bluuebunny/crossref_metadata_2025 huggingface dataset:
EMBR-21. CLINICALLY TRACTABLE OUTCOME PREDICTION OF GROUP 3/4 MEDULLOBLASTOMA BASED ON TPD52 IMMUNOHISTOCHEMISTRY: A MULTICOHORT STUDY
Authors: Delaidelli Alberto , Dunham Christopher , Santi Maria Rita , Negri Gian Luca , Triscott Joanna , Zheludkova Olga , Golanov Andrey , Ryzhova Marina , Okonechnikov Konstantin , Schrimpf Daniel , Stichel Damian , von Deimling Andreas , Kool Marcel , Pfister Stefan , Ramaswamy Vijay , Korshunov Andrey , Taylor Michael , Sorensen Poul
Published: June 2021

Background

International consensus and the 2021 WHO classification recognize eight molecular subtypes among Group 3/4 medulloblastoma (representing ~60% of tumors). However, very few clinical centers worldwide possess the technical capabilities to determine DNA-methylation patterns or other molecular parameters of high-risk for Group 3/4 tumors. As a result, biomarker-driven risk stratification and therapy assignment constitutes a major challenge in medulloblastoma research. Here, we identify an immunohistochemistry (IHC) marker as a clinically tractable method for improved medulloblastoma risk-stratification.

Patients and Methods

We bioinformatically analyzed published medulloblastoma transcriptomes and proteomes identifying as a potential biomarker TPD52, whose IHC prognostic value was validated across three Group 3/4 medulloblastoma clinical cohorts (n = 387) treated with conventional therapies. Risk stratification and prediction capability were computed utilizing uni- and multivariate survival analysis. Newly developed risk classifiers including TPD52 IHC were compared to state-of-the-art risk stratification schemes in terms of prediction error, area under the time-dependent receiver operating characteristic (ROC) curves and C-statistic. Biomarker-driven prognostic stratification models identified were cross validated in different cohorts.

Results

TPD52 IHC positivity represents a significant independent predictor of early relapse and death for Group 3/4 medulloblastoma (HRs between 3.67–26.7 [95% CIs between 1.00–706.23], p = 0.05, 0.017 and 0.0058). Cross-validated survival models incorporating TPD52 IHC with clinical features outperformed existing disease risk-stratification schemes, and reclassified ~50% of patients into more appropriate risk categories. Finally, TPD52 immunopositivity is a predictive indicator of poor response to chemotherapy (HR 12.66 [95% CI 3.53–45.40], p < 0.0001), suggesting important implication for therapeutic choices.

Conclusion

The current study redefines the approach to risk-stratification in Group 3/4 medulloblastoma. Integration of TPD52 IHC in classification algorithms significantly improves outcome prediction and can be rapidly adopted for risk stratification on a global scale, independently of advanced but technically challenging molecular profiling techniques.
EMBR-28. CNS DISTRIBUTIONAL KINETICS OF PANOBINOSTAT IN THE MOUSE
Authors: Zhang Wenqiu , Mohammad Afroz , Sirianni Rachael , Elmquist William
Published: June 2021

Medulloblastoma (MB) is the most common malignant pediatric brain tumor and has the potential for leptomeningeal spread, often responsible for the preponderance of morbidity and mortality in MB patients. Adverse sequelae following high dose craniospinal radiation and aggressive chemotherapies, including neurocognitive deficits and endocrine disorders, impairs patient quality of life and thus call for new therapeutic strategies. Panobinostat is a pan-histone deacetylase (HDAC) inhibitor that has been shown to be effective against MBs in vitro. The objective of the current study is to evaluate the key pharmacokinetic (PK) parameters and the mechanisms influencing the brain penetration of panobinostat in the mouse model, often used in MB preclinical efficacy trials. Pharmacokinetic studies were conducted using FVB wild-type (WT) mice and triple-knockout (TKO, Mdr1a/b–/–Bcrp1–/–) transporter-deficient mice. Panobinostat was administered intravenously (10 mg/kg) and concentrations at selected time points in plasma, brain and spinal cord were determined using LC-MS/MS. PK parameters, including systemic clearances, volumes of distribution, half-lives, and areas-under-the-concentration time curve (AUC) were calculated by using non-compartmental analysis. In WT mice, the CNS penetration was initially limited; however, it increased with time and the brain-to-plasma (B/P) and spinal cord-to-plasma (SC/P) distributional partition coefficients (AUC ratios) were 3.7 and 1.4, respectively. The B/P and SC/P ratios of panobinostat in BBB transporter-deficient TKO mice were 1.7- and 2.3-fold higher than those in WT mice, respectively. Our data suggest that P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp) efflux transporters, localized in BBB, may influence the delivery of panobinostat to tumors in the brain following systemic intravenous administration. These systemic and CNS distributional parameters will inform future preclinical efficacy studies of panobinostat for MB. As such, studies investigating other administration routes that can bypass the BBB, such as the intrathecal injection of novel formulations, are ongoing.
EPEN-07. SINGLE-CELL RNA SEQUENCING IDENTIFIES A UNIQUE MYELOID SUBPOPULATION ASSOCIATED WITH MESENCHYMAL TUMOR SUBPOPULATION IN POOR OUTCOME PEDIATRIC EPENDYMOMA
Authors: Griesinger Andrea , Riemondy Kent , Donson Andrew , Willard Nicholas , Prince Eric , Harris Faith , Amani Vladimir , Grimaldo Enrique , Hankinson Todd , Grundy Richard , Jackson Andrew , Foreman Nicholas , Ritzmann Timothy
Published: June 2021

We have previously shown immune gene phenotype variations between posterior fossa ependymoma subgroups. PFA1 tumors chronically secrete IL-6, which induces secretion of myeloid cell IL-8 and pushes the infiltrating myeloid cells to an immune suppressive function. In contrast, PFA2 tumors have a more immune activated phenotype associated with a better prognosis. The objective of this study was to use single-cell(sc) RNAseq to descriptively characterize the infiltrating myeloid cells. We analyzed approximately 8500 cells from 21 PFA patient samples. Using advanced machine learning, we identified eight myeloid cell subpopulations with unique gene expression profiles. Interestingly, only one subpopulation was significantly enriched in PFA1 tumors. This subpopulation, denoted as the hypoxia myeloid subpopulation, was defined by genes associated with angiogenesis, response to hypoxia, wound healing, cell migration, neutrophil activation and response to oxygen levels. These myeloid cells also share similar gene expression profile to a mesenchymal tumor subpopulation (MEC) enriched in PFA1 and associated with poor outcome in EPN patients. This tumor subpopulation was the only population expressing IL-6. Using immunohistochemistry, we found the hypoxia myeloid located in regions of tumor necrosis and perivascular niches. The MEC cells were also more abundant in these regions. In an independent single-cell cytokine release assay, we identified eight subpopulations of functional myeloid cells. One subpopulation significantly secreted IL-8, which represented the hypoxia subpopulation based on IL-8 gene expression in the scRNAseq dataset. This data suggests the tumor necrosis resulting in the development of MEC tumor subpopulation is driving the immune suppressive myeloid phenotype in PFA1 tumors through polarization of myeloid cells to the hypoxia subpopulation. Further studies are needed to determine how these myeloid cells interact with the lymphocyte subpopulations and whether they contribute to the progression of PFA1 EPN.
EPEN-08. THE TREM1 POSITIVE HYPOXIC MYELOID SUBPOPULATION IN POSTERIOR FOSSA EPENDYMOMA
Authors: Willard Nicholas , Donson Andrew , Ritzmann Timothy , Grundy Richard , Jackson Andrew , Hankinson Todd , Griesinger Andrea , Foreman Nicholas
Published: June 2021

We have previously shown the importance of immune factors in posterior fossa ependymoma (PF EPN). Recently, we found eight transcriptionally unique subpopulations of myeloid cells infiltrating PF EPN with one population particularly enriched in PFA1 tumors. This subpopulation, denoted as hypoxia myeloid subpopulation, is defined by genes associated with angiogenesis, hypoxia response, wound healing, cell migration, neutrophil activation, and response to oxygen levels. TREM1 (Triggering receptor expressed on myeloid cells 1) was found to be expressed almost exclusively within this hypoxia myeloid subpopulation. TREM1 encodes for a receptor belonging to the immunoglobulin superfamily that is expressed on myeloid cells, and stimulates neutrophil and monocyte inflammatory responses. However, single-cell RNAseq give little data suggesting location of cells within the tumor microenvironment. We performed immunohistochemistry (IHC) on our bank of ~90 FFPE PFA EPN samples using TREM1 to characterize and identify the location of the hypoxia myeloid cells. The TREM1 positive cells have an ambiguous cytomorphology reminiscent of a monocyte with modest cytoplasm and a mono-lobated nucleus. IHC also showed that TREM1+ myeloid cells are largely localized to the interface of necrosis and viable tissue, most frequently in a perivascular and intravascular distribution. The latter finding suggests that the TREM1+ cells are derived from the bone marrow and that they may be associated with the mesenchymal tumor population (MEC), which we have previously described as being enriched in PFA1 tumors and localizing to perinecrotic zones. This is supported by parallel IHC analysis of subpopulation-specific markers in the same cohort of PFA EPN which showed the highest TREM1 correlation was with CAIX, a marker of MEC. In PFA matched primary/recurrent pairs, the proportion of TREM1+ cells were increased at recurrence in the majority of cases, suggesting an evolving interaction between this TREM1+ hypoxia myeloid subpopulation and neoplastic cells over the disease course.
EPEN-12. MULTIPLE RECURRENCES OF ANAPLASTIC EPENDYMOMA WITH EXTRA AXIAL AND EXTRA NEURAL METASTASIS IN A PEDIATRIC PATIENT
Authors: Hoerig Clay , Goldberg Aaron , Xu Jordan , Pathare Jody , Williams Venita , Loudon William , Plant-Fox Ashley , Muhonen Michael , Abongwa Chenue
Published: June 2021

Background

Ependymomas are the third most common CNS tumor in the pediatric population, accounting for 10% of all CNS tumors. Co-occurring extraneural and extracranial metastasis of ependymomas are extremely rare, with only 1 reported adult case in current literature.

Case Description

We describe the case of a patient with multiple reoccurrences of anaplastic ependymoma. Initial imaging showed a 5 x 8 x 8 cm complex cystic mass with nodular enhancing components within the left occipital lobe. The 4th ventricle was intact and imaging was negative for metastasis. Pathology following resection demonstrated perivascular pseudorosettes, areas of calcification, and increased mitotic activity. Biopsy revealed GFAP, EMA, neurofilament, INI-1, and was negative for CAM5.2, confirming anaplastic ependymoma. Methylation studies for PFA or PFB subgroup differentiation were not available. The patient had recurrences at 4-, 5-, and 6-years after his initial diagnosis. Seven years from his initial diagnosis, the patient underwent resection of four nodular lesions from the occipital lobe and surrounding soft tissue. Pathology of these lesions and the lymph nodes/soft tissue confirmed anaplastic ependymoma. A PET scan showed increase uptake in the supraclavicular lymph nodes and had multiple bilateral pulmonary nodules. Scans at 3 months post-surgery were negative for leptomeningeal metastases but showed further lymph node involvement with progression of pulmonary disease.

Conclusion

Co-occurring extraneural and extracranial metastasis of ependymoma is a rare occurrence across all populations. To our knowledge, this would be the first published pediatric case of anaplastic ependymoma with lymph node, soft tissue, and pulmonary involvement. Treatment of ependymoma itypically local and the utility of chemotherapy remains unclear. Treatment options for extraneural mets is very limited, illustrating the need for new therapies and further studies directed at understanding the biology of these tumors and the factors that could influence their ability to metastasize to extraneural and extracranial sites.
GERM-04. PRIMARY INTRACRANIAL GERM CELL TUMORS ARE MORE PREVALENT AMONG PEDIATRIC PATIENTS OF ASIAN/PACIFIC ISLANDER RACE/ETHNICITY IN THE UNITED STATES
Authors: Lamba Nayan , Iorgulescu Bryan
Published: June 2021

Introduction

Primary intracranial germ cell tumors (GCTs) appear to be more prevalent among pediatric patients in eastern Asia than in the U.S. Herein we use cancer registry data to evaluate whether GCT prevalence differs by race/ethnicity among U.S. pediatric patients.

Methods

Pediatric patients (age≤14) presenting between 2004–2017 with a primary intracranial GCT were identified by ICD-O-3 histological and topographical coding from the National Cancer Database (comprising >70% of cancers newly-diagnosed cancers in the U.S.), and categorized by NICHD age stages. Patients’ age, sex, race/ethnicity, and overall survival, and tumor location and size were evaluated.

Results

889 pediatric patients with primary intracranial GCTs were identified, which were overwhelmingly male (64.8%) and pure germinomas (64.0%). Non-germinomatous (24.5%) and mixed (11.5%) tumor types were in the minority. Overall, primary GCTs comprised 4.9% of intracranial tumors in pediatric males and 2.9% of intracranial tumors in pediatric females. Asian/Pacific Islander pediatric patients in the U.S. had a notably higher prevalence of GCTs: among Asian/Pacific Islander males, 10.6% of all brain tumors were GCTs, compared to only 4.5% in White non-Hispanic patients, 2.8% in Black non-Hispanic patients, and 6.0% in Hispanic patients. Despite the much lower prevalence of GCTs among female patients overall, this predominance also persisted for Asian/Pacific Islander females, among whom 7.5% of brain tumors were GCTs, compared to only 2.5% in White non-Hispanic patients, 2.4% in Black non-Hispanic patients, and 4.1% in Hispanic patients. Overall, 9.4% of pediatric primary intracranial GCTs occurred in patients of Asian/Pacific Islander race/ethnicity, in contrast to 4.0% of diffuse astrocytic/oligodendroglial tumors, 2.8% of other astrocytic tumors, or 4.6% of embryonal tumors.

Conclusions

Primary intracranial GCTs affect a substantially larger proportion of both male and female pediatric patients of Asian/Pacific Islander race/ethnicity in the United States.
HGG-04. TARGETING GABAERGIC NEURON-GLIOMA SYNAPSES IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) THROUGH ANTI-EPILEPTIC DRUG REPURPOSING
Authors: Barron Tara , Mehta Vilina , Woo Pamelyn , Monje Michelle
Published: June 2021

Pediatric high-grade gliomas, including diffuse intrinsic pontine glioma (DIPG), are the leading cause of brain cancer-related death in children. While enormous progress has been made in recent years for many forms of cancer, high-grade gliomas remain seemingly intractable, indicating that fundamental aspects of glioma growth are not yet sufficiently understood. Neuronal activity drives glioma growth both through paracrine signaling and through direct neuron-to-glioma synapses. Recently glutamatergic, AMPA receptor-dependent synapses were discovered between microenvironmental neurons and malignant glioma cells. The depolarizing current that results from synaptic and other forms of electrical neuron-glioma signaling promotes pediatric high-grade glioma proliferation and regulates growth. Neuron-glioma cell synapses mediated by other neurotransmitters remain largely unexplored, though glioma cells express genes encoding neurotransmitter receptors such as GABAA receptor subunits. Using whole-cell patch clamp electrophysiology in patient-derived DIPG xenografts, we have identified functional GABAergic neuron-to-glioma synapses mediated by GABAA receptors. GABAergic input has a depolarizing effect on glioma cells, but the magnitude of depolarization is heterogeneous between high-grade glioma subtypes and between patient-derived DIPG xenograft models. As membrane depolarization increases glioma proliferation, depolarizing GABAergic inputs to glioma cells could promote DIPG progression. Drugs that stimulate GABA signaling, such as benzodiazepines, are often given to pediatric glioma patients to treat nausea, seizures or anxiety. In patient-derived DIPG xenograftn models, lorazepam, a benzodiazepine that increases GABAA receptor conductance, increases glioma growth. Conversely, levetiracetam, an anti-epileptic drug that reduces synaptic transmission including at GABAergic neuron-glioma synapses, reduces glioma proliferation in patient-derived DIPG xenografts. This emerging understanding of brain cancer neurophysiology reveals new therapeutic targets and highlights commonly used drugs about which more study is required in this disease context.
HGG-05. NEURONAL ACTIVITY PROMOTES PEDIATRIC HIGH-GRADE GLIOMA GROWTH THROUGH A NLGN3-CSPG4 SIGNALING AXIS
Authors: Gillespie Shawn , Kim Yoon , Geraghty Anna , Woo Pamelyn , Monje Michelle
Published: June 2021

High-grade gliomas, including diffuse intrinsic pontine glioma (DIPG), are a lethal group of cancers whose progression is strongly regulated by neuronal activity {Venkatesh 2015}{Venkatesh 2017}{Venkatesh 2019}. One way in which glioma cells sense neuronal activity is via interaction with the ectodomain of post-synaptic adhesion protein neuroligin-3 (NLGN3), which is cleaved and released into the tumor microenvironment (TME) by the sheddase ADAM10. This interaction drives glioma growth, but the relevant binding partner of shed NLGN3 (sNLGN3) on glioma cells is currently unknown. Here, we report that sNLGN3 binds to chondroitin sulfate proteoglycan 4 (CSPG4), in turn inducing regulated intramembrane proteolysis (RIP) of CSPG4, and initiating a signaling cascade within DIPG cells to promote tumor growth. CSPG4 RIP involves activity-regulated ectodomain shedding by ADAM10 and subsequent gamma secretase-mediated release of the intracellular domain in healthy oligodendroglial precursor cells (OPCs), putative cells of origin for several forms of high-grade glioma {Sakry 2014}{Nayak 2018}. Incubation of high-grade glioma cells or healthy OPCs with recombinant NLGN3 is sufficient to augment ADAM10-mediated ectodomain release of CSPG4 and subsequent gamma secretase-mediated cleavage of the CSPG4 intracellular domain (ICD). Pre-treatment of glioma cells or OPCs with an ADAM10 inhibitor entirely blocks NLGN3-induced CSPG4 shedding. Acute depletion of CSPG4 via CRISPR gene editing renders glioma cells insensitive to the growth-promoting effects of NLGN3 application in vitro. We are now performing experiments to better discern how the CSPG4 ICD regulates signaling consequences downstream of sNLGN3 binding. In addition, we are using surface plasmon resonance to investigate whether the shed ectodomains of NLGN3 and CSPG4 remain in complex or only transiently interact. Altogether, our data form a critical missing link in understanding how glioma cells sense, translate and respond to neuronal activity in the TME and identify a new therapeutic target to disrupt neuron-glioma interactions.
HGG-10. THE BLOOD-BRAIN BARRIER IN DIPG: INVESTIGATING REGION-SPECIFIC DIFFERENCES IN PERMEABILITY
Authors: Ung Caitlin , Upton Dannielle , Tsoli Maria , Ziegler David
Published: June 2021

Diffuse Intrinsic Pontine Glioma (DIPG) is the most aggressive pediatric high-grade glioma with median survival of only 12 months from diagnosis. Current therapies are essentially palliative. The blood-brain barrier (BBB) is a major obstacle, limiting delivery of effective chemotherapeutics into the brain. We hypothesized that tumors in the brainstem region have a BBB less permeable than tumors in other brain regions. We have confirmed the presence of an intact BBB in three orthotopic models of DIPG by Evans Blue extravasation assay. Immunohistochemical staining of CD13+ pericytes and CD34+ endothelial cells in healthy mouse brain compared to orthotopic DIPG model showed higher levels of both components in brainstem compared to cortical region. Single-cell RNA sequencing experiments are currently being undertaken to investigate region-specific differences in BBB cell populations and the impact of DIPG on signaling pathways that govern permeability. To determine if tumor location impacts therapeutic outcome, we performed in vivo efficacy studies with DIPG orthotopically injected into cortical region or brainstem region and treated with SAHA, HDAC inhibitor, or temsirolimus, mTOR inhibitor. Temsirolimus or SAHA was ineffective at extending survival in mice injected with DIPG in the brainstem compared to control. However, temsirolimus led to a significant improvement in survival in mice injected with DIPG cells in cortical region (median survival 85 days) compared to control (median survival 69 days (P≤0.01)). This suggests that the same tumor in cortical region may respond to systemic therapy that is ineffective in the brainstem and that the intact BBB in the brainstem is a major reason for treatment failure in DIPG. In conclusion, the BBB in the brainstem and in the presence of DIPG may be altered, changing signaling pathways that affect permeability. Understanding the brainstem cerebrovasculature may potentially lead to a novel strategy to treat DIPG as well as other brain tumors.
HGG-16. DISSECTING THE EFFECT OF ATM DELETION ON RADIOSENSITIVITY IN DIFFUSE MIDLINE GLIOMAS WITH H3K27M MUTATION
Authors: Guerra Garcia Maria , Deland Katherine , Luo Lixia , Ma Yan , Williams Nerissa , Stewart Connor , Kirsch David , Reitman Zachary
Published: June 2021

Diffuse midline gliomas (DMGs) are responsible for a large proportion of childhood brain tumor deaths. Currently, radiation therapy is thought to be one of the most effective treatment options, but more than 90% of children still die within 2 years of diagnosis. DMGs are defined by somatic histone 3 K27M (H3K27M) mutations that have been shown to promote the G0/G1 to S cell cycle transition. The majority of DMGs also contain loss-of-function mutations in TP53. Prior research demonstrated that orthotopic xenograft and primary mouse models of non-H3K27M-mutated gliomas with inactivation of p53 are preferentially radiosensitized by inactivation of Ataxia Telangiectasia Mutated (ATM), a kinase that mediates DNA repair in response to DNA damage caused by radiation. The high frequency of mutations that deregulate p53 in DMGs raises the possibility that H3K27M-mutant DMGs may also be radiosensitized by ATM inhibition, representing a unique therapeutic opportunity. Here, we hypothesize that H3K27M-mutant DMGs that have loss of function of p53 will be radiosensitized by loss of ATM. To test this hypothesis, we used the RCAS-TVA viral gene delivery system to generate genetically-faithful primary mouse models of H3K27M-mutant DMG with p53 deletion, and we used Cre recombinase to delete Atm in the tumor cells of these mice and generated littermate controls that retained Atm. Mice were imaged weekly via luciferase-based bioluminescence to track tumor development and irradiated with three daily fractions of 10 Gy after tumor detection. We subsequently quantified the survival of mice without neurological decline following irradiation. In separate cohorts, we collected primary tumors after irradiation to verify H3K27M expression and to assess cell cycle arrest and mechanisms of cell death. These studies will elucidate mechanisms by which ATM inactivation can radiosensitize H3K27M-mutant DMGs with nonfunctioning p53, which will guide the design of clinical trials testing ATM inhibitors in DMG patients.
HGG-25. PRMT5 PROMOTES TUMOR GROWTH BY MAINTAINING STEMNESS OF PEDIATRIC HIGH-GRADE GLIOMA CELLS
Authors: DeSisto John , Knox Aaron , Chatwin Hannah , Balakrishnan Ilango , Venkataraman Sujatha , Vibhakar Rajeev , Green Adam
Published: June 2021

Background

Pediatric high-grade gliomas (pHGG) are aggressive tumors that together constitute the most common cause of childhood cancer mortality. Tumor stem cells that drive proliferation of pHGG resist chemotherapy and radiation, complicating treatment. The arginine methyltransferase PRMT5 maintains self-renewal in neural stem cells through epigenetic modifications. We hypothesized that PRMT5, which we identified as a potential driver of diffuse midline glioma (DMG) through an shRNA screen, plays a similar role in pHGG.

Methods

Using lentiviral delivery of shRNA, we knocked down (KD) PRMT5 in cortical pHGG and DMG cell lines and performed phenotypic, mechanistic and self-renewal assays. We irradiated PRMT5 KD and control cells to study sensitization. We orthotopically injected mice with PRMT5 KD pHGG cells, and with DMG cells in which PRMT5 was knocked out (KO) using CRISPR-Cas.

Results

In cellular models of cortical pHGG and DMG, PRMT5 KD significantly reduced proliferation, inhibited cell cycle progression, increased apoptosis resistance, and decreased self-renewing cell frequency. A relative shift of PRMT5 from the cytoplasm to the nucleus accompanied differentiation induced by PRMT5 KD. Epigenetic changes accompanying PRMT5 KD included increased H3K27me3, a global transcription inhibitor, and decreased H3K27M expression in DMG. PRMT5 KD sensitized pHGG cells to radiation, increasing cell death 17–30%. PRMT5 KD/KO significantly increased survival in mice and decreased tumor aggressiveness and proliferation, but mice still died of tumor-related effects.

Conclusions

PRMT5 maintains self-renewal and drives proliferation in preclinical pHGG models. In cellular and in vivo models, PRMT5 KD/KO produces epigenetic changes, including increased H3K27me3 levels and diminished H3K27M, that may reduce proliferation and self-renewal. Future work includes elucidation of the mechanisms by which PRMT5 produces the observed changes. Because PRMT5 KD/KO does not eliminate tumor growth, we plan to further study combining PRMT5 KD/KO and clinical-grade small molecule PRMT5 inhibitors with radiation and chemotherapeutic agents.
HGG-35. IDENTIFICATION OF G PROTEIN-COUPLED RECEPTOR 17 (GPR17) UP-REGULATION IN PAEDIATRIC H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA AND EXAMINATION OF ITS ROLE IN DIFFUSE MIDLINE GLIOMA CELL LINES
Authors: Loveson Katie , Fillmore Helen
Published: June 2021

Paediatric H3 K27M-mutant diffuse midline glioma (pDMG) is an incurable, aggressive childhood brain malignancy, that arises in a region- and age-specific nature. The underlying pathophysiology suggests dysregulation of postnatal neurodevelopmental processes causing aborted cell differentiation. The cell of origin is unclear, but data suggests an oligodendrocytic lineage (OPC), supported by the over-expression of transcription factors such as Olig1 and Olig2 in 80% of DIPG cases. In-depth bioinformatics and principal component analyses (PCA) of genes involved in brain development and pDMG support reports of OPC gene dysregulation and led to the identification of the G-protein coupled receptor 17 (GPR17) and its association with pDMG. GPR17, a rhodopsin-like orphan GPCR (unknown ligand), has the typical features of the GPCR superfamily, seven transmembrane domains (TM1-TM7), eight amphipathic helices, an extracellular N-terminal domain, and an intracellular C-terminus. GPR17 has been implicated in several normal physiological and pathological processes, such as oligodendrocyte differentiation, spinal cord injury and brain injury. GPR17 mRNA and protein expression was confirmed in all pDMG cell lines tested. Using a well-characterised agonist (MDL 299,51) and antagonist (HAMI3379) to modulate GPR17 function in pDMG cell lines resulted in phenotypic and genomic changes as well as in cell growth and migration. HAMI3379, a GPR17 specific antagonist resulted in a significant reduction in GPR17 mRNA and protein expression (p<0.006) and a significant reduction in migration (p<0.0025). When pDMG cells were pretreated with HAMI3379 in combination with known cytotoxic agents (Bleomycin, a radiation mimic, Panobinostat or Vincristine), there was a decrease in cell viability compare to cytotoxic agent alone. There are no current effective therapies for pDMG patients and the ability of blocking GPR17 function to enhance sensitivity to standard therapies is appealing and warrants further investigation.
HGG-41. CHARACTERIZATION OF THE IMMUNE RESPONSE FOLLOWING VARIOUS RADIOTHERAPY TREATMENTS IN GLIOBLASTOMA
Authors: Cocito Carolina , Branchtein Mylene , Gongora Tatyana , Dahmane Nadia , Greenfield Jeffrey
Published: June 2021

Malignant gliomas represent 6.5% of all childhood brain neoplasms with a 5-years survival rate of less than 20%. Current standard of care for these tumors include radiotherapy; recent data in solid tumors indicate that adequate radiation protocols may synergize with immunotherapy strategies for better outcomes. Nonetheless, a great discrepancy between preclinical studies and clinical trials outcomes persists, the basis of which is not fully understood. One hypothesis may be due to different radiation protocols used. We used the GL261 syngeneic mouse model of glioma to test this hypothesis and characterize the immune response to radiotherapy, with either a single dose of 10Gy, a dose often used in preclinical models, or a fractionated treatment of 2Gy for five consecutive days (2Gyx5), as fractioned radiotherapy is most often used in patients. The immune content of the brain and the blood was assessed by flow cytometry in un-irradiated (control), 10Gyx1 and 2Gyx5 treated mice for three weeks after radiation. In the brain, both radiation regimens drastically reduced the number of CD45+ cells for the first two weeks after treatment. When compared to controls, 10Gyx1 but not 2Gyx5 treated mice showed a significant increase in tumor infiltrating lymphocytes (CD3+) starting from the second week following treatment. This effect persisted until three weeks post treatment. The 10Gyx1 dose was better tolerated by the resident microglia (CD45lowCD11b+) when compared to the 2Gyx5 treatment. Our data describe the dynamics through which the immune microenvironment responds to two radiation regimens over time. Our results show that 10Gyx1 is the most effective regimen to impede tumor growth and to induce lymphocyte infiltration once the system recovers from the treatment. Our work suggests that, in the GL261 model, the fractionated radiation treatment we tested may be less optimal in priming glioma cells to the immune system.
IMMU-01. THE ONCOLYTIC VIRUS DELTA-24-RGD IN COMBINATION WITH AN AGONISTIC CD40 MAB INDUCES A DURABLE AND SYNERGISTIC ANTI-TUMOR IMMUNE EFFECT IN DIPG PRECLINICAL MODELS
Authors: Labiano Sara , Laspidea Virginia , Garcia-Moure Marc , Puigdelloses Montserrat , Becher Oren J , Gomez-Manzano Candelaria , Fueyo Juan , Patiño-Garcia Ana , Alonso Marta M
Published: June 2021

With a 2-year survival less than 20%, Diffuse Intrinsic Pontine Glioma (DIPG) is the principal cause of pediatric death. Despite recent advances in the current treatments, the outcome for children with DIPGs remains dismal. Since the approval of T-VEC for melanoma by the FDA, oncolytic adenoviruses have emerged as a promising therapeutic strategy for brain tumors. Thus, our group launched the first world clinical trial phase I with the oncolytic adenovirus Delta-24-RGD (DNX-2401 in the clinic) for newly diagnosed DIPG (NCT03178032), which has shown safety and feasibility. Despite DNX-2401 increases the recruitment of T cells into the tumor, they usually become inactive due to the suppressive tumor microenvironment evidencing the urgent need to improve this strategy focusing on the generation of effective long-term immune responses. Therefore, we decided to combine the Delta-24-RGD with the targeting of the costimulatory molecule CD40 in two unique orthotopic immunocompetent mouse models of DIPG. The activation of the CD40 receptor, which is expressed by antigen presenting cells (APC) such as microglia, macrophages and dendritic cells, is known to increase antigen presentation and enable T-cell priming and activation. Here, we have observed that in addition to Delta-24-RGD anti-tumor effects, the stimulation of CD40 on the tumor APCs results in a remodeling of the tumor immune compartment with a more efficient T-cell tumor infiltration. Of importance, there is an increase of the survival of mice treated with the combination as compared to single treatments or non-treated mice. In addition, the combination therapy induced a complete regression of tumors in 25% of treated mice indicating the development of long-term anti-tumor immunity. We believe that these results provide a translational breakthrough in the treatment of these lethal tumors and open the door for a future innovative clinical trial.
LGG-09. SENOLYTIC AGENT NAVITOCLAX TARGETS VINBLASTINE- AND MAPK INHIBITORS-INDUCED SENESCENT TUMOUR CELLS IN PAEDIATRIC LOW GRADE GLIOMAS
Authors: Guiho Romain , Selt Florian , Stone Thomas , Jacques Thomas , Hargrave Darren , Gil Jesús , Witt Olaf , Milde Till , Barbera Juan Pedro Martinez
Published: June 2021

Pilocytic astrocytoma (PA, WHO grade I), the most common paediatric brain tumour, is characterized by constitutive activation of the MAPK pathway. PA tumours show a slow growth, without tendency to progress to high-grade malignancies. However, a significant group of patients for whom a total resection is not feasible require additional therapy. The typical proliferative index of a PA, measured by Ki-67 staining, is 1–2%, whereas a large part of the tumour is Ki-67 negative and expresses markers of oncogene-induced senescence (OIS) such as SA-β-Gal positivity and the cell cycle inhibitors p16INK4a (CDKN2A) and p21Cip1 (CDKN1A). Conventional treatments (i.e. chemotherapy) tend to target only proliferative cells and the effect of new molecularly targeted therapies (e.g., MAPK pathway inhibitors) on senescent cells remains unclear. Here, we discuss the opportunities to combine these therapies with new compounds targeting the senescent cells, referred to as senolytics, using three different PA models. (1) Ex vivo culture of human PA tumours (2) Two cell lines: the DKFZ-BT66 PA human cell line, carrying the oncogenic driver KIAA1549:BRAF-fusion, used as a model of OIS; and the proliferative BT40 cell line harbouring the BRAFV600E mutation; (3) In vivo xenograft model induced by orthotopic transplantation of BT40 cells. We have previously shown that OIS cells exhibit an increased sensitivity to senolytic compounds, such as navitoclax, a clinically approved BCL2/XL inhibitor, relative to proliferative controls (Buhl et al, Clin Cancer Res. 2019). Our current research demonstrates that treatments with low doses of chemotherapy (e.g., vinblastine) or MAPK inhibitors (e.g., dabrafenib or trametinib) triggers a therapy-induced senescence response in proliferative cells (e.g., abolished proliferation, SA-β-Gal positivity, SASP production), making these senescent cells sensitive to senolytic compounds, including navitoclax. Together, our research provides a strong rationale supporting the combined use of senolytics with current conventional and targeted therapies against human PA.
LGG-10. AN UNUSUAL PRESENTATION OF BILATERAL OPTIC NERVE GLIOMA IN CROUZON SYNDROME
Authors: Na Brian , Wang Anthony C , Watterson Christopher Travis , Martinez-Agosto Julian A , Saitta Sulagna , Dutra-Clarke Marina , Hinkamp Franceska , Pineles Stacy L , de Blank Peter , Chang Vivian Y , Moore Theodore
Published: June 2021

Optic pathway gliomas (OPGs) are low grade gliomas intrinsic to the visual pathway, frequently associated with Neurofibromatosis Type 1 (NF-1). Bilateral OPGs without chiasmatic involvement are almost pathognomonic for NF-1. We report an unusual case of bilateral optic nerve glioma without chiasmatic involvement in a 17-month old male patient with craniosynostosis and Crouzon Syndrome, an autosomal dominant disorder caused by activating FGFR2 mutations associated with craniosynostosis and optic atrophy. The patient’s c.1032 G>A pathogenic variant in FGFR2 is a variant known to affect splicing and results in a protein that lacks part of an important domain involved in ligand binding. Although FGFR1 mutations have been implicated in low-grade glioma through MAPK activation, FGFR2 mutations have not yet been described in OPGs, although they have been described in epileptogenic low-grade gliomas and mixed neuronal-glial tumors. Our patient presented with worsening vision in the setting of known Crouzon Syndrome. An eye examination revealed bilateral primary optic atrophy. Brain and orbital MRIs demonstrated fusiform dilation and STIR hyperintensity of the intraorbital segments of the optic nerves bilaterally with normal pre-chiasmatic optic segments. There were no other radiographic or physical stigmata suggestive of NF-1. Next generation sequencing and copy number analysis from peripheral blood were negative for variants in NF1. RNA based studies for NF1 aberrations are pending. Although follow up MRI scans demonstrated stable size of his OPGs, the risk of further visual deficit was considered significant due to his pre-existing optic atrophy and poor baseline visual acuity. Therefore, he was started on vincristine and carboplatin chemotherapy according to A9952, and induction therapy has been well tolerated. To our knowledge, this is the first patient with Crouzon Syndrome who has developed bilateral optic pathway gliomas. Orbital MRIs should be considered for these patients with worsening visual acuity not explained by other causes.
OMIC-05. PHOSPHOPROTEOMIC ANALYSIS IDENTIFIES SUBGROUP ENRICHED PATHWAYS AND KINASE SIGNATURES IN MEDULLOBLASTOMA
Authors: Leskoske Kristin , Garcia-Mansfield Krystine , Krishnan Aparna , Sharma Ritin , Rusert Jessica , Mesirov Jill , Wechsler-Reya Robert , Pirrotte Patrick
Published: June 2021

Medulloblastoma (MB) is classified into four molecular subgroups: wingless (WNT), sonic hedgehog (SHH), Group 3 (G3) and Group 4 (G4), each with different molecular profiles and patient outcomes. Subgroup heterogeneity and low mutational burdens have hindered the identification of actionable therapeutic targets, especially in G3 MB which has a particularly poor prognosis. Therefore, we took a (phospho)-proteomics approach to identify active pathways and potential therapeutic opportunities in twenty orthotopic patient-derived xenograft (PDX) models of MB comprising SHH, G3 and G4 subtypes. Through our enrichment analysis, we identified processes and pathways specifically upregulated in each MB subgroup. We also utilized neural network derived kinase-substrate predictions and kinase activity scores inferred by a heuristic machine learning algorithm to further characterize phosphosignaling activity. We found that MB PDX models recapitulate many features of primary MB tumors including two distinct proteomic subtypes of G3. G3a was enriched for transcription, translation and MYC target genes while G3b was enriched for axon guidance and neurotrophin signaling pathways. Notably, both G3a and G3b contained higher abundance of mitochondrial proteins, suggesting altered tumor metabolism in G3 MB. SHH PDXs displayed increased NFκB and JNK-MAPK signaling. Group 4 MBs most closely resembled differentiated neuronal cells and were enriched for PKC and AMPK signaling as well as DNA repair pathways. In conclusion, we have provided a comprehensive proteomic and phosphoproteomic characterization of commonly studied MB PDX models and revealed new insights into subgroup enriched pathways and kinase activity in MB.
OMIC-07. FEASIBILITY AND UTILITY OF EPIGENOMIC PROFILING FOR CHILDHOOD CNS TUMORS IN HONG KONG
Authors: P.Y. Liu Anthony , Ho Ronnie S L , Li Kay K W , Chan Shing , Ku Dennis T L , Fu Eric , Luk Chung-Wing , Yau Jeffrey P W , Ling Siu-Cheung , Chung Brian H Y , Yang Wanling , Kan Amanda , Shing Matthew M K , Ng Ho-Keung , Chan Godfrey C F
Published: June 2021

Genome-wide DNA methylation profiling has emerged as an important diagnostic tool that complements histopathology for CNS tumors in children and adults. Literature describing its application in Asian countries is nonetheless limited. Herein, we report the feasibility and utility of adopting such platform for children diagnosed with CNS tumors in Hong Kong. A multi-institutional cohort (n=94, 97% of Chinese ethnicity) with CNS embryonal or high grade neuroepithelial tumors (HGNET) diagnosed in Hong Kong from 1996–2020 was assembled based on tissue availability. DNA was extracted from FFPE tumor material (median 301ng, range 13-1000ng), bisulfite converted and profiled with the Infinium Methylation EPIC BeadChip kit. Raw data were analyzed on the German Cancer Research Center MNP 2.0 classifier and through unsupervised dimensionality-reduction analysis (t-SNE) referencing a published CNS tumor reference dataset (GSE90496). The radio-histologic diagnosis included medulloblastoma (n=65), ATRT (n=9), pineal parenchymal tumors (n=7), ETMR (n=5), CNS-PNET (n=4) and other embryonal tumors/HGNETs (n=4). Methylation class could be assigned based on results from MNP 2.0 (calibrated score ≥ 0.9) in 62 patients (66%, including 2 clustering with control) and t-SNE in 22 (23%), while no-match was encountered in 10 (11%). Methylation-based analysis allowed confirmation of diagnosis and assignment of molecular subgroup in 64 patients (68%), confirmation of histologic diagnosis alone in 5 (5%) and resulted in revision/reassignment of diagnosis in 13 (14%). Among medulloblastoma samples that were assigned molecular tumor classes (n=57), 8 clustered with WNT-activated medulloblastoma, 13 with SHH-activated medulloblastoma, 10 with Group 3 medulloblastoma, 21 with Group 4 medulloblastoma, and 5 with non-medulloblastoma entities (high-grade gliomas=3, ETMR=1, ATRT=1). In conclusion, epigenomic profiling allowed refinement of disease classification for pediatric CNS tumors. Availability of such methodology in Asia sets the stage for international collaborations in molecularly-driven trials.
OMIC-11. SINGLE CELL RNA SEQUENCING FROM THE CSF OF SUBJECTS WITH H3K27M+ DIPG/DMG TREATED WITH GD2 CAR T-CELLULAR THERAPY
Authors: Mochizuki Aaron , Ramakrishna Sneha , Good Zina , Patel Shabnum , Chinnasamy Harshini , Yeom Kristen , Schultz Liora , Richards Rebecca , Campen Cynthia , Reschke Agnes , Mahdi Jasia , Toland Angus , Baggot Christina , Mavroukakis Sharon , Egeler Emily , Moon Jennifer , Landrum Kayla , Erickson Courtney , Rasmussen Lindsey , Barsan Valentin , Tamaresis John , Marcy Anne , Kunicki Michael , Celones Michelle , Ehlinger Zach , Kurra Sreevidya , Cornell Timothy , Partap Sonia , Fisher Paul , Grant Gerald , Vogel Hannes , Davis Kara , Feldman Steven , Sahaf Bita , Majzner Robbie , Mackall Crystal , Monje Michelle
Published: June 2021

Introduction

We are conducting a Phase I clinical trial utilizing chimeric antigen receptor (CAR) T-cells targeting GD2 (NCT04196413) for H3K27M-mutant diffuse intrinsic pontine glioma (DIPG) and spinal cord diffuse midline glioma (DMG). Cerebrospinal fluid (CSF) is collected for correlative studies at the time of routine intracranial pressure monitoring via Ommaya catheter. Here we present single cell RNA-sequencing results from the first 3 subjects.

Methods

Single cell RNA-sequencing was performed utilizing 10X Genomics on cells isolated from CSF at various time points before and after CAR T-cell administration and on the CAR T-cell product. Output was aligned with Cell Ranger and analyzed in R.

Results

As detailed in the Majzner et al. abstract presented at this meeting, three of four subjects treated at dose-level one exhibited clear radiographic and/or clinical benefit. We have to date completed single cell RNA-sequencing for three of these four subjects (two with benefit, one without). After filtering out low-quality signals and doublets, 89,604 cells across 3 subjects were analyzed. Of these, 4,122 cells represent cells isolated from CSF and 85,482 cells represent CAR T-cell product. Two subjects who demonstrated clear clinical and radiographic improvement exhibited fewer S100A8+S100A9+ myeloid suppressor-cells and CD25+FOXP3+ regulatory T-cells in the CSF pre-infusion compared to the subject who did not derive a therapeutic response. In one subject with DIPG who demonstrated improvement, polyclonal CAR T-cells detectable in CSF at Day +14 demonstrated enrichment of CD8A, GZMA, GNLY and PDCD1 compared to the pre-infusion CAR T-cells by trajectory analysis, suggesting differentiation toward a cytotoxic phenotype; the same subject exhibited increasing numbers of S100A8+S100A9+ myeloid cells and CX3CR1+P2RY12+ microglia over time. Further analyses will be presented as data become available.

Conclusions

The presence of immunosuppressive myeloid populations, detectable in CSF, may correlate to clinical response in CAR T cell therapy for DIPG/DMG.
OMIC-12. PREVALENCE AND SPECTRUM OF GERMLINE PATHOGENIC VARIANTS IN CANCER PREDISPOSITION GENES ACROSS THE CHILDREN’S BRAIN TUMOR NETWORK (CBTN)
Authors: Vaksman Zalman , McQuaid Shelly , Bornhorst Miriam , Zhu Yuankun , Heath Allison , Waanders Angela , Cole Kristina , MacFarland Suzanne , Diskin Sharon
Published: June 2021

Germline variants are known to contribute to the pathogenesis of specific central nervous system (CNS) tumor subtypes; however, a large pan-pediatric brain and nervous system cancer germline susceptibility study has not been performed. To define the prevalence and spectrum of pathogenic variants in known cancer predisposition genes (CPGs; n=200), we analyzed whole genome sequencing (WGS) data from 880 pediatric subjects across 19 different cancer types in the Children’s Brain Tumor Network (CTBN). Data were aligned using BWA. Variants were called using GATK and annotated with SnpEff and ANNOVAR. After quality control, variants with a minor allele frequency (MAF) < 0.1% in Gnomad 2.11 or ExAC were retained. Pathogenicity was assessed with American College of Medical Genetics (ACMG) guidelines using a lab-developed modification of ClinVar and InterVar. Automated pathogenic/likely pathogenic (P-LP) calls were manually reviewed by two cancer predisposition clinicians and a bioinformatician. Frequency of P-LP variants was assessed and gene burden testing was performed against Gnomad3.1 (without cancer samples) using Fisher’s exact test with Bonferroni adjustment. We observed 214 P-LP variants involving 190 unique individuals (21.6% of cohort). As expected, the most frequent variants were observed in NF1, NF2, and TP53(n=40 variants in 21% of individuals). ATM, TSC2 and CHEK2 variants (n=23) were observed in another 12% of individuals. An increased burden of P-LP variants was observed for 5 of these 6 genes (p = 1.7x10-25 to 1.4x10-2, CHEK2 p=5.5x10-2). We also identified 5 variants in BRCA2 (3 in high-grade glioma), 7 in REQC helicases (BLM, WRN, REQL4), and 16 variants in Fanconi anemia genes. Overall, cases harbored increased burden in P-LP variants in CPG genes (p=8.8x-18) and the subset of DNA repair genes (p=4.7x10-4). In conclusion we confirmed the association of variants in established predisposition genes while potentially identifying novel variants and genes associated in CNS tumors.