bluuebunny/crossref_metadata_2025 huggingface dataset:
DIPG-16. Evaluation of myeloid component of DIPG microenvironment
Authors: Mishra Deepak , Rajendran Sakthi , Zhu Xiaoting , Nazzaro Matthew , Kumar Shiva Senthil , Mchugh Todd , Rajappa Prajwal , Drissi Rachid
Published: June 2022

Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable disease of the central nervous system in children with median overall survival of less than one year. In recent years, several immunotherapy strategies have emerged as an option to treat DIPG. However, the low mutational burden and rare infiltration of T lymphocytes, render these tumors immunologically “cold” and therefore pose challenges for general immunotherapy. The myeloid component was implicated in the immunosuppression in other solid tumors. Previous data have shown that DIPG tumors are enriched in macrophages, but their role in tumor growth and progression have not been elucidated. Specifically, it remains unclear whether the myeloid cells are recruited to the tumor microenvironment from the peripheral circulation. Here, we examined the recruitment of myeloid cell populations to the tumor microenvironment and further delineated their role in tumor progression in a syngeneic mouse model of DIPG. We showed that this DIPG mouse model displays an immune microenvironment similar to that of patients’ DIPGs. DIPG tumors harbored rare tumor infiltrating lymphocytes and are enriched in myeloid cells. To further characterize the phenotype and functions of these myeloid populations, we evaluated the changes in proportions of myeloid cell subsets using flow cytometry (CD11b, Ly6c, Ly6G, MHCII, F4/80, CD206, Arg1) in the bone marrow, peripheral blood, and in the tumor microenvironment during tumor progression. Also, we investigated the role of these myeloid cells in angiogenesis and immune suppression by performing histological and expression analyses of endothelial markers and chemokines (CD31, CD34, KDR, IL-10, IL-13, IL-4, CCL2, CCL5). Furthermore, decitabine (DNA methyltransferase inhibitor) treated tumors showed a decrease in myeloid population associated with a reduction in tumor growth, suggesting an important role of myeloid populations in tumor growth and progression.
DIPG-26. Targeted Protein Degradation of LSD1 synergizes with HDAC inhibitors in Diffuse Intrinsic Pontine Glioma
Authors: Groves Andrew , Poetschke Rebecca , Mire Hafsa , Panditharatna Eshini , Guzman Maria Tarazona , Qi Jun , Filbin Mariella
Published: June 2022

Diffuse intrinsic pontine glioma (DIPG) remains one of the most lethal brain tumors in all of childhood with no effective treatments besides radiation, which only extends survival a few months. Against this backdrop, our lab recently executed a focused CRISPR negative selection screen in DIPG cell lines after treatment with the histone deacetylase (HDAC) inhibitor panobinostat and discovered a strong co-dependence with the histone demethylase LSD1. To further explore the therapeutic potential of this synergistic interaction, we tested a drug library of HDAC- and LSD1- targeting drugs with the goal of identifying a combination with optimal synergy and blood brain barrier (BBB) penetration suitable for clinical translation. We were surprised to find that traditional catalytic LSD1 inhibitors had minimal effect in isolation and did not seem to synergize with HDAC inhibitors, while a recently described CoREST/LSD1 degrader named UM171 phenocopied the effects seen in our CRISPR screen. Degraders are a class of compounds that recruit an E3 ubiquitin ligase to a protein-of-interest and cause target ubiquitination and proteasomal degradation. Given our unexpected finding, we hypothesized that UM171 induces synergy with HDAC inhibitors through elimination of a scaffolding function of LSD1. To prove this, we knocked out LSD1 using CRISPR/Cas9 and subsequently treated with a panel of HDAC inhibitors, which showed a signification sensitization of DIPG cells to HDAC inhibitors compared to standard controls. We also confirmed that UM171 interacts with the CoREST complex (members include LSD1, RCOR1, HDAC1/2) by performing streptavidin bead pull down with a newly synthesized biotin-conjugated UM171 probe. In summary, our results show that targeting LSD1 for degradation in combination with HDAC inhibition is a synergistic strategy in DIPG worthy of further translational study.
DIPG-36. The brain-gut-microbiota axis to predict outcome in pediatric diffuse intrinsic pontine glioma
Authors: De Cecco Loris , Biassoni Veronica , Schiavello Elisabetta , Carenzo Andrea , Iannò Maria Federica , Licata Armando , Marra Manuela , Carollo Maria , Boschetti Luna , Massimino Maura
Published: June 2022

OBJECTIVE: Diffuse intrinsic pontine glioma (DIPG) is a rare childhood brain tumour with poor prognosis. Radiotherapy (RT) remains the only palliative intervention. In this scenario, it is essential to investigate tumor and patient microinvironment. Microbiota plays a critical role in human health and a functional link between the central nervous system and gut microbiota has been reported: the microbiota-gut-brain axis. With these premises, we have investigated the gut microbiota in DIPG. METHODS: A cohort of 18 patients was enrolled and we collected stool specimens at diagnosis (pre-RT) and after radiotherapy. Microbiota content was analysed through 16S rRNA sequencing on IONS5xl; base calling and demultiplexing were performed by Torrent Suite (ThermoFisher). Association to progression free survival (PFS) and overall survival (OS) were assessed by cox-regression univariate analyses and differential abundance (DA) analyses identified differences following RT. RESULTS: The Firmicutes/Bacteroidetes (F/B) ratio in pre-RT specimens has a median of 0.757 (range= 0.243-1.19). Having PFS as endpoint and at family classification level, an increased risk of disease progression was disclosed for Flavobacteriaceae and Bacillales with HR of 1.57 (p=0.00913) and 1.57 (p=0.215), respectively. The anaerobic bacteria Synergistaceae is found related to a decreased risk of progression with HR 0.662 (p=0.00718). These findings were also confirmed with OS as endpoint. DA analyses pointed out a number of families belonging to Firmicutes and Bacteroidetes phyla highly differentiated (log2|fold-change|>2) between pre- and post-RT. CONCLUSIONS: This study represents the first report of the microbiota-gut-brain axis role in DIPG patients. The F/B ratio, a parameter of normal intestinal homeostasis, show the presence of inflammatory patterns and some family members are associated to increased disease progression. These preliminary findings needs further validations. However, 16S microbiota has the potential ability to stratify patients and probiotic administration could restore a normal F/B ratio.
DIPG-44. H3K27-altered diffuse midline gliomas with secondary driver molecular alterations
Authors: Gestrich Catherine , Grieco Kristina , Lidov Hart , Ligon Keith , Santagata Sandro , Yeo Kee Kiat , Alexandrescu Sanda , Meredith David
Published: June 2022

INTRODUCTION: Large-scale sequencing led to the identification of driver molecular alterations such as FGFR1 and BRAF in occasional diffuse midline gliomas (DMGs) H3K27-altered, but their significance is not completely explored. We evaluated these associations in our institutional cohorts. MATERIALS AND METHODS: We searched our sequencing data base (2013-2020) for H3K27M-mutant gliomas and analyzed the co-occurring genetic alterations. The demographics, clinical information, and pathology were reviewed. Copy number profiles were evaluated using BioDiscovery's Nexus Copy Number software package. Oncoplots and Kaplan-Meier survival curves were generated with the maftools R package. RESULTS: We identified 77 patients (age range 2-68, median 26). The diagnosis was DMG (n=55), anaplastic astrocytoma/glioblastoma (n=19), low-grade glioma (n=1), low-grade glioneuronal tumor (n=1), and ganglioglioma (n=1). Recurrent alterations were seen in TP53 (n=42), ATRX (n=17), NF1 (n=15), PDGFRA (n = 4). Five cases had BRAF V600E (1 ganglioglioma; 4 DMG); twelve had FGFR1 mutations (9 DMG; 3 anaplastic astrocytoma/glioblastoma). The most common location in the BRAF group was the brainstem and in the FGFR1 was the thalamus. Survival ranged from 0 to 97 months, median 12.9 months (28.8 months for FGFR1 and 22.8 for the BRAF V600E). This was not significantly different from OS reported in the literature for DMG.The BRAF V600E ganglioglioma patient is alive 37 months after diagnosis. CONCLUSION: There was no significant difference in outcomes for patients with secondary molecular drivers when compared with conventional H3K27M DMG. The outcome of the BRAF V600E tumors seemed to correlate with the histology. These findings and the possibility of targeted therapy argue for comprehensive sequencing of H3K27-altered gliomas.
DIPG-52. Activators of the integrated stress response synergize to kill DIPG
Authors: Novak Orlandi , Arnold Antje , Eberhart Charles , Raabe Eric
Published: June 2022

DIPG has elevated baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that allows cells to tolerate various forms of stress. Increased expression of activating transcription factor 4 (ATF4) indicates activation of the ISR. Intermediate levels of ATF4 protect cells from stress, while sustained high levels result in cell death. The imipridone drug ONC201 binds to and activates the mitochondrial protease ClpP, leading to increased mitochondrial stress and persistent ATF4 activation. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003, ONC201, and fenretinide would synergize to kill DIPG. Sal003 inhibits dephosphorylation of ATF4 upstream regulator, eIF2α. The retinoic acid derivative fenretinide induces ATF4, increases reactive oxygen species, and has clinical activity in pediatric patients with neuroblastoma. After determining the IC25 of Sal003, fenretinide, and ONC201, we treated patient-derived DIPG cell lines with low micromolar doses. The combination of Sal003 and ONC201 significantly increased apoptosis as measured by CC3 immunofluorescence in comparison to DMSO (p<0.0001, ANOVA). Combination therapy also significantly increased CC3 positivity compared to single treatment. Western blots for cleaved PARP expression detected induction of apoptosis in DIPG treated with both Sal003 and ONC201 over DMSO and monotherapy treated cells. In some cell lines, the combination increased ATF4 expression. Since Sal003 is not yet available for clinical testing in humans, we treated DIPG cells with ONC201 and fenretinide. CC3 immunofluorescence indicated synergistically elevated apoptosis in the combination of ONC201 and fenretinide vs. DMSO (p<0.0001, ANOVA). Western blots showed increased cleaved PARP, ATF4, and CHOP expression in DIPG treated with ONC201 and fenretinide. We are currently testing the efficacy of this combination in orthotopic DIPG xenografts. Our results suggest the combination of ONC201 with fenretinide could potentially serve as a therapy for DIPG.
DIPG-59. Blood Brain Barrier (BBB) modulation using Cadherin peptides in the treatment of Diffuse Midline Glioma of the Pons (DMG-P)
Authors: Line Stacey , Sajesh Babu Velayuthan , Schwinghamer Kelly , Yathindranath Vinith , Tsoli Maria , Ziegler David , Siahaan Teruna , Miller Donald , Vanan Magimairajan Issai
Published: June 2022

BACKGROUND: Diffuse midline glioma of the Pons (DMG-P) is the most common brainstem tumor of childhood and is universally fatal. The BBB is frequently intact in DIPG and restricts entry of systemically administered conventional and biological therapies. We have synthesized and validated synthetic cadherin peptides (HAV6, HAVN1) that cause transient opening of the BBB by preventing the dimerization of the extracellular membrane bound VE-Cadherin in the vascular endothelium. METHODS: We used DMG-P tumor conditioned media (TCM) and co-cultures in two in-vitro model systems (trans well inserts, Blood Brain Tumor Barrier microfluidics model (BBTB-DMG-P microchip)-using Human Brain microvascular endothelial cells (HBMVECs) / SF8628 (DMG-P PDX) and validated the in-vitro findings in a DMG-P orthotopic murine model using RA055. The permeability of different molecular weight markers [(Gd-DTPA, IRDye 800CW PEG, rhodamine 800 (R800)] were examined in presence / absence of HAV6 / HAVN1 peptides. RESULTS: The BBTB integrity is maintained in-vitro as evidenced by decreased permeability of all the agents across the barriers in the TCM / co-culture models. Addition of HAV6/ HAVN1 peptides in-vitro increased the permeability of all the agents across the barriers. The permeability of R800 increased in the presence of Elacridar (P-gp inhibitor). The BBB was intact in the DMG-P murine model, as evidenced by minimal Gd-DTPA contrast enhancement in T1 weighted MRI images. Administration of HAV6 / HAVN1 peptides resulted in Gd-DTPA contrast enhancement of the tumor at 5- and 10-minutes post injection. CONCLUSION: Our data confirms that the BBTB is intact in DMG-P and can be transiently modulated using synthetic cadherin peptides. Transient BBTB opening using synthetic peptides will help BBB non-penetrant chemotherapy drugs like Vincristine/Mitoxantrone and biological therapies to reach intra-tumoral therapeutic levels and improve therapeutic efficacy.
DDEL-05. Intraventricular therapy with topotecan is feasible and safe: Experience in 50 pediatric patients with various malignant brain tumors
Authors: Peyrl Andreas , Stepien Natalia , Mayr Lisa , Azizi Amedeo , Gojo Johannes , Baumgartner Alicia , Hedrich Cora , Lippolis Maria Aliotti , Slavc Irene
Published: June 2022

BACKGROUND: Malignant brain tumors carry a high risk for leptomeningeal dissemination, but the CSF compartment is often not affected by systemic therapy. Intraventricular therapy via an Ommaya reservoir is one possibility to increase the cytotoxic drug concentration in the CSF. Unfortunately, the number of drugs that can be administered directly into the CSF is limited. We report on our experience with topotecan administered via an Ommaya reservoir. PATIENTS AND METHODS: Between 2015 and 2021, 50 patients aged 1 to 22 years (mean and median both 8 years) with various malignant brain tumors received intraventricular topotecan via an Ommaya reservoir. Topotecan was administered at 0.4mg twice a week (>1 and <2 years 0.25mg, >2 and <3 years 0.32mg). RESULTS: In total, 1168 doses of topotecan (1-87/patient, mean 23, median 18) were administered at our department. Treatment was given over a period of 0-65 months (mean and median 33 months). Intraventricular treatment with topotecan was generally well tolerated. Two patients reported side effects. One boy with multiple recurrences of an ependymoma in the posterior fossa showed increased tremor after intraventricular administration of topotecan, another girl with recurrent medulloblastoma reported fatigue. CONCLUSION: Intraventricular therapy with topotecan is feasible and generally well tolerated. Topotecan can be an important addition for patients with recurrent malignant brain tumors to increase cytotoxic drug concentrations in CSF.
EPCT-01. Pediatric Brain Tumor Consortium (PBTC)-055: A phase I study of trametinib and hydroxychloroquine (HCQ) for BRAF-fusion or Neurofibromatosis type-1 (NF1)-associated pediatric gliomas
Authors: Hoffman Lindsey M , Levy Jean Mulcahy , Kilburn Lindsay , Billups Catherine , Stokes Vanetria , McCourt Emily , Poussaint Tina Young , Campagne Olivia , Partap Sonia , Dorris Kathleen , Sait Sameer Farouk , Robinson Giles , Baxter Patricia , Stewart Clinton F , Fangusaro Jason , Onar-Thomas Arzu , Dunkel Ira
Published: June 2022

INTRODUCTION: Autophagy is a highly conserved process by which intracellular components are degraded and recycled promoting cell survival. Preclinically, autophagy has been implicated as a resistance mechanism in BRAF-mutant glioma cells treated with MAPK-pathway inhibitors. HCQ, an oral autophagy inhibitor, has been evaluated preclinically and clinically to overcome resistance. METHODS: PBTC-055 (NCT04201457) is a phase I/II trial of HCQ combined with trametinib (BRAF-fusion or NF1-associated gliomas) or trametinib and dabrafenib (BRAFV600E gliomas) in patients < 30 years with progressive glioma. Prior treatment with RAF and/or MEK inhibitor with sub-optimal response (no response or response followed by progression on therapy) was required. Here, we present phase I data combining trametinib with HCQ utilizing a rolling-6 design. HCQ was administered at escalating dose levels (8, 15, or 20 mg/kg/day divided BID) in combination with standard pediatric trametinib dosing. All patients received prior MEK inhibitor therapy; 5/18 (28%) exhibited no response and 13/18 (72%) progressed on active therapy. RESULTS: Eighteen eligible/evaluable subjects were enrolled. Median age was 9.6 years (2.5-20.4 years); 10 were male. There were 2 dose-limiting toxicities (both grade 3 rash one each at DL1 and DL3). The highest dose level of HCQ (20 mg/kg/day divided BID) was declared the RP2D. Grade 3 adverse events possibly related to therapy included skin infection, rash, cardiac ejection fraction decrease, weight loss, and anorexia. There were no grade 4 or 5 attributable toxicities. Preliminarily, combination pharmacokinetic assessment revealed similar metabolism of trametinib to that reported as a single agent; HCQ demonstrated more rapid clearance compared to adults. Pharmacodynamic assessments are ongoing. CONCLUSIONS: The combination of trametinib and HCQ is safe with a RP2D of HCQ of 20 mg/kg/day divided BID. Currently, subjects are enrolling on the phase II portion evaluating the efficacy of this novel combination.
EPCT-03. Working together to accelerate the preclinical to clinical translation of drug delivery systems for children’s brain tumours
Authors: Campbell Emma , Aquilina Kristian , Dandapani Madhumita , Walker David , Rahman Ruman
Published: June 2022

Children's brain tumours are the biggest cancer killer in children and young adults. Several techniques, such as intra-cerebrospinal fluid chemotherapy, ultrasound-mediated blood-brain barrier disruption, convection enhanced delivery, polymer delivery systems, electric field therapy, and intra-arterial and intra-nasal chemotherapy, have the potential to transform the treatment of brain tumours in children. However, there have been very few clinical trials to evaluate these. In 2021, the CBTDDC (Children’s Brain Tumour Drug Delivery Consortium) and the ITCC (Innovative Therapies for Children with Cancer) brain tumour group established a Clinical Trials Working Group comprising international researchers and clinicians to address this issue. This partnership highlighted the main challenges in preclinical to clinical translation of paediatric CNS drug delivery as: (1) a lack of specific funding for prototype development and/or scale-up for clinical trials; (2) difficulties in navigating the regulatory landscape; (3) lack of accurate preclinical models; and (4) increased need for multi-centric working. In response to this, we ran a hybrid workshop in November 2021 on ‘Clinical Trial Readiness for CNS Drug Delivery’. At this workshop, around 50 delegates (comprising clinicians, researchers, trial regulatory experts, policy makers, and representatives from funding organisations, brain tumour charities and industry) came together to discuss issues around funding, preclinical models and regulatory processes. We have established speciality-specific working groups to build on the workshop discussions, with the aim of producing recommendations around the use of preclinical models and drug delivery techniques according to brain tumour type. We have also used the workshop presentations and discussions to create a ‘Roadmap’ document for preclinical to clinical translation, which will be freely shared with the neuro-oncology research community. We continue to liaise with funders and regulatory bodies to address the changes that are needed in these areas. If you would like to join our network, contact: cbtddc@nottingham.ac.uk
EPCT-05. Phase Ib study of unesbulin (PTC596) in children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) and high-grade glioma (HGG): A report from the COllaborative Network for NEuro-Oncology Clinical Trials (CONNECT)
Authors: Lazow Margot A , Baxter Patricia , Stanek Joseph , Lane Adam , Rodriguez Diana P , Kumar Shiva Senthil , Leach James L , Mikael Leonie , Fuller Christine , Boué Daniel R , Pierson Christopher R , Thomas Diana , Breneman John , Palmer Joshua , Li Xiao-Nan , Salloum Ralph , Ashley David , de Blank Peter , Hwang Eugene , Leary Sarah E S , Plant Ashley , Crabtree Dorothy , Wahba Mona , Weetall Marla , Baird John , Leonard Jeffrey , Stewart Clinton F , Mardis Elaine , Fouladi Maryam , Drissi Rachid
Published: June 2022

BACKGROUND: The B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) protein, implicated in self-renewal and DNA-damage signaling, is highly expressed in DIPG and HGG. Preclinically, BMI-1 modulation by unesbulin (PTC596 [which mediates hyperphosphorylation and subsequent degradation of BMI-1]) leads to DIPG/HGG cell proliferation blockade, mitotic abnormalities, and tumor cell sensitization to radiation-induced DNA damage. METHODS: This phase Ib study sought to determine the maximally tolerated dose/ recommended phase 2 dose (RP2D) of unesbulin administered concurrently with radiotherapy and adjuvantly in children with newly diagnosed DIPG or HGG. Patients were enrolled according to a Rolling-6 design and received oral unesbulin twice weekly during radiotherapy and as maintenance therapy. RESULTS: Twenty-seven patients enrolled (median age: 8.5 years [range: 2-18]), including 18 patients with DIPG and nine patients with HGG. Unesbulin was administered in capsule formulation in the first nine patients, then tablet formulation for subsequent patients. Within the capsule formulation group, three dose-limiting toxicities (DLTs) were observed in two patients on dose level 2 (grade 4 neutropenia). Within the tablet formulation group, four DLTs were experienced by three patients on dose level 2 (grade 3 ALT elevation, grade 3 dehydration/vomiting, grade 3 decreased ejection fraction, grade 4 neutropenia). Dose level 1 was declared the RP2D, and six additional patients enrolled in the expansion cohort at this dose without DLTs. Most common drug-related grade 3/4 toxicities were neutropenia (48%), leucopenia (35%), and elevated ALT (26%). Similar pharmacokinetic profiles were observed for capsule and tablet formulations, consistent with adult data. Survival outcomes and genomics results will be shared at time of presentation. CONCLUSIONS: The RP2D of unesbulin in children newly diagnosed with DIPG or HGG is 200mg/m2 twice weekly, concurrent with and following radiotherapy. The recently opened surgical cohort will assess intratumoral pharmacokinetics and inhibition of tumor BMI-1 signaling, with results forthcoming.
EPEN-01. Response assessment in pediatric intracranial ependymoma: recommendations from the Response Assessment in Pediatric Neuro-Oncology (RAPNO) working group
Authors: Lindsay Holly , Massimino Maura , Avula Shivaram , Stivaros Stavros , Grundy Richard , Metrock Katie , Bhatia Aashim , Fernández-Teijeiro Ana , Chiapparini Luisa , Bennett Jeffrey , Wright Karen , Hoffman Lindsey , Smith Amy , Pajtler Kristian , Poussaint Tina Young , Warren Katherine , Foreman Nicholas , Mirsky David
Published: June 2022

INTRODUCTION: Ependymomas remain a major cause of cancer-related death in childhood and adolescence, with recurrence occurring in up to 50% of patients. Despite exciting molecular advances in understanding ependymoma tumorigenesis and recurrence, MRI remains the mainstay for assessing objective response to therapy and duration of disease stability. Standardized response assessment criteria for clinical trials studying pediatric intracranial ependymoma are critically needed in order to accurately compare results between studies. METHODS: To generate these standardized response criteria in pediatric intracranial ependymoma, a multidisciplinary team of pediatric neuro-oncologists, neuroradiologists, neurosurgeons, radiation oncologists, and molecular biologists formed the Response Assessment in Pediatric Neuro-Oncology (RAPNO) working group. The expert members reviewed relevant published literature, assessed current clinical practices, and engaged in iterative discussions to provide consensus recommendations for objective response assessment in pediatric intracranial ependymoma for use in prospective clinical trials. RECOMMENDATIONS AND CONCLUSIONS: The primary sequences for detecting and measuring disease and assessing radiologic response to therapy should be the contrast-enhanced T1-weighted sequence or T2-weighted sequence (T2 or T2-FLAIR) depending on which sequence the tumor is best visualized. When metastatic disease is present, only the three largest lesions will be followed in addition to any residual disease at the primary tumor focus. Importantly, the RAPNO working group notes that radiologic response to therapy is of limited value in clinical trials of patients with ependymoma, since most patients enroll on clinical trials with either no evidence of disease or only minimal disease. In recurrent or progressive disease that cannot be resected, true radiologic disease response to therapy is less clinically meaningful as a study endpoint than event-free and/or overall survival (representing prolonged stable disease) but may provide a signal of efficacy worthy of future exploration in patients with complete to near complete resections.
EPEN-03. USP7 is an interaction partner of EZHIP and potential druggable target in PFA ependymomas
Authors: Jenseit Anne , Camgöz Aylin , Mauermann Monika , Pfister Stefan , Kool Marcel
Published: June 2022

Ependymomas (EPN) arise in the supratentorial brain (ST-EPN), posterior fossa (PF-EPN), or the spinal cord (SP-EPN), in children and adults. Molecular profiling has identified distinct subgroups in each location. Among the three molecular subgroups of PF-EPN, PFAs are characterized by young median age at diagnosis, an overall balanced genome and bad clinical outcome (56 % 10-year OS). Therapy involves tumor resection and radiotherapy, but the role of chemo- or targeted therapies remains to be defined. Recently, we and others identified enhancer of zeste inhibiting protein (EZHIP) as potential driver of PFAs. By inhibiting EZH2, catalytic subunit of the polycomb repressive complex 2 (PRC2), EZHIP prevents the distribution of the epigenetic repressor mark H3K27me3. However, since EZHIP does not possess any known enzymatic functions, it does not seem to be the druggable target urgently searched for in PFA. We therefore focused on essential and potentially druggable interactions. Here, we present ubiquitin-specific protease 7 (USP7), a known cancer regulator with multiple inhibitors available, which has been shown to interact with EZHIP in non-PFA cells. We confirmed this interaction in PFA cells by co-immunoprecipitation and mass spectrometry, and showed that the EZHIP-USP7 interaction is independent of EZH2, a separate interactor of both. Functionally, we show that USP7 de-ubiquitinates EZHIP, preventing its degradation and thus stabilizing it. As EZHIP is essential for PFA cell survival, we aim to target EZHIP indirectly by affecting its stability regulation via USP7. Knockdown experiments showed a susceptibility of PFA cells to a USP7 loss, focusing on proliferation, apoptosis, and expressional changes induced by altered USP7 levels. Moreover, USP7 inhibitors highly affected the survival of different PFA cell lines at a low micromolar IC50 in vitro and in vivo treatments of PFA patient-derived xenografts with USP7 inhibitors are ongoing, hopefully helping to improve targeted therapies for PFA ependymoma patients.
EPEN-06. Comprehensive profiling of myxopapillary ependymomas identifies a distinct molecular subtype with relapsing disease
Authors: Bockmayr Michael , Harnisch Kim , Pohl Lara , Schweizer Leonille , Mohme Theresa , Körner Meik , Alawi Malik , Suwala Abigail , Dorostkar Mario , Monoranu Camelia , Hasselblatt Martin , Wefers Annika , Capper David , Hench Jürgen , Frank Stephan , Richardson Timothy , Tran Ivy , Liu Elisa , Snuderl Matija , Engertsberger Lara , Benesch Martin , von Deimling Andreas , Obrecht Denise , Mynarek Martin , Rutkowski Stefan , Glatzel Markus , Neumann Julia , Schüller Ulrich
Published: June 2022

Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients’ clinical course are unknown. We assembled a cohort of 185 tumors classified as MPE based on DNA methylation from pediatric, adolescent, and adult patients. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPEB, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. Tumors in the methylation class MPE were histologically diagnosed as WHO grade I (59%), WHO grade II (37%), or WHO grade III tumors (4%). 75/77 analyzed tumors expressed HOXB13, which is a diagnostic feature not detected in other spinal ependymal tumors. Based on DNA methylation, our series split into two subtypes. MPE-A occurred in younger patients (median age 27 vs. 45 years, p=7.3e-05). They were enriched with WHO grade I tumors and associated with papillary morphology and MGMT promoter hypermethylation (all p<0.001). MPE-B included most tumors initially diagnosed as WHO grade II and cases with tanycytic morphology. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. 15/30 MPE-A could not be totally resected compared to 1/58 MPE-B (p=6.3e-08), and progression-free survival was significantly better for MPE-B (p=3.4e-06, 10-year relapse rate 33% vs. 85%). We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.
EPEN-29. Spatial transcriptomic analysis of ependymoma implicates unresolved wound healing as a driver of tumor progression
Authors: Donson Andrew , Fu Rui , Norris gregory , Willard Nicholas , Griesinger Andrea , Riemondy Kent , Amani Vladimir , Grimaldo Enrique , Harris Faith , Hankinson Todd , Mitra Siddhartha , Ritzmann timothy , Grundy Richard , Foreman Nicholas
Published: June 2022

Ependymoma is a childhood brain tumor that remains incurable in approximately 50 percent of cases, most commonly in posterior fossa subgroup A (PFA). Uncovering how heterogeneous cell types within the tumor microenvironment (TME) interact is crucial to a complete understanding of PFA disease progression. The underlying cellular components of the PFA TME have been revealed by single-cell RNAseq (scRNAseq), identifying divergent epithelial differentiation and epithelial-mesenchymal transition (EMT) lineages. Here, we utilize spatial transcriptomics (Visium) of 14 PFA samples, integrated with scRNAseq, to chart neoplastic and immune cell architecture, with a higher resolution of cellular heterogeneity than scRNAseq alone. At a gross level, all PFA were primarily comprised of neoplastic epithelial and mesenchymal transcriptomic spatial zones, each containing a diversity of hierarchical cellular stages. In all samples we revealed spatially and transcriptomically-distinct mesenchymal zone-associated subclusters, including a quiescent undifferentiated progenitor-like subpopulation and clusters with characteristics of early and late stage EMT. Two early stage EMT clusters were distinguished by signatures of either myeloid cell interaction or hypoxia, and both were demonstrated to be EMT-initiating processes in in-vitro PFA experimental models. Myeloid cell interaction is the predominant initiating stage of EMT in PFA, occurring in zones that are spatially distinct from hypoxia induced EMT. Other mesenchymal clusters represent later EMT stages characterized by wound repair and tissue remodeling. Increased proliferation was a general characteristic of epithelial zone clusters, which included a second undifferentiated progenitor-like population that showed a particularly high mitotic rate and was associated with histologically hypercellular areas. Given the biological parallels with normal wound healing, we propose that mesenchymal and epithelial zones interact to create a cycle of persistent tissue damage response and mitogenic re-epithelialization signals. Unresolved wound repair is therefore a potential driver of PFA progression, a new concept that could provide novel targets for effective therapeutic intervention.
EPID-05. A novel, clinically-relevant classification of pediatric CNS tumors for cancer registries using a clustering analysis
Authors: Moreira Daniel , Chen Yichen , Qaddoumi Ibrahim , Cioffi Gino , Waite Kristin , Ostrom Quinn , Kruchko Carol , Barnholtz-Sloan Jill , Devidas Meenakshi , Bhakta Nickhill
Published: June 2022

To accurately evaluate the burden of pediatric central nervous system (CNS) tumors, estimate resources for cancer control, and monitor outcomes, a classification system that segregates tumors into clinically relevant groups is essential. The current classification of CNS tumors included in the third revision of the International Childhood Cancer Classification does not identify key clinical groups, such as low- and high-grade gliomas. To address this need, a novel classification was embarked upon using ICD-O-3 codes, CBTRUS grouping, incidence, survival, and treatment modalities as inputs. For each ICD-O-3 code with >50 new cases/year in CBTRUS from 2000 to 2016, 2 clinicians reached consensus defining the efficacy of three treatment modalities: surgical resection, radiotherapy, and chemotherapy. Then, patient level 5-year overall survival (OS) times were simulated based on total incidence and 5-year OS for each code. Subsequently, 5 factors were included as potential classifiers: tumor behavior, CBTRUS sub-group, and efficacy of the three treatment modalities. A “survival tree” was developed by using partitioning. Starting with the patient cohort (root), univariate cox proportional hazards model was used to identify statistically significant (P < 0.05) factors. The factors with the largest hazard ratio were selected manually to create child nodes. Within each child node, the partitioning process was repeated on remaining factors until no statistically significant factor remained. This clustering yielded 4 main groups (low-, intermediate-, high-, and very high-risk tumors) and 11 subgroups, including “embryonal tumors” and “low-risk glial and glioneuronal tumors”. Further validation of the classification will be sought through a structured consensus process using multidisciplinary experts. This systematic method to develop a classification for pediatric CNS tumors will allow for more relevant estimations of outcomes and better estimation of resource utilization. Furthermore, this strategy could be replicated for other disease groups.
ETMR-02. Overexpression of Lin28A in neural progenitor cells in vivo does not lead to brain tumor formation but results in reduced spine density
Authors: Middelkamp Maximilian , Ruck Lisa , Krisp Christoph , Sumislawski Piotr , Mohammadi Behnam , Dottermusch Matthias , Meister Valerie , Küster Lukas , Schlüter Hartmut , Windhorst Sabine , Neumann Julia E
Published: June 2022

The RNA binding protein LIN28A is a stem- and progenitor marker and one of the factors necessary to induce pluripotent stem cells. An overexpression of LIN28A has been identified in malignant brain tumors called embryonal tumors with multilayered rosettes (ETMR) but its specific role during brain development remains largely unknown. Radial glia cells of the ventricular zone (VZ) are proposed as a cell of origin for ETMR. We asked whether an overexpression of LIN28A in such cells might affect brain development or result in the formation of brain tumors. Constitutive overexpression of LIN28A in hGFAP-cre::lsl-Lin28A (GL) mice led to a transient increase of proliferation in the cortical VZ at embryonic stages but no postnatal brain tumor formation. Postnatally, GL mice displayed a pyramidal cell layer dispersion of the hippocampus and altered spine and dendrite morphology, including reduced dendritic spine densities in the hippocampus and cortex. GL mice displayed hyperkinetic activity and differential quantitative MS-based proteomics revealed altered time dependent molecular functions regarding mRNA processing and spine morphogenesis. Phosphoproteomic analyses indicated a downregulation of mTOR pathway modulated proteins such as Map1b being involved in microtubule dynamics within a crosstalk of Gsk3b/Rho-Rac/Map1b signaling. In conclusion, we show that Lin28A overexpression transiently increases proliferation of neural precursor cells but it is not sufficient to drive brain tumors in vivo. In contrast, Lin28A impacts on protein abundancy patterns related to spine morphogenesis and phosphorylation levels of proteins involved in microtubule dynamics, resulting in decreased spine densities of neurons in the hippocampus and cortex as well as in altered behavior. Our work provides new insights into the role of LIN28A for neuronal morphogenesis and development and may reveal future targets for treatment of ETMR patients.
GCT-03. MonoGerm: A proposed phase II trial of carboplatin or vinblastine monotherapy induction prior to radiotherapy for intracranial germinoma
Authors: Murray Matthew , Apps John , Lawson Anna , Kirton Laura , Peet Andrew , Arvanitis Theodoros , Fern Lorna , Mitra Dipayan , Coleman Nicholas , Ajithkumar Thankamma , Bison Brigitte , Veal Gareth , Stark Daniel , Morana Giovanni , Nicholson James , Billingham Lucinda
Published: June 2022

BACKGROUND: Intracranial germinoma is chemosensitive but radiotherapy (RT) is needed for cure. In localised disease, three-drug standard-of-care (SOC) inpatient chemotherapy is used to reduce RT fields/dose. Concomitant diabetes insipidus is common, making chemotherapy delivery challenging. Small studies have demonstrated benefits from single-agent carboplatin or vinblastine in germinoma as an alternative to SOC. However, this needs prospective evaluation in a clinical trial. METHODS: We developed a trial, with a patient-public-involvement workstream, primarily evaluating whether single-agent chemotherapy (carboplatin or vinblastine) is non-inferior to SOC for inducing radiological complete response (CR) in localised disease, and is associated with reduced toxicity and improved quality-of-life (QoL), evaluated through patient-reported-outcome-measures (PROMs). RESULTS: The resultant proposed multi-centre, phase II proof-of-principle trial will investigate, in parallel, two single agents as monotherapy induction, in children/teenagers/adults with intracranial germinoma. Trial features include: a) Bayesian statistical design determining whether CR rate for either agent is sufficiently non-inferior to SOC; b) ‘Flip-flop’ design with alternating, continuous enrolment to the two single-agents, interim assessments after each recruited cohort, and early stopping rules for inferiority; c) Safety MRI, after 6-weeks of chemotherapy with real-time central-radiological-review; d) Proof-of-principle vinblastine monotherapy arm for metastatic patients awaiting definitive craniospinal-irradiation; e) State-of-the-art integrated imaging acquisition, QoL/PROM, pharmacokinetics and circulating microRNA studies to maximise information/learning; f) European and North American neuroradiological response criteria comparison and prospective evaluation of new consensus criteria. CONCLUSIONS: Trial results will: a) establish whether monotherapy is a treatment option in this setting, which may be practice-altering; b) use QoL/PROM data to inform on optimal treatment if results similar; c) use embedded radiological assessments to develop intracranial germinoma trials and facilitate European/US study comparisons; d) describe vinblastine pharmacokinetic data to inform future dosing schedules in this and other malignancies; and e) quantify circulating microRNAs, facilitating future non-invasive diagnosis/risk-stratification.
HGG-42. Evolutionary selection of key oncogenic alterations in patient-derived models of paediatric diffuse high grade glioma (PDHGG) subtypesin vitro andin vivo
Authors: Mackay Alan , Temelso Sara , Carvalho Diana , Kessler Ketty , Boult Jessica , Izquierdo Elisa , Pereira Rita , Potente Elisabet Fernandez , Burford Anna , Molinari Valeria , Bjerke Lynn , Grabovska Yura , Rogers Rebecca , Crampsie Shauna , Das Molina , Robinson Simon , Clarke Matthew , Jones Chris
Published: June 2022

PDHGG are a diverse group of childhood brain tumours comprising multiple subgroups carrying distinct molecular drivers. Patient-derived models accurately recapitulating this underlying biology are critical for mechanistic/preclinical studies aimed at improving patient outcome, however their behaviour over time in the environments in which they are propagated, and how this relates to the human disease, is largely unknown. To explore this, we collected 94 models of PDHGG established as 2D/3D stem cell cultures in vitro, and generated patient-derived xenografts (PDX) in 33/62 specimens implanted orthotopically in vivo. We carried out exome/targeted sequencing, methylation profiling and RNAseq to profile cells through their first 25 passages in culture, and sequential implantation from p0-p2 in mice. In 15/83 cultures, we observed enrichment of gene expression signatures of non-malignant cells over the first 5 passages, with concurrent depletion of somatic mutations/CNAs, excluding them from further study. Validated models retained tumour-matched genotypes, CNAs and driver alterations including H3.3G34R, H3.3/H3.1K27M, BRAF and ACVR1 over time, however subclonal alterations underwent selection in culture which profoundly altered their response to targeted drug treatment. In 6/7 PDGFRA-mutant models, activating mutations were selected against between p5-20 in 2D and/or 3D, whilst MAPK pathway mutations in NF1/PIK3R1 similarly diverged over 15 passages under different growth conditions, resulting in isogenic models with differential signalling, in vivo tumorigenicity, and in vitro sensitivity to multiple MEK inhibitors. In PDXs, serial xenografting reduced the time to tumour formation by up to half, with a concomitant shift in clonal architecture. Multi-region sequencing of diffusely-infiltrating tumours showed selection for alterations such as PIK3CA/NF1 at distant sites, with evidence for convergent evolution of subclonal mutations, as in human tumours. Understanding the evolutionary dynamics of targetable/predictive alterations in PDHGG model systems is key to developing new and effective therapeutic interventions in this highly heterogenous disease.
HGG-45. Characterization of spinal diffuse midline gliomas, H3 K28M-mutant
Authors: Stegat Lotte , Thomas Christian , Schweizer Leonille , Neyazi Sina , Pohl Lara , Frank Stephan , Dorostkar Mario M , Schüller Ulrich , Wefers Annika K
Published: June 2022

Diffuse midline gliomas (DMGs) are malignant gliomas that arise in the midline structures of the central nervous system. Due to their aggressive and diffuse growth and a two-year survival rate of less than 10%, DMGs are assigned to CNS WHO grade 4. Depending on the localization, median age of patients is about 11‒20 years. Genetically, most tumors are defined by a K28M-mutation in one of the highly homologous genes encoding histone protein H3. Since DMGs most frequently occur in pons and thalamus, comparatively little is known about spinal DMGs. Therefore, we histologically, molecularly, and clinically characterized spinal DMGs and analyzed, in which aspects they differ from DMGs of other localizations. Our cohort currently consists of 25 spinal DMGs and 40 pontine/thalamic reference cases. Histological, immunohistochemical and molecular analyses (DNA methylation, DNA panel sequencing) were done from FFPE tissue. Spinal DMGs were histologically very heterogeneous, both regarding different areas of single tumors as well as in comparison to other spinal and reference cases. First cluster analyses of DNA methylation data indicated a separation into three main clusters enriched for pontine, thalamic or spinal cases. The cluster enriched for spinal cases contained many tumors from elderly patients. Overall, mean age of patients with spinal DMGs was 28 years. Patients were significantly older than those with pontine DMGs. 19/20 spinal DMGs were H3-3A K28M-mutant, while one tumor had an H3-2B mutation. 4/19 (21%) spinal DMGs had mutations in FGFR1, and 6/10 (60%) in NF1. Three tumors had KRAS or BRAF mutations. In summary, first analyses suggest slight histological differences of spinal DMGs compared to DMGs of other localizations. Preliminary cluster analyses of DNA methylation data showed an enrichment of clusters for different localizations. About one third of spinal DMGs had mutations in a gene associated with the MAPK-signaling pathway.
HGG-46. Inter and Intra-tumor Heterogeneity of Pediatric-type Diffuse High-Grade Glioma Revealed by High-Dimensional Single-Cell Proteomics
Authors: Petrilli Lucia Lisa , Fuoco Claudia , Pasquini Luca , Palma Alessandro , Pericoli Giulia , Grabovska Yura , Mackay Alan , Rossi Sabrina , Camassei Francesca Diomedi , Carcaboso Angel Montero , Carai Andrea , Mastronuzzi Angela , Jones Chris , Cesareni Gianni , Locatelli Franco , Vinci Maria
Published: June 2022

Paediatric-type diffuse high-grade gliomas (PDHGG) are aggressive brain tumors, affecting children and young adults, with no effective treatments. A main constraint to the development of effective treatment is associated with their highly heterogeneous nature. In order to further dissect their intra and inter tumor heterogeneity, we exploited the mass cytometry technology, an advanced -OMIC approach that, by using metal-tagged antibodies, allows the simultaneous measurement of more than 40 markers, at single-cell level. Here we characterized 8 primary cell lines derived from diffuse pediatric-type high-grade glioma H3-wildtype (DHGG-WT), Diffuse hemispheric glioma H3G34-mutant (DHG-G34) and Diffuse midline glioma H3K27-altered (DMG-K27) patients. The adopted antibody panel was set to recognize antigens expressed by brain and tumor cells, including H3K27M and H3.3G34R variants, and it highlighted important intra- and inter- tumor heterogeneity in the expression of the 16 considered markers. Of these, CD56, CD44, CD29 and NESTIN were more expressed in the hemispheric cell lines, while CD90 was more expressed in the pontine. Even if there was not always a concordance between CyTOF and mRNA expression data from cell lines and tumor samples (e.g. CD90 and GFAP), CyTOF data were in line with the immunohistochemistry analysis for GFAP, whose expression was significantly higher in H3.1K27 compared to H3.3K27. The UMAP analysis allowed us to identify 10 cell clusters, with very minimal overlap between hemispheric and pontine location subgroups and with a peculiar antigenic profile, whose abundance strongly varied according to the mutational subgroups. For example, while the G34 subgroup was enriched for cluster 9 (CD29/CD63/CD56/PDGRFa), the H3.1K27 was enriched for cluster 3 (H3K27M/CD90/CD63/CD56) and cluster 4 (H3K27M/CD63/CD90/CD56/GFAP). In conclusion, single-cell mass cytometry reveals a significant inter and intra-tumoral heterogeneity at protein level, dependent on the molecular alterations. This approach could contribute to the identification of new clinically relevant biomarkers for PDHGG.