Pediatric Glioblastoma Research Articles

CNSC-70. THE ROLE OF MAP4K4 IN TUMOR CELL INVASION OBSERVED IN A 3D IN VITRO MODEL OF PEDIATRIC GLIOBLASTOMA

Abstract Several CNS tumors such as pediatric glioblastoma (pGBM), diffuse midline glioma (DMG), and medulloblastoma (MB) are characterized by local invasive spread and dissemination of tumor cells into surrounding brain tissue. As identified by our group in a genome-wide CRISPR screen and several others, MAP4K4 is a mediator of cell invasion in several cancers, including glioblastoma and medulloblastoma. Recently, we validated the pro-invasive role of MAP4K4 in a panel of pGBM cell lines. To further validate this finding in the context of pGBM cellular invasion, we performed 3D Matrigel timelapse invasion assays. We performed single- and multi-spheroid encapsulation to track spheroid invasion and cell-to-cell interactions between multiple spheroids. Tumor spheroids were cultured in a round-bottom, non-adherent well plate encapsulated in a Matrigel matrix and treated with a MAP4K4 small molecule inhibitor (PF06260933 dihydrochloride) or had a MAP4K4 knockout performed with Cas9 editing. Cell invasion was recorded over 72 hours and three-dimensional structures were stained for actin and imaged 10 days post-initial seeding. We show that MAP4K4 KO (p ~ 0.00033), small-molecule inhibition in the presence of a TGFB receptor agonist (SRI-011381), (p ~ 0.001) and with drug alone (p ~ 0.013) decreased spheroid invasion and prevented single-cell invasion of pGBM cells (SF188) into the matrix. More surprisingly, abrogation of MAP4K4 prevented individual spheroids in a multi-spheroid co-culture, from forming integrated glioma networks through the formation of tube-like structures in the matrix. Based on our initial findings, it appears that MAP4K4 is necessary for single-cell invasion into a three-dimensional matrix. It also appears that MAP4K4 is required for cell-to-cell connections to form between spheroids in the 3D environment to allow for the formation of higher-ordered and spatially organized structures. Future directions include studying how MAP4K4 allows for the formation of glioma tumor networks in pediatric GBM.

Cell Fate Dynamics Reconstruction Identifies TPT1 and PTPRZ1 Feedback Loops as Master Regulators of Differentiation in Pediatric Glioblastoma-Immune Cell Networks.

Pediatric glioblastoma is a complex dynamical disease that is difficult to treat due to its multiple adaptive behaviors driven largely by phenotypic plasticity. Integrated data science and network theory pipelines offer novel approaches to studying glioblastoma cell fate dynamics, particularly phenotypic transitions over time. Here we used various single-cell trajectory inference algorithms to infer signaling dynamics regulating pediatric glioblastoma-immune cell networks. We identified GATA2, PTPRZ1, TPT1, MTRNR2L1/2, OLIG1/2, SOX11, FXYD6, SEZ6L, PDGFRA, EGFR, S100B, WNT, TNF , and NF-kB as critical transition genes or signals regulating glioblastoma-immune network dynamics, revealing potential clinically relevant targets. Further, we reconstructed glioblastoma cell fate attractors and found complex bifurcation dynamics within glioblastoma phenotypic transitions, suggesting that a causal pattern may be driving glioblastoma evolution and cell fate decision-making. Together, our findings have implications for developing targeted therapies against glioblastoma, and the continued integration of quantitative approaches and artificial intelligence (AI) to understand pediatric glioblastoma tumor-immune interactions.

Glioma oncogenesis in the Constitutional mismatch repair deficiency (CMMRD) syndrome.

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition due to biallelic mutations in one of the mismatch repair (MMR) genes associated with early onset of cancers, especially high-grade gliomas. Our aim was to decipher the molecular specificities of these gliomas. Clinical, histopathological, and whole exome sequencing data were analyzed in 12 children with genetically proven CMMRD and a high-grade glioma. PDL1 expression was present in immunohistochemistry in 50% of the samples. In 9 patients, the glioma harbored an ultra-hypermutated phenotype (104-635 coding single nucleotide variants (SNV) per Mb, median 204). Driver mutations in POLE and POLD1 exonuclease domains were described for 8 and 1 patients respectively and were always present in the mutation burst with the highest variant allele frequency (VAF). The mutational signatures were dominated by MMR-related ones and similar in the different mutation bursts of a same patient without subsequent enrichment of the mutation signatures with POL-driven ones. Median number of coding SNV with VAF above one of the driving polymerase mutation per Mb was 57 (17-191). Our findings suggest that somatic polymerase alterations does not entirely explain the ultra-hypermutant phenotype. SETD2, TP53, NF1, EPHB2, PRKDC, and DICER1 genes were frequently mutated with higher VAF than the deleterious somatic polymerase mutation. CMMRD-associated gliomas have a specific oncogenesis that does not involve usual pathways and mutations seen in sporadic pediatric or adult glioblastomas. Frequent alterations in other pathways such as MAPK may suggest the use of other targeted therapies along with PD1 inhibitors.

IMMU-21. B7-H3 NANOBODY CAR-T CELL THERAPY IN PEDIATRIC GLIOBLASTOMA

Abstract BACKGROUND Immunotherapy is a developing but challenging field of research for the treatment of central nervous system malignancies. Chimeric antigen receptor (CAR)-T cell therapy has been effective in treating hematologic malignancies, but that success has not been clinically translated to solid tumors in children. B7-H3 has been identified as a promising immunotherapy target as it is highly expressed in pediatric glioblastoma and correlates with tumor progression and poor prognosis. We aim to evaluate a nanobody-based CAR-T cell therapy that binds B7-H3 with high affinity and hypothesize that anti-B7-H3 nanobody CAR-T cells can effectively kill B7-H3-expressing tumor cells. METHODS B7-H3 nanobodies were isolated from camel phage libraries and cloned into lentiviral CAR-T cell constructs. Human T cells were transduced by lentivirus to express CAR on the surface. Nanobody CAR-T cell functionality was evaluated by an impedance-based cytotoxicity assay and cytokine release was measured by ELISA. CAR-T avidity was quantified by the Lumicks z-Movi assay. In vivo studies are evaluating the efficacy of B7-H3 CAR-T cells in an orthotopic glioblastoma model in immunodeficient mice. Mice were treated with B7-H3 CAR-T cells by intracerebroventricular or intravenous delivery. RESULTS We confirmed high expression of B7-H3 on the surface of three pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. We confirmed effective killing of B7-H3-expressing tumor cells and the release of high levels of cytokines. B7-H3 nanobody CAR-T cells were observed to have high avidity compared to control CAR-T cells. In vivo studies are currently ongoing and treatment response will be evaluated by luminescence imaging and endpoint histologic analysis. CONCLUSION We have shown that B7-H3 nanobody CAR-T cells demonstrate significant preclinical efficacy. It is critical to study CAR-T cell therapy in immunocompetent in vivo models for further development and clinical application.

DIPG-75. PHARMACOLOGICAL INHIBITION OF LSD1/KDM1A IN DIFFUSE MIDLINE GLIOMA MODELS AND IMPACT ON KINASE SIGNALING

Abstract BACKGROUND High grade gliomas (HGGs) in both adults and children confer poor prognosis, with median survival rates under two years post-diagnosis. Diffuse midline gliomas (DMG) are a subset of HGG characterized by histone mutations, typically occurring in children. Genetic alterations in chromatin modifiers and receptor tyrosine kinases (RTKs) are seen in both adult and pediatric glioblastoma, however the interplay between these pathways requires further investigation to design therapeutic strategies. Our past work has evaluated KDM1A/ LSD1 (Lysine-Specific Demethylase 1) inhibition through gene silencing and small molecules; here we assessed crosstalk between LSD1 and kinase signaling networks to uncover potential compensatory resistance mechanisms using clinically relevant pharmacological inhibitors of LSD1. METHODS RNA-Seq and GSEA in high grade glioma cells bearing LSD1 knockdown were evaluated for kinase signaling pathway enrichment. A panel of DMG lines (SU-DIPGIV (MDM4, ACVR1), SU-DIPGVI (TP53), VUMC-DIPG10 (NF1, MYCN), and SU-DIPGXIII and DMG data sets were probed for LSD1 expression, activity and kinase expression and treated with pharmacological inhibitors of LSD1 in vitro and in vivo. RESULTS Pharmacological inhibition of LSD1 using clinically relevant agents revealed IC50s in the micromolar range across DMG lines. DMG lines varied with respect to LSD1 expression and baseline activation of ERK and AKT pathways. With LSD1 inhibition at subtoxic doses, increased phospho-ERK/ERK and phospho-AKT/AKT was noted in several DMG models and co-inhibition of these pathways suggests improved efficacy. CONCLUSIONS Our data reveals that LSD1 inhibition causes an increase in kinase signaling at subtoxic doses which may alter drug response. These results urge the assessment of combination treatments with kinase inhibitors and additional in vivo analysis, which are currently underway.

DIPG-25. RELATIONSHIP BETWEEN MOLECULAR CLASSIFICATION, ORIGIN AND PROGNOSIS IN DIFFUSE MALIGNANT GLIOMA IN CHILDREN

Abstract INTRODUCTION Diffuse malignant gliomas that occurs in childhood is recognized as a biologically different disease from that in adults. Furthermore, with the recent development of molecular diagnostics, diffuse midline glioma (DMG), which occurs mainly in the thalamus and brainstem, frequently occurs in childhood and have poor prognosis. We report the relationship between imaging findings, origin, and prognosis of diffuse malignant glioma in children or young adult based on our case series. PATIENTS AND METHODS 43 cases of malignant glioma that occurred from childhood to young adult (below 30 years old) and were treated at our hospital since 2004 were registered in the analysis. Of the 42 cases, 24 cases were in brain stem, 11 cases in cerebral hemisphere, 6 cases in thalamus, and 1 case in cerebellar hemisphere. Histological diagnosis was possible in 31 cases, but without histological verification in 11 brain stem lesions. Histological diagnosis included 13 high-grade glioma (GBM, including NOS and G34R mutations), 11 DMG, and 7 anaplastic astrocytoma. RESULTS Of 13 cases of hemispheric high-grade gliomas, 2 cases had long-term survival (both cases received WT1 peptide vaccination), and 1 case survived for more than 5 years (total resection, MGMT methylation). Among the DMGs, 7 cases were found in brain stem (5 pons, 2 pontomedullary junction, 1 cerebellar peduncle, and 1 midbrain), 3 cases in the thalamus. Two cases of onset at the pontomedullary junction were found incidentally, and the symptoms gradually increased during follow-up, and multidisciplinary treatment was performed. Bilateral thalamus DMG with HIST1H3B mutation showed slowly growing and invasion. SUMMARY There were cases of pediatric hemisphere glioblastoma with long-term survival. In addition, the origin and imaging findings of DMG were various, and tumors that originate at the pontomedullary junction may be relatively favorable.

DIPG-55. DISSECTING THE ECOSYSTEM OF PEDIATRIC DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Pediatric gliomas are heterogeneous tumors encompassing high grade (pHGG) and low grade (pLGG) subtypes. For tumors not amenable to surgery, in particular high grade H3K27 mutant diffuse midline glioma (DMG), no curative options exist. DMG is characterized by 1) extensive heterogeneity of tumor cells that can exist in different interchangeable cellular states mimicking normal development and 2) extensive heterogeneity of the tumor microenvironment (TME). A better understanding of the DMG ecosystem is needed for therapeutic target identification. METHODS Publicly available single cell RNA seq data of DMG (n=17, Jessa et al. Nat. Genet. 2022), high grade pediatric glioblastoma (GBM, n=16, scPCA) and low grade tumors (ganglioglioma, n=5; pilocytic astrocytoma, n=14, scPCA) were analyzed to define key molecular characteristics and differences between these pediatric brain tumors. Doublet cells were removed from each dataset and tumor and TME cells were discriminated based on marker gene expression and inferred CNVs. RESULTS In DMG, glioma cellular states and TME heterogeneity differed depending on the tumor location. DMG arising in the pons harbored a bigger fraction of astrocyte-like glioma cells whereas oligodendrocyte-like cells were more abundant in thalamic tumors. In line with this observation, Liana cellular communication analysis inferred more intratumoral (autocrine) signaling involving oligodendrocyte-like glioma cells in thalamic tumors (e.g. NCAM1-PTPRZ1, NLGN4X-NRXN1, NTN1-DSCAM) than in pons tumors. Notably, the TME of thalamic tumors contained a bigger fraction of immunotolerant macrophages expressing CD163 or VSIG4. In contrast, pons tumors harbored a unique cluster of VEGFA expressing astrocytes, not found in GBM or pLGG. Liana analysis inferred COPA/APP-CD74 interaction between VEGFA+ astrocytes and CD163/VSIG4+ macrophages and microglia, potentially contributing to an immunotolerant TME in pons DMG. CONCLUSIONS In DMG, patterns of tumor and TME composition associated with tumor location were uncovered. Furthermore, unique TME celltypes could contribute to the particularly aggressive nature of these tumors.

Characterization of cellular heterogeneity in recurrent pediatric glioblastoma: Machine learning-enhanced single-cell RNA-Seq unveils regulatory signatures

The most common oncologic cause of mortality in children is pediatric glioblastoma, an extremely dangerous brain tumor. The tumor progress is almost inevitable and recurs after first-line standard care. Because surgical resection is often more effective when tumors are localized and smaller, early identification and action may be essential to assure favourable outcomes for the recurring disease. This study aims to employ single-cell RNA-Sequencing data (scRNA-Seq data) for clustering and explainable Artificial intelligence framework to find gene biomarkers and signature cell types for the diagnosis and prognosis of reoccurring pediatric glioblastoma. Distinct cell types and statistically significant DEGs were found using scRNA-Seq data retrieved from the Gene Expression Omnibus database. Random forest (RF) and extreme gradient boosting (XGBoost) machine learning (ML) classifiers were constructed to select genes significantly contributing to the disease using Shapley (SHAP) values, an explainable artificial intelligence (EAI) framework. Potential biomarkers were chosen based on the shared genes among statistically discovered DEGs and SHAP-based relevance. B cells, macrophages, CD8+ T cells, T cells, and NK cells were identified as distinct cell types, which played an essential role in disease recurrence. Also, five significant genes, namely HMGB2, H2AFZ, HIST1H4C, KIAA0101, and DUT, were screened and in silico validated through survival analysis and feature plot, hence, proposed as biomarkers for recurring pediatric glioblastoma. Utilising these five genes may improve disease prognosis and provide a crucial understanding of the molecular causes of recurrent pediatric glioblastoma.

Unraveling the Landscape of Pediatric Glioblastoma Biomarkers: A Comprehensive Review of Enhancing Diagnostics and Therapeutic Insights.

Glioblastoma, the most common and aggressive form of primary brain tumor, poses significant challenges to patients, caregivers, and clinicians alike. Pediatric glioblastoma is a rare and aggressive brain tumor that presents unique challenges in treatment. It differs from its adult counterpart in terms of genetic and molecular characteristics. Its incidence is relatively low, but the prognosis remains grim due to its aggressive behavior. Diagnosis relies on imaging techniques and histopathological analysis. The rarity of the disease underscores the need for effective treatment strategies. In recent years, the quest to understand and manage pediatric glioblastoma has seen a significant shift towards unraveling the intricate landscape of biomarkers. Surgery remains a cornerstone of glioblastoma management, aiming to resect as much of the tumor as possible. Glioblastoma's infiltrative nature presents challenges in achieving a complete surgical resection. This comprehensive review delves into the realm of pediatric glioblastoma biomarkers, shedding light on their potential to not only revolutionize diagnostics but also shape therapeutic strategies. From personalized treatment selection to the development of targeted therapies, the potential impact of these biomarkers on clinical outcomes is undeniable. Moreover, this review underscores the substantial implications of biomarker-driven approaches for therapeutic interventions. All advancements in targeted therapies and immunotherapy hold promise for the treatment of pediatric glioblastoma. The genetic profiling of tumors allows for personalized approaches, potentially improving treatment efficacy. The ethical dilemmas surrounding pediatric cancer treatment, particularly balancing potential benefits with risks, are complex. Ongoing clinical trials and preclinical research suggest exciting avenues for future interventions.

Glioblastoma stem cell metabolism and immunity.

Despite enormous efforts being invested in the development of novel therapies for brain malignancies, there remains a dire need for effective treatments, particularly for pediatric glioblastomas. Their poor prognosis has been attributed to the fact that conventional therapies target tumoral cells, but not glioblastoma stem cells (GSCs). GSCs are characterized by self-renewal, tumorigenicity, poor differentiation, and resistance to therapy. These characteristics represent the fundamental tools needed to recapitulate the tumor and result in a relapse. The mechanisms by which GSCs alter metabolic cues and escape elimination by immune cells are discussed in this article, along with potential strategies to harness effector immune cells against GSCs. As cellular immunotherapy is making significant advances in a variety of cancers, leveraging this underexplored reservoir may result in significant improvements in the treatment options for brain malignancies.

Abstract 7578: BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes

Abstract DIPGs (diffuse intrinsic pontine gliomas) are a particularly aggressive and malignant type of pediatric brain tumor. Given the tumor's location, surgery has a restricted role, and it is insensitive to most chemotherapies. Although fractionated radiation is the standard of therapy for DIPG, the cancer invariably worsens in 6-12 months. TP53 tumor suppressor protein mutations are found in 70-80% of DIPG tumors. These TP53 mutations have been linked to radiation resistance in DIPG patients. Our goal was to find new pharmacological compounds that would improve the radiation sensitivity of p53 mutant DIPG cell lines and to determine their molecular mechanism of action. Numerous bromodomain-containing protein 4 (BRD4) inhibitors have entered clinical trials in recent years, yielding promising outcomes in tumor treatment since BRD4 expression in DIPG is substantially higher than in neighboring normal brain tissue. So consequently, BRD4 is a target for DIPG treatment. ARV-825 is an emerging PROTAC (Proteolysis Targeting Chimera) compound which can cause rapid and persistent BRD4 protein degradation. However, delivering ARV-825 to pons is a major challenge owing to poor oral bioavailability of PROTACS. In this study, we used camel milk derived exosomes (CME) for delivery of ARV-825 to pons by both nasal and oral routes. To begin with, goat milk exosomes were also considered but CME was chosen because of its innate cytotoxicity against DIPG cell lines. CME were isolated using ultracentrifugation and nanoparticle tracking analysis and revealed a size distribution of 112.5 ± 2.8 nm, zeta potential of -26.38 ± 0.12 mV. CME (1*1011) were cytotoxic to both SF8628 (with a H3.3 K27M mutation) and SF188 (grade IV pediatric glioblastoma with wild type H3.3) cell lines and had 51% and 44% cell viability respectively. ARV-825 showed IC50 of 546.58 ± 22.14 nM and 608.94 ± 30.04 nM in SF8628 cells and SF188 cells respectively. ARV-825’s IC50 was lowered when ARV-825 loaded CME were used. ARV-825 was loaded using sonication and entrapment efficiency was found to be 42.96 ± 2.14 % by HPLC analysis. CME were labelled with Vybrant DiO (CME-DIO) and instilled into each nostril of Sprague Dawley rats daily. Brain sections were viewed with fluorescent microscope and CME-DIO were seen in olfactory region after 3 days and in the pons region after 5 days demonstrating the ability of CME to reach the target site. In-vitro permeability studies using MDCK cells showed approximately 9µg/mL (60%), 15µg/mL (80%) and 16µg/mL (100%) of ARV-825 permeation in 2, 4 and 24 hours respectively when loaded into CME. The results of the present study suggest the possibility of using CME as a viable delivery strategy for ARV-825 against DIPG. Further in vitro and in vivo studies are currently undergoing to validate the results and understand kinetics and the molecular mechanisms of ARV-825 loaded CME. Citation Format: Aakash Nathani, Mounika Aare, Li Sun, Yan Li, Mandip Singh. BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7578.

Myeloid cells as potential targets for immunotherapy in pediatric gliomas.

Pediatric high-grade glioma (pHGG) including pediatric glioblastoma (pGBM) are highly aggressive pediatric central nervous system (CNS) malignancies. pGBM comprises approximately 3% of all pediatric CNS malignancies and has a 5-year survival rate of approximately 20%. Surgical resection and chemoradiation are often the standard of care for pGBM and pHGG, however, even with these interventions, survival for children diagnosed with pGBM and pHGG remains poor. Due to shortcomings associated with the standard of care, many efforts have been made to create novel immunotherapeutic approaches targeted to these malignancies. These efforts include the use of vaccines, cell-based therapies, and immune-checkpoint inhibitors. However, it is believed that in many pediatric glioma patients an immunosuppressive tumor microenvironment (TME) possess barriers that limit the efficacy of immune-based therapies. One of these barriers includes the presence of immunosuppressive myeloid cells. In this review we will discuss the various types of myeloid cells present in the glioma TME, including macrophages and microglia, myeloid-derived suppressor cells, and dendritic cells, as well as the specific mechanisms these cells can employ to enable immunosuppression. Finally, we will highlight therapeutic strategies targeted to these cells that are aimed at impeding myeloid-cell derived immunosuppression.

Deposition of onco-histone H3.3-G34W leads to DNA repair deficiency and activates cGAS/STING-mediated immune responses.

Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.

Critical domains for NACC2-NTRK2 fusion protein activation.

Neurotrophic receptor tyrosine kinases (NTRKs) belong to the receptor tyrosine kinase (RTK) family. NTRKs are responsible for the activation of multiple downstream signaling pathways that regulate cell growth, proliferation, differentiation, and apoptosis. NTRK-associated mutations often result in oncogenesis and lead to aberrant activation of downstream signaling pathways including MAPK, JAK/STAT, and PLCγ1. This study characterizes the NACC2-NTRK2 oncogenic fusion protein that leads to pilocytic astrocytoma and pediatric glioblastoma. This fusion joins the BTB domain (Broad-complex, Tramtrack, and Bric-a-brac) domain of NACC2 (Nucleus Accumbens-associated protein 2) with the transmembrane helix and tyrosine kinase domain of NTRK2. We focus on identifying critical domains for the biological activity of the fusion protein. Mutations were introduced in the charged pocket of the BTB domain or in the monomer core, based on a structural comparison of the NACC2 BTB domain with that of PLZF, another BTB-containing protein. Mutations were also introduced into the NTRK2-derived portion to allow comparison of two different breakpoints that have been clinically reported. We show that activation of the NTRK2 kinase domain relies on multimerization of the BTB domain in NACC2-NTRK2. Mutations which disrupt BTB-mediated multimerization significantly reduce kinase activity and downstream signaling. The ability of these mutations to abrogate biological activity suggests that BTB domain inhibition could be a potential treatment for NACC2-NTRK2-induced cancers. Removal of the transmembrane helix leads to enhanced stability of the fusion protein and increased activity of the NACC2-NTRK2 fusion, suggesting a mechanism for the oncogenicity of a distinct NACC2-NTRK2 isoform observed in pediatric glioblastoma.

Glioma synapses recruit mechanisms of adaptive plasticity

The role of the nervous system in the regulation of cancer is increasingly appreciated. In gliomas, neuronal activity drives tumour progression through paracrine signalling factors such as neuroligin-3 and brain-derived neurotrophic factor1–3 (BDNF), and also through electrophysiologically functional neuron-to-glioma synapses mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors4,5. The consequent glioma cell membrane depolarization drives tumour proliferation4,6. In the healthy brain, activity-regulated secretion of BDNF promotes adaptive plasticity of synaptic connectivity7,8 and strength9–15. Here we show that malignant synapses exhibit similar plasticity regulated by BDNF. Signalling through the receptor tropomyosin-related kinase B16 (TrkB) to CAMKII, BDNF promotes AMPA receptor trafficking to the glioma cell membrane, resulting in increased amplitude of glutamate-evoked currents in the malignant cells. Linking plasticity of glioma synaptic strength to tumour growth, graded optogenetic control of glioma membrane potential demonstrates that greater depolarizing current amplitude promotes increased glioma proliferation. This potentiation of malignant synaptic strength shares mechanistic features with synaptic plasticity17–22 that contributes to memory and learning in the healthy brain23–26. BDNF–TrkB signalling also regulates the number of neuron-to-glioma synapses. Abrogation of activity-regulated BDNF secretion from the brain microenvironment or loss of glioma TrkB expression robustly inhibits tumour progression. Blocking TrkB genetically or pharmacologically abrogates these effects of BDNF on glioma synapses and substantially prolongs survival in xenograft models of paediatric glioblastoma and diffuse intrinsic pontine glioma. Together, these findings indicate that BDNF–TrkB signalling promotes malignant synaptic plasticity and augments tumour progression.

P11.66.A COMBINATION OF TROFOSFAMIDE AND ETOPOSIDE IN RECURRENT GLIOBLASTOMA

Abstract BACKGROUND Standard of care treatment options at glioblastoma relapse are still not well defined. Few studies indicate that the combination of trofosfamide plus etoposide may be feasible in pediatric glioblastoma patients. In this retrospective analysis, we determined tolerability and feasibility of combined trofosfamide plus etoposide treatment at disease recurrence of adult glioblastoma patients. MATERIAL AND METHODS We collected clinicopathological data from adult recurrent glioblastoma patients treated with the combination of trofosfamide and etoposide for more than four weeks (one course). A cohort of patients receiving empiric treatment at the investigators’ discretion balanced for tumor entity and canonical prognostic factors served as control. RESULTS A total of n=22 recurrent glioblastoma patients were eligible for this analysis. Median progression-free survival (3.1 versus 2.3 months, HR: 1.961, 95% CI: 0.9724-3.9560, p=0.0274) and median overall survival (9.0 versus 5.7 months, HR: 4.687, 95% CI: 2.034-10.800, p=0.0003) were significantly prolonged compared to the control cohort (n=17). In a multivariable Cox regression analysis, treatment with trofosfamide plus etoposide emerged as a significant prognostic marker regarding progression-free and overall survival. We observed high-grade adverse events in n=16/22 (73%) patients with hematotoxicity comprising the majority of adverse events (n=15/16, 94%). Lymphopenia was by far the most commonly observed hematotoxic adverse event (n=11/15, 73%). CONCLUSION This study provides first indication that the combination of trofosfamide plus etoposide is safe in adult glioblastoma patients and may be associated with prolonged survival in recurrent glioblastoma. Our data provide a reasonable rationale for follow-up of a larger cohort in a prospective controlled trial.

Citrullination Post-Translational Modification: State of the Art of Brain Tumor Investigations and Future Perspectives.

The present review aims to describe the state of the art of research studies investigating the citrullination post-translational modification in adult and pediatric brain tumors. After an introduction to the deimination reaction and its occurrence in proteins and polypeptide chains, the role of the citrullination post-translational modification in physiological as well as pathological states, including cancer, is summarized, and the recent literature and review papers on the topic are examined. A separate section deals with the specific focus of investigation of the citrullination post-translational modification in relation to brain tumors, examining the state of the art of the literature that mainly concerns adult and pediatric glioblastoma and posterior fossa pediatric tumors. We examined the literature on this emerging field of research, and we apologize in advance for any possible omission. Although only a few studies inspecting citrullination in brain tumors are currently available, the results interestingly highlighted different profiles of the citrullinome associated with different histotypes. The data outlined the importance of this post-translational modification in modulating cancer invasion and chemoresistance, influencing key factors involved in apoptosis, cancer cell communication through extracellular vesicle release, autophagy, and gene expression processes, which suggests the prospect of taking citrullination as a target of cancer treatment or as a source of potential diagnostic and prognostic biomarkers for potential clinical applications in the future.

A feasibility study of ultra-high dose rate mini-GRID therapy using very-high-energy electron beams for a simulated pediatric brain case

Ultra-high dose rate (UHDR, >40 Gy/s), spatially-fractionated minibeam GRID (mini-GRID) therapy using very-high-energy electrons (VHEE) was investigated using Monte Carlo simulations. Multi-directional VHEE treatments with and without mini-GRID-fractionation were compared to a clinical 6 MV volumetric modulated arc therapy (VMAT) plan for a pediatric glioblastoma patient using dose-volume histograms, volume-averaged dose rates in critical patient structures, and planning target volume D98s. Peak-to-valley dose ratios (PVDRs) and dose rates in organs at risk (OARs) were evaluated due to their relevance for normal-tissue sparing in FLASH and spatially-fractionated techniques. Depths of convergence, defined where the PVDR is first ≤1.1, and depths at which dose rates fall below the UHDR threshold were also evaluated. In a water phantom, the VHEE mini-GRID treatments presented a surface (5 mm depth) PVDR of (51±2) and a depth of convergence of 42 mm at 150 MeV and a surface PVDR of (33±1) with a depth of convergence of 57 mm at 250 MeV. For a pediatric GBM case, VHEE treatments without mini-GRID-fractionation produced 25% and 22% lower volume-averaged doses to OARs compared to the 6 MV VMAT plan and 8/9 and 9/9 of the patient structures were exposed to volume-averaged dose rates >40 Gy/s for the 150 MeV and 250 MeV plans, respectively. The 150 MeV and 250 MeV mini-GRID treatments produced 17% and 38% higher volume-averaged doses to OARs and 3/9 patient structures had volume-averaged dose rates above 40 Gy/s. VHEE mini-GRID plans produced many comparable dose metrics to the clinical VMAT plan, encouraging further optimization.

IMMU-20. EVALUATION OF B7-H3 NANOBODY CAR-T CELLS IN PEDIATRIC GLIOBLASTOMA

Abstract Chimeric antigen receptor (CAR)-T cell therapies are an undoubted success in childhood acute lymphoblastic leukemia; however, this success has not translated clinically to any childhood solid tumor. B7-H3 has been found to be highly expressed in pediatric brain tumors and correlates with tumor progression and poor prognosis. We utilized a novel nanobody based CAR-T cell which can bind tumor antigens with high affinity. We hypothesize that anti-B7-H3 nanobody CAR-T cells mediate more potent antitumor effects both in vitro and in vivo compared to existing antibody-based B7-H3 CAR-T cells. High expression of B7-H3 was confirmed on the surface of two pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. B7-H3 nanobodies isolated from camel phage libraries were cloned into lentiviral CAR-T cell constructs. We manufactured CAR-T cells by lentiviral transduction of human T cells. Glioblastoma tumor cells were co-cultured with B7-H3 CAR-T cells vs. control T cells. We confirmed B7-H3 specific cytokine release by CAR-T cells via ELISA and effective tumor cell killing via two different cytotoxicity assays (impedance or luciferase based). Nanobody based B7-H3 CAR-T cells exhibited statistically significant higher cytokine release and cytotoxicity compared to antibody based B7-H3 CAR-T cells. In vivo studies are currently evaluating the efficacy of intracerebroventricular delivery of B7-H3 CAR-T cells against two orthotopic glioblastoma models utilizing multiple human donor T cells in immunocompromised mice. We are simultaneously developing a syngeneic orthotopic glioblastoma model to evaluate the nanobody based B7-H3 CAR-T cell cross-reactive in mice utilizing an immunocompetent model. Careful investigation of CAR-T cell therapy in appropriate immunocompetent in vivo models is needed to develop effective immunotherapy for clinical application.

Giant-cell glioblastoma in a child: A rare case report and literature review

In Pediatric Giant Cell Glioblastoma (GCG) as a sub type of Glioblastoma (GBM), is a rare variant. However, it has a better prognosis.