Xu , Yeh Rebecca , Zhang Zhiguo , Szalontay Luca , Pavisic Jovana , Garty Guy , Garvin James , Zacharoulis Stergios , Canoll Peter , Vanpouille-Box Claire I , Menon Vilas , Olah Marta , Rabadan Raul , Wu Cheng-Chia , Gartrell Robyn D
Published: June 2022

Diffuse Midline Glioma (DMG), H3K27M altered, confers a dismal survival of 9-15 months and has a non-inflammatory tumor immune microenvironment (TIME). Radiation therapy (RT) is the mainstay treatment for DMG and has been shown in other cancers to recruit an immune component. However, the effect of RT on the DMG TIME has not been explored. In a syngeneic murine model of pontine DMG (PDGFB+, H3.3K27M, p53−/−), mice were treated with single fraction 15Gy RT or sham control, four mice per group. We performed single cell sequencing after CD45 isolation to evaluate the TIME 4 days post RT and compare to untreated tumor (sham control). Unsupervised clustering of 14,848 CD45+ cells revealed 16 immune cell subsets, most abundantly microglia at 75% of cells, with four subtypes representing a spectrum of homeostatic to activated. Microglia from RT are more concentrated in the activated subtypes with an upregulation of interferon response (i.e. Isg15, Ifit3) compared to untreated tumor with an increase in several interferon pathways using REACTOME. Consistent with RT response, RT treated tumors have increase in cell cycle regulatory genes such as Cdkn1a, across all clusters. In non-resident myeloid cells, compared to untreated tumor, RT is associated with a robust upregulation of interferon response genes in both macrophages (Isg15 Fold Change (FC) 2.30; Ifit1 FC 1.64; Ifit3 FC 2.02; Cxcl10 FC 2.29) and dendritic cells (Isg15 FC 2.67; Ifit1 FC 1.72; Ifit3 FC 2.06; Cxcl10 FC 1.50). We also find differential expression of immune checkpoints in RT-treated versus untreated tumor with decreased expression of Lag3, Tim3 (Havcr2), and Csf1R and increased expression of Cd47, Sirpa and Gitr (Tnfrsf18) post RT. In summary, RT stimulates a pro-inflammatory TIME response and alters immune checkpoints in DMG, highlighting the potential for combining RT and immunotherapy in these tumors.
DIPG-48. MRI volumetric and machine learning based analyses predict survival outcome in pediatric diffuse midline glioma
Authors: Bonner Erin R , Liu Xinyang , Tor-Diez Carlos , Kambhampati Madhuri , Eze Augustine , Packer Roger J , Nazarian Javad , Linguraru Marius George , Bornhorst Miriam
Published: June 2022

INTRODUCTION: Diffuse midline glioma (DMG) is a fatal childhood CNS tumor. Magnetic resonance imaging (MRI) is the gold standard for DMG diagnosis and monitoring of response to therapy. Leveraging novel MRI analytical approaches, including volumetric and machine learning based analyses, may aid in the prediction of patient overall survival (OS) and help to identify high-risk cases. METHODS: T1- and T2-weighted MR images were retrospectively collected from children and young adults diagnosed with DMG (n=43). MRI features, including manually determined 3D tumor volume (T2), T1 contrast-enhancing tumor volume, T1 relative to T2 volume (T1/T2), tumor relative to whole brain volume, tumor average intensity, and tumor heterogeneity (i.e., intensity skewness and kurtosis), were evaluated at upfront diagnosis. MRI features were analyzed to identify significant predictors of OS outcome, which was defined as OS shorter, or longer, than one year from diagnosis. A support vector machine was used to predict OS outcomes using combinations of these features. RESULTS: The presence of T1 contrast-enhancing tumor at diagnosis (p=0.01), and a high T1/T2 ratio (>25%, p=0.009), predicted significantly shorter median OS. Moreover, feature selection identified T2 mean intensity (p<0.001), T2 image intensity skew (p=0.006), T1/T2 ratio (p=0.02), and T1 volume relative to whole brain (p=0.03) as significant predictors of OS outcome (short versus long). Combining T2 mean intensity, T2 image skew, T1 segment kurtosis and patient gender resulted in OS outcome prediction accuracy of 83.3% (sensitivity=85%, specificity=81.8%, n=42 cases).CONCLUSION: We have identified MRI volume and imaging features that significantly predict OS outcome in children diagnosed with DMG. Our findings provide a framework for incorporating MRI volumetric and machine learning analyses into the clinical setting, allowing for the customization of treatment based on tumor risk characteristics.
DIPG-51. Hydrocephalus Treatment and the Effect on Survival in Diffuse Intrinsic Pontine Glioma
Authors: Baugh Joshua , Mohammed Nada Ben , van Zanten Sophie Veldhuijzen , Kramm Christof , Biassoni Veronica , Massimino Maura , van Vuurden Dannis
Published: June 2022

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG), can cause hydrocephalus and if symptomatic, leads to rapid changes in consciousness requiring surgical intervention. The effect of cerebrospinal fluid (CSF) diversion on overall survival and the clinical factors influencing outcome remain unclear. The aim of this study was to evaluate the impact of the treatment of hydrocephalus on survival in DIPG patients. METHODS: The study was retrospective in design using data from the SIOPE-European Society of Pediatric Oncology DIPG Registry. Hydrocephalus was determined based on a centrally reviewed diagnostic MRI. The Kaplan-Meier method was used for survival statistics. Clinical prognostic factors including: duration of symptoms, age and cranial nerve palsy at diagnosis were evaluated for confounding and effect modification. The effect of hydrocephalus treatment (CSF diversion) on survival was examined using Cox regression. RESULTS: Among 582 patients from the SIOPE-DIPG Registry, 86 (14%) had hydrocephalus at diagnosis. Median OS for hydrocephalus patients treated with CSF diversion (n=43) was 13 months (95% CI, 10.2-17.7) and 9 months (95% CI, 7.4-10.6) for hydrocephalus patients without a CSF diversion (n=43). Survival rates were not significantly different (p=.41). On adjusted Cox regression, correcting for duration of symptoms, hydrocephalus patients with signs of cranial nerve palsy at diagnosis and a CSF diversion had a hazard ratio 0.476 (p=0.004). CONCLUSION: Survival among DIPG patients presenting with hydrocephalus at diagnosis was not influenced by CSF diversion. Hydrocephalus patients with signs of cranial nerve palsy at diagnosis, had a significantly reduced risk after undergoing CSF diversion. There is an indication this subgroup of DIPG patients may benefit more from CSF diversion, although the relationship between hydrocephalus and cranial nerve palsy requires further investigation.
DIPG-54. p53 pathway reactivation as a therapeutic strategy in diffuse intrinsic pontine glioma
Authors: Lupien Leslie , Rendo Veronica , Morin Eric , Khuu Nicholas , DiGiacomo Jeromy , Khadka Prasidda , Chacon Madison , Lu Sophie , Pelton Kristine , Elcavage Lara , Langhnoja Jaldeep , Ligon Keith , Beroukhim Rameen , Phoenix Timothy , Bandopadhayay Pratiti
Published: June 2022

TP53 is the most frequently mutated tumor suppressor with somatic alterations found in approximately 50% of all human cancers. In the remaining TP53 wild-type (WT) tumors, functional inactivation of the p53 pathway may be achieved through a variety of other mechanisms, including gene deletion, epigenetic silencing, and/or alterations in prominent negative regulators, including MDM2/MDM4 and PPM1D. These alterations block p53 activity and lead to uncontrolled cell proliferation and oncogenesis in a majority of cancers, including the highly aggressive, universally fatal glial cell tumors of childhood, known as Diffuse Intrinsic Pontine Gliomas (DIPGs). DIPGs are inoperable due to their location within the brainstem. Available treatment options, including radiotherapy, have had a palliative effect at best, with almost all children succumbing to the disease within 18 months of diagnosis. Recent advances have led to an improved understanding of the biological underpinnings of this disease and identification of recurrent genetic alterations that represent potential therapeutic targets for these patients. Prominent among these targets in DIPGs with WT p53 status (50%) are MDM2/4 and PPM1D, whose suppression lead to p53 reactivation specifically in the WT p53 context. We have undertaken a combination of approaches to better understand the therapeutic potential of MDM2 and PPM1D inhibition in DIPG, characterizing the genomic, transcriptomic, and cell-state changes that drive resistance, and identifying novel vulnerabilities that can be exploited with combination therapies towards a cure.
DIPG-57. A systems biology approach to defining and targeting master regulator dependencies from bulk and single-Cell RNA-seq in diffuse midline glioma (DMG)
Authors: Fernández Ester Calvo , Wang Junqiang , Griffin Aaron , Minns Hanna , Wei Hong-Jian , Zhang Xu , Szalontay Luca , Mundi Prabhjot , Wu Cheng-Chia , Gartrell Robyn , Zacharoulis Stergios , Califano Andrea , Pavisic Jovana
Published: June 2022

Diffuse midline glioma (DMG) are fatal pediatric brain tumors with no effective systemic therapies. Molecular profiling demonstrates epigenetic dysregulation and heterogeneity, and novel approaches are needed to identify promising drugs and drug combinations. We used network-based computational analysis of RNA-seq to discover Master Regulator (MR) proteins that represent targetable, mechanistic determinants of distinct DMG cell states. We reverse-engineered the first DMG-specific regulatory network from 122 publicly available DMG RNA-seq profiles with ARACNe. Using this network, we measured sample-specific protein activity based on differential expression of their targets via VIPER. Activity-based clustering identified two clusters showing a trend in survival differences (>1 year, by χ2). The most activated MRs (i.e., TOP2A, CENPF, BUB1B, FOXM1, GTSE1, MKI67, E2F8), relative to normal caudate tissue, were enriched in cell cycle regulation members. The cluster with worse outcomes had significantly higher activity. Targetable MRs activated in subsets of patients () included TOP2A, CHEK1, CDK2, and EZH2. RNA-seq profiles were generated in two DMG cell lines following perturbation with ~400 oncology drugs, and used to identify drugs that invert MR activity profiles with the OncoTreat algorithm. We identified four pharmacotypes, and predicted sensitivity to HDAC, proteosome, EGFR, MEK, and PI3K inhibitors (), amongst others, consistent with published DMG high-throughput drug screens. To dissect intra-tumor heterogeneity, we measured protein activity from published single-cell RNA-seq profiles of 6 DMG patients, using single-cell based regulatory networks. Preliminary activity-based analysis identified 6 cell states representing distinct stages of differentiation/proliferation, including an oligodendrocyte precursor cell-like proliferative state whose MRs overlapped with bulk data (i.e. TOP2A, CENPF, FOXM1, E2F8, ZWINT, CCNA2), suggesting this as a key regulatory module. We are identifying cell state-specific MR-inverter drugs with OncoTreat to ultimately suggest drugs and drug combinations that collapse the transcriptional program and induce tumor kill for validation in MR-matched preclinical DMG models.
DDEL-01. The role of key pharmacodynamic and pharmacokinetic parameters in drug response prediction of pediatric tumors in the precision oncology study INFORM
Authors: Jamaladdin Nora , Peterziel Heike , ElHarouni Dina , Jäger Natalie , Holland-Letz Tim , Kopp-Schneider Annette , Sigaud Romain , Witt Olaf , Oehme Ina , Milde Till
Published: June 2022

INTRODUCTION: The INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study is a European pediatric precision oncology program. Using state of the art molecular assays, INFORM aims for identification of targetable genetic alterations, matching drugs and clinical trials. High evidence targets were associated with doubling of progression free survival when patients received a matching drug. However, the fraction of tumors with high evidence drug targets remains low requiring functional layers of information such as drug sensitivity profiling. The aim of this project is to identify and investigate the role of key pharmacodynamic and pharmacokinetic parameters to improve the predictivity of ex vivo drug response of pediatric tumors. METHODS: Positive control cell lines harboring specific mutations (n=7) and primary tumors (n=121) from INFORM, including 10% ependymomas, 7% high grade gliomas, 5% neuroblastomas and 4% medulloblastomas, were profiled ex vivo using a library of n=76 clinically relevant oncology drugs in a 384 well plate format. Metabolic activity was measured after 72h of treatment. Quality control (QC) was done using the robust z-factor, correlation of replicates and mean negative control. Hit selection was based on maximum percentage inhibition, normalized AUC metric (DSSasym) and maximum serum concentration (Cmax) of the drug. Clinical follow-up was collected using a questionnaire. RESULTS: A linear mixed model revealed the DSSasym to be the strongest pharmacodynamic parameter in drug prediction in cell lines. Drug screens of n=105 INFORM cases passed QC. Application of the filtering parameters resulted in prediction of n=1-16 drugs/case (min-max). A data base of published pediatric pharmacokinetic parameters of the drug library was generated. Analysis of predictive parameters and clinical follow-up of clinical samples is ongoing. CONCLUSION: Including pharmacodynamic as well as clinical pharmacokinetic parameters is paramount to identify potentially clinically active compounds from ex vivo drug screen data. Further algorithm development is warranted.
EPCT-06. Phase I study of ribociclib and everolimus post-radiotherapy in children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) and high-grade glioma (HGG): Updated report from the COllaborative Network for NEuro-Oncology Clinical Trials (CONNECT)
Authors: Lazow Margot A , DeWire Mariko , Campagne Olivia , Leach James L , Fuller Christine , Kumar Shiva Senthil , Stanek Joseph , de Blank Peter , Hummel Trent R , Pillay-Smiley Natasha , Salloum Ralph , Stevenson Charles B , Baxter Patricia , Gass David , Goldman Stewart , Leary Sarah E S , Carle Adam , Lane Adam , Drissi Rachid , Stewart Clinton , Fouladi Maryam
Published: June 2022

BACKGROUND: Dual inhibition of CDK4/6 and mTOR in DIPG and pediatric HGG has strong biologic rationale, given prevalent genetic alterations resulting in upregulated cell cycle and PI3K/mTOR pathways in these diseases, as well as non-overlapping agent toxicities. This study sought to evaluate safety/tolerability and determine the recommended phase 2 dose (RP2D) of ribociclib and everolimus among children with newly diagnosed DIPG and HGG post-radiotherapy. METHODS: Patients were enrolled according to a Rolling-6 design and received oral ribociclib and everolimus once daily for 21 and 28 days, respectively, starting 2-4 weeks post-completion of radiotherapy. All HGG and biopsied DIPG patients were screened for RB protein presence by immunohistochemistry. Pharmacokinetics and survival data were analyzed.

RESULTS: Nineteen patients enrolled (median age: 8 years [range: 2-18]). Three patients enrolled at each of dose levels 1 and 2 without dose-limiting toxicities (DLTs). Thirteen patients enrolled at dose level 3, with one patient experiencing a DLT (grade 3 infection). One patient came off therapy prior to cycle 9 due to cardiac toxicity. The most common grade 3/4 toxicities were neutropenia (33%), leucopenia (17%), and lymphopenia (11%). Steady-state everolimus exposures in combination were 1.9±0.9-fold higher than single-agent administration. Median overall survival (OS) for 15 patients with DIPG was 13.9 months, with 12-, 24-, and 36-month OS of 53.3%, 38.9%, and 38.9%. Median event-free survival for four patients with HGG was 10.5 months. Among patients with tumor molecular profiling, two longer survivors (OS: 20, >37 months) had evidence of cell cycle upregulation with CDKN2A/B deletion and CDK4 overexpression identified. CONCLUSIONS: The combination of ribociclib and everolimus was well-tolerated post-radiotherapy in children with newly diagnosed DIPG and HGG, with a RP2D of ribociclib 170 mg/m2 days 1-21 and everolimus 1.5 mg/m2 days 1-28. Results will inform a molecularly-guided phase II study currently underway to evaluate efficacy.
EPEN-02. Adaptive Convergence of Methylomes Reveals Epigenetic Drivers and Boosters of Repeated Relapses in Patient-matched Childhood Ependymomas and Identifies Targets for Anti-Recurrence Therapies
Authors: Zhao Sibo , Li Jia , Zhang Huiyuan , Qi Lin , Du Yuchen , Kogiso Mari , Braun Frank K , Huang Yulun , Teo Wan-Yee , Baxter Patricia , Adesina Adekunle , Song Yongcheng , Sun Deqiang , Li Xiao-Nan
Published: June 2022

Ependymoma (EPN) is the third most common brain tumor in children and frequently recurs. Here, we report an integrated longitudinal analysis of epigenetic, genetic and tumorigenic changes in 30 patient-matched repeated relapses obtained from 10 pediatric patients to understand the mechanism of recurrences. Genome-wide DNA methylation analysis revealed stable molecular subtypes and convergent epigenetic reprogramming during serial relapses of the 5 RELA and 5 PFA EPNs that paralleled with elevated patient-derived orthotopic xenograft (PDOX) (13/27) formation in the late relapses. Differentially methylated CpGs (DMCs) preexisted in the primary tumors and persisted in the relapses (driver DMCs) were detected, ranging from 51 hypo-methylated in RELA to 148 hyper-methylated DMCs in PFA tumors; while newly acquired DMCs sustained in all the relapses but was absent in the primary tumors (booster DMCs) ranged from 38- 323 hyper-methylated DMCs in RELA and PFA EPNs, respectively. Integrated analysis of these DMC associated DNA methylation regions (DMRs) and RNAseq in both patient and PDOX tumors identified a small fraction of the differentially expressed genes (4.6±4.4% in RELA and 4.5±1.1% in PFA) as regulated by driver DMRs (e.g., up-regulated CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and booster DMRs (including the sole upregulated PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). Most these genes were novel to EPN relapses. Seven DMCs in RELA and 22 in PFA tumors were also identified as potential relapse predictors. Finally, integrating DNA methylation with histone modification identified LSD1 as a relapse driver gene. Combined treatment of a novel inhibitor SYC-836 with radiation significantly prolonged survival times in two PDOX models of recurrent PFA. This high-resolution epigenetic and genetic roadmap of EPN relapse and our 13 new PDOX models should significantly facilitate biological and preclinical studies of pediatric EPN recurrences.
EPEN-05. Adenosine receptor expression in paediatric ependymoma
Authors: Spanoudakis Chelsea , Chapman Rebecca , Paine Simon , Grundy Richard , Franks Hester , Ritzmann Timothy
Published: June 2022

PURPOSE: Paediatric ependymoma is associated with dismal outcomes. Whilst understanding of its underlying biology has advanced, there has been little progress in treatment and clinical outcomes. Acting through four G protein-coupled receptors (encoded by ADORA1, ADORA2A, ADORA2B and ADORA3), adenosine is a signalling molecule often present at high levels in tumours. Adenosine signalling can aid tumour proliferation and invasiveness via mechanisms including suppression of tumour-infiltrating immune cells. Adenosine receptors therefore represent a potential therapeutic target in paediatric ependymoma, however neither levels nor patterns of expression have been previously reported. We hypothesised that adenosine receptors would be expressed in paediatric ependymoma and that this expression would vary between molecular subgroups. METHODS: Three publicly available gene expression datasets were analysed for adenosine receptor expression using Kruskal-Wallis, Mann-Whitney U and chi-square tests. RNAscope assays for adenosine receptors and CD68 were then performed on ten full-face ependymoma FFPE sections from posterior fossa A (PFA1 and PFA2) tumours to understand patterns of expression within ependymomas with the highest levels of expression identified by the gene expression datasets. RESULTS: Statistically significant differences were identified between adenosine-related genes across ependymoma subgroups of differing anatomical origin (supratentorial ZFTA-positive versus posterior fossa A and B (PFA/PFB)), with median adenosine-related gene levels generally higher in the PFA subgroup. Particularly, ADORA1, 2A, 2B and 3 gene expression was higher in PFA tumours than other subgroups. Analysis of the ten cases demonstrated measurable expression of all four adenosine receptors by RNAscope and patterns in the distribution and relative levels of expression of the adenosine receptors across PFA1 and PFA2 tumours were described. CONCLUSION: Using two different techniques we demonstrated that adenosine receptors are expressed in paediatric ependymomas. There are significant differences in level of expression between tumour subgroups. Adenosine receptors therefore represent a potential therapeutic target which should be explored further.
EPEN-11. Phase 0/I Study of GM-CSF and Intrathecal Trastuzumab In Children With Recurrent Posterior Fossa Ependymoma
Authors: Norris Gregory , Widener Melissa , Amani Vladamir , Donson Andrew , Schissel Debra , Bruno Courtney , Mettetal Ashley , Ramirez Dominique , Gustafson Daniel , Hankinson Todd , Handler Michael , Macy Margaret , Foreman Nicholas , Dorris Kathleen
Published: June 2022

BACKGROUND: Posterior fossa ependymoma (PF EPN) is a pediatric central nervous system malignancy that has a poor outcome to standard therapeutic approaches. The majority of PF EPN have been shown to harbor increased HER2 expression, making it a logical therapeutic target. Trastuzumab is a monoclonal antibody that targets HER2, and sargramostim (GM-CSF) stimulates hematopoietic progenitor cell proliferation. The combination of trastuzumab and GM-CSF has been shown to trigger antibody-dependent cell cytotoxicity in-vitro in patient-derived PF EPN cell lines. METHODS: Children aged 1–21 years with relapsed PF EPN, no ventriculoperitoneal shunt, and no CSF obstruction are eligible for the Phase 0/I single-institution clinical trial at Children’s Hospital Colorado. Stratum 1 involves intrathecal (IT) trastuzumab and subcutaneous (subQ) GM-CSF prior to standard-of-care surgical resection. Stratum 2 involves a 3 + 3 phase I design with serial IT trastuzumab doses, each preceded by three days of GM-CSF, to establish the maximum tolerated dose for IT trastuzumab. RESULTS: Trastuzumab was detected in a sufficient number of tumors after presurgical IT delivery in Stratum 1 to open Stratum 2. Seven patients (3 female) have been enrolled in Stratum 2. Median age at enrollment is 8.1 years (range, 3–20 years). CSF pharmacokinetic analysis demonstrate detectable trastuzumab up to 14 days after IT doses. No dose-limiting toxicities or grade 3 or 4 adverse events have occurred. Four patients completed all planned study therapy and remain progression-free post-therapy (median, 23 months, range, 6-42 months). Three patients progressed on therapy (median, 4 cycles). Biologic correlative studies are in process. CONCLUSIONS: IT trastuzumab penetrates PF EPN tumor tissue and demonstrates an excellent safety profile. Stratum 2 remains open to accrual at Dose Level 2. IT trastuzumab+GM-CSF warrants consideration for a multi-institutional Phase II trial.
EPEN-12. Sunitinib induces apoptosis-mediated cytotoxicity in PFA ependymoma
Authors: O'Shaughnessy Bohdan , Donson Andrew , Amani Vladimir , Griesinger Andrea , Grimaldo Enrique , Harris Faith , Foreman Nicholas
Published: June 2022

Ependymoma (EPN) has been previously shown to be selectively targeted by three classes of FDA-approved chemotherapy drugs: fluorinated pyrimidines, retinoids, and a subset of small molecule receptor tyrosine kinase inhibitors (RTKIs). We have identified sunitinib malate as an RTKI with ependymoma selectivity, in addition to previously identified ependymoma-selective RTKIs axitinib, imatinib, and pazopanib. Sunitinib—which targets VEGFR, PDGFR and c-kit—elicited unexpectedly high levels of cytotoxic apoptosis (caspase 3/7 cleavage) in EPN PFA cell lines harboring high-risk chromosome 1q gain and 6q loss. In this in-vitro screen of 100+ FDA-approved chemotherapy drugs, sunitinib was the only RTKI in the top five most cytotoxic drugs, a group that included idarubicin HCl, topotecan HCl, daunorubicin HCl, and doxorubicin. Additionally, sunitinib exhibited comparable caspase 3/7 cleavage levels in normoxia and hypoxia, suggesting that this therapy would be as effective in treatment of tumors with potentially resistant hypoxic necrotic cores. The effect of sunitinib on EPN cellular proliferation was tracked visually using Incucyte cell imaging technology, demonstrating consistent dose-dependent inhibition of proliferation. Sunitinib achieved more acute upregulation of apoptosis than axitinib, an EPN-selective RTKI currently being studied in preclinical models. A prior phase II trial of sunitinib in pediatric EPN and high-grade glioma showed that treatment was well tolerated but with no clinical benefit as a monotherapy. However, given encouraging clinical results of combining sunitinib with radiation, our ongoing preclinical studies of sunitinib in EPN are being conducted in the context of radiation which is standard treatment for EPN. Sunitinib presents a promising treatment to an intractable pediatric brain tumor that exhibits high rates of relapse and morbidity in affected individuals.
EPEN-15. Radiotherapy with helium ions has the potential to improve both endocrine and neurocognitive outcome in pediatric patients with ependymoma
Authors: Wickert Ricarda , Adeberg Sebastian , Deng Maximilian , Tessonnier Thomas , Hoeltgen Line , Debus Jürgen , Herfarth Klaus , Harrabi Semi
Published: June 2022

BACKGROUND: Ependymoma are the third most frequent pediatric brain tumors. To prevent local recurrence, the resection site should be irradiated. Especially the treatment of pediatric patients requires precise dose application and optimal protection of organs at risk. Compared to photon radiation treatment, proton therapy often achieves better results regarding target coverage and organ-sparing. Due to their physical properties, helium ions can further reduce side effects, providing better protection of healthy tissue despite similar target coverage. PATIENTS AND METHODS: In our in-silico study, 15 pediatric patients (median age six years) with ependymoma located in the posterior cranial fossa were considered. All patients underwent adjuvant radiotherapeutic treatment with active scanned protons at Heidelberg Ion-Beam Therapy Center (HIT). Both helium ion and highly conformal IMRT plans were calculated to evaluate the potential dosimetric (and clinical) benefit of ion beam therapy compared to the current state of the art photon-based treatments. RESULTS: Target coverage was comparable in all three modalities (He, H+, Ph): homogeneity indices (HI) and inhomogeneity coefficients (IC) of HIHe=5.8±3.8, HIH+=7.5±4.0, HIPh=6.4±4.3, ICHe=0.2±0.1, ICH+=0.2±0.1, ICPh=0.2±0.1. As expected, the integral dose absorbed by the healthy brain tissue could be reduced significantly using helium ions versus IMRT (-45.3%±15.1%). Based on our preliminary results, even compared to active scanned protons – currently the most precise radiation technique worldwide – the relative dose reduction for critical neuronal structures using helium ions on average amounts to -27.2%±14.4%. CONCLUSION: Previous studies could clearly demonstrate that the dosimetric advantage of protons translates into a measurable clinical benefit for pediatric patients with brain tumors. Given the dose response relationship of critical organs at risk, the results of our in-silico study provide a strong rationale that the use of helium ions has the potential to even further reduce the risk for treatment related sequelae.
EPEN-16. Epithelial Progenitor Cell Abundance and Copy Number Variant Gains and Losses Impact the Biology of Recurrent Ependymoma
Authors: Norris Gregory , Fu Rui , Riemondy Kent , Willard Nicholas , Griesinger Andrea , Amani Vladamir , Grimaldi Enrique , Harris Faith , Gilani Ahmed , Hankinson Todd , Hesselberth Jay , Foreman Nicholas , Donson Andrew
Published: June 2022

Ependymoma (EPN) is a common pediatric brain tumor that is fatal in approximately 50% of cases. Posterior fossa A (PFA) EPN has the highest rate of recurrence and the worst prognosis of all EPN subtypes. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent ependymoma remains largely unknown, which hinders clinical advances. In this study, we use paired samples of primary and recurrent disease from the same patient to investigate the drivers of recurrence. DNA methylation studies reveal frequent copy number variants at recurrence that were not present at primary presentation. We report a frequent gain of chromosome 1q and loss of 6p at recurrence, which has not been previously reported and may be a driver of recurrent disease. We have previously shown that PFA EPN is comprised of 4 main neoplastic cell populations, two well-differentiated populations termed ciliated and transportive ependymal cells, a mesenchymal cell population, and an undifferentiated population. Using spatial transcriptomics (Visium) integrated with single-nuclei RNA-seq (Chromium), we discovered that a highly proliferative EPN progenitor population of epithelial lineage is significantly upregulated at recurrence which we hypothesize drives refractory disease. Accordingly, we found higher expression of EPN progenitor gene signatures in bulk RNA transcriptomes of primary tumors that later recurred compared to tumors that never recurred. Together, these findings highlight the biologic differences between primary and recurrent disease and add to our understanding of treatment resistance in childhood ependymoma.
EPEN-24. Biological markers of ependymoma in children and adolescents (BIOMECA): Systematic comparison of methods for the precise evaluation of biomarkers for ependymoma diagnosis and prognostication
Authors: Chapman Rebecca , Ghasemi David R , LeBlond Pierre , Witt Hendrik , Grill Jacques , Kilday John-Paul , Modena Piergiorgio , Varlet Pascale , Tauziede-Espariat Arnault , Haberler Christine , Buttarelli Francesca , Giangaspero Felice , Paine Simon M L , Jacques Thomas S , Scott Ian , Andreiuolo Felipe , Pietsch Torsten , Massimino Maura , Grundy Richard , Pajtler Kristian W , Ritzmann Timothy A
Published: June 2022

The identification and validation of prognostic and diagnostic biomarkers is a key element of The SIOP Ependymoma II trial, realised through the Biomarkers of Ependymoma in Children and Adolescents study (BIOMECA). BIOMECA aims to identify and validate biomarkers for prediction of outcome whilst enhancing stratification for the next generation of ependymoma trials. We outline our findings from the first 147 consecutive BIOMECA cases (posterior fossa, PF=111; supratentorial, ST=32; spinal, SP=4). We compared various methods for biomarker assessment, across six European laboratories to determine key analysis methods. Methods included: methylation-based classification (EPIC 850K DNA methylation array) (n=141); immunohistochemistry (IHC) for nuclear p65-RELA (n=32), H3K27me3 (n=115), and Tenascin-C (TNC) (n=147); copy number (CN) analysis by FISH, MLPA (1q, CDKN2A) (n=147), and MIP (molecular inversion probe) and DNA methylation array (1q, CDKN2A, 6q, 11q, 13q, 22q) (n=141); analysis of ZFTA- and YAP1-fusions by RT-PCR, sequencing, Nanostring assays and break-apart FISH (n=32). Using DNA methylation-based classification, 91% (n=101/111) of PF cases classified as PF ependymoma group A (PFA) and 69% (n=22/32) of ST cases as ST ependymoma, ZFTA fusion-positive (ZFTA). Most PFAs demonstrated inter-centre agreement for loss of H3K27me3, and were TNC positive, representing surrogate markers for PFA identification. Combinations of p65-RELA IHC, FISH analysis, and RNA-based methods were suitable to identify ZFTA- and YAP1- fused ST ependymomas. Predictive CN alterations were identified by high-resolution, quantitative MIP technology.The integration of histopathology assessment and molecular typing is now critical as the updated 2021 WHO CNS5 classification of ependymomas lists seven molecularly distinct entities. This study highlights the importance of evaluating different methods in a prospective trial cohort. Here, advanced molecular techniques represent powerful tools for the classification of ependymoma entities (DNA methylation array) and for the detection of CN alterations (MIP) and specific fusions, enabling the correct classification and identification of prognostic markers.
EPEN-25. A novel spontaneous model of ZFTA-RELA fusion ependymoma
Authors: Bell Sigourney , King Claire , Rahrmann Katherine Wickham , Jassim Amir , Taylor Jessica , Gilbertson Richard
Published: June 2022

Ependymomas driven by the ZFTA-RELA fusion account for >70% of all supratentorial ependymomas. These tumours are now recognised in the WHO classification of CNS tumours and have been associated with a poor prognosis. Seven ZFTA-RELA fusion variants have been described: around two thirds of cases are fusion 1. No spontaneous genetically modified mouse models (GEMMS) have been described and current models require invasive intracranial injection (of transduced cells or RCAS-TVA system). Here we describe the first spontaneous GEMM of ZFTA-RELA fusion-driven ependymoma. Nestin-Flx-STOP-Flx-ZFTA-RELA (Fusion 1) or E1alpha-Flx-STOP-Flx-ZFTA-RELA open reading frames were targeted together with luciferase to the Rosa-26 locus. Breeding these mice with Nestin-CreERT2 or Blbp-Cre lines that drive recombination in neural progenitor cells resulted in forebrain tumours that could be tracked with bioluminescence imaging from P20. Tumours displayed NF-kB and L1CAM expression and ZFTA-RELA protein was detected using a novel in-house antibody. Tumours display expression of a known ZFTA-RELA fusion ependymoma transcriptomic signature. ZFTA-RELA tumours can be grown as neurospheres and passaged as allografts in nude mice. We provide the first spontaneous GEMM of this important group of ependymomas. We are now characterising these tumours histologically and transcriptomically relative to the human disease and using these to understand the lineage origins of ependymoma and plan use of conventional and novel treatments.
EPEN-28. Oncogenic dependency of pediatric ependymomas on extracellular vesicle pathways
Authors: Maass Kendra K , Roosen Mieke M , Mueller Torsten , Senfter Daniel , Benzel Julia , Wedig Tatjana , Kalxsdorf Mathias , Krijgsveld Jeroen , Pfister Stefan M , Pajtler Kristian W
Published: June 2022

INTRODUCTION: The majority of pediatric ependymoma (EPN) comprise either supratentorial EPN characterized by ZFTA-fusions (ST-EPN-ZFTA) or posterior fossa group A EPN (PF-EPN-A), for both of which only limited therapeutic options are available. Because pediatric EPNs have a relatively low mutational burden, identification and characterization of tumor-associated pathways and molecular processes are of critical importance to reveal potential therapeutic targets. Data from previous transcriptional studies and a cross-species in vivo screen implied aberrant vesicular pathways in ST-EPN-ZFTA, prompting further investigation of their putative role in EPN pathogenesis. METHODS: We investigated EPN group-specific differences in extracellular vesicle (EV) biogenesis pathways in human EPN transcriptome and proteome datasets. In addition, we characterized isolated EPN EVs by mass spectrometry. EPN-specific EV cargo was further investigated by immunofluorescence staining and western blotting. This enhanced understanding of EPN vesicular signaling allowed for a pre-selection of inhibitors targeting specific EV biogenesis pathways. In vitro proliferation and invasion assays as well as in vivo treatment studies were performed on EPN model systems. RESULTS: Integration of multi-omic data from both EPN tissues and EPN-EV-associated proteome led to the identification of ST-EPN-ZFTA-specific EV populations. We could spatially map specific EV markers to the perivascular niche that primarily harbors undifferentiated ST-EPN-ZFTA cell populations. Targeting EV biogenesis pathways by inhibiting factors of the lipid metabolism reduced the abundance of released EVs resulting in altered growth behavior and decreased invasion of tumor cells in vitro. In vivo validation of EV release inhibitors in an orthotopic ST-EPN-ZFTA PDX model significantly reduced tumor growth and increased survival. OUTLOOK: In summary, we have leveraged ST-EPN-ZFTA-specific EV pathways as a potential therapeutic vulnerability. Further mechanistic investigations on EPN EV biogenesis, release, or uptake are expected to improve our understanding of the cross-talk between tumor cells and cells of the microenvironment and may lead to potential new therapeutic avenues.
EPEN-30. 5FU with Radiation Followed by Maintenance of 5FU and ATRA Significantly Improves Survival of 1q+/6q- PFA Ependymoma Xenograft Models
Authors: Grimaldo Enrique , Donson Andrew , Harris Faith , Amani Vladimir , Norris Gregory , Steiner Jenna , Serkova Natalie , Dorris Kathleen , Foreman Nicholas , Griesinger Andrea
Published: June 2022

In a screen of over 100 FDA approved drugs on PFA 1q+ EPN cells, 5-fluorouracil (5FU) and All-Trans-Retinoic Acid (ATRA) were identified as inhibitors of EPN cell line growth. We performed in-vitro cell growth assays combining increasing doses of radiation and 5FU and found a significant synergistic effect on cell growth and apoptosis in 1q+ PFA EPN cell lines. Further growth attenuation was seen when ATRA was added 48 hours following radiation and 5FU treatment. This led us to development of preclinical studies in the 1q+ PFA orthotopic xenograft models MAF-811_XF and MAF-928_XF. In the initial cohort, tumors were allowed to establish prior to treatment start confirmed by MRI. In both MAF-811 and MAF-928, chemotherapy improved survival compared to no treatment. As consistent with standard of care, radiation significantly improved survival (p=0.0016) but there was no added benefit to combining 5FU or 5FU+ATRA with radiation. A second cohort was treated using the same treatment approach, however radiation and 5FU were started with minimal to no visible tumors by MRI. Interestingly, we found a significant increase in survival between vehicle control and combination 5FU+ATRA (HR 5.121, 95% CI: 0.2506, 2.409, p=0.048) in MAF-811 mice. However, again with radiation, there was no significant change in survival with only a single cycle of 5FU+ATRA. This led to continued maintenance of 5FU+ATRA cycles of 6 weeks with 2 weeks off for 4 cycles post radiation in mice with minimal tumor. When 5FU with radiation is followed by 5FU+ATRA and is continued in mice with minimal disease, survival significantly improved when compared to radiation alone (HR 9.020, 95% CI: 1.933 to 42.09, p=0.007). These studies highlight the importance of chemotherapy in minimal disease and is the rationale for a Phase I/II study in relapsed PFA EPN and in upfront 1q+ PFA EPN.