bluuebunny/crossref_metadata_2025 huggingface dataset:
HGG-48. A comparative proteomic analysis of the cell membrane of histone 3 mutated paediatric high-grade gliomas in order to identify potential immunotherapeutic targets
Authors: Suresh Sneha , Khan Alina , Snary Anya , Pickering James , Rahman Ruman , Grundy Richard , Layfield Robert , Haque Farhana
Published: June 2022

Improvements in the treatments for childhood and adolescent brain tumours, High-Grade Glioma (pHGG) and Diffuse Intrinsic Pontine Glioblastoma (DIPG), have not advanced much and they continue to carry a very poor prognosis. These brain tumours are now defined by mutations affecting histone 3 proteins, indeed 80% of DIPGs harbour histone H3.1 and H3.3 K27M somatic mutations whilst 30% of pHGGs exhibit H3.3 G34R or G34V mutations. We hypothesized that the histone 3 mutant tumours will have distinct mutation specific surfactome or cell surface proteins. We therefore analysed the cell surface proteomics of H3.3 G34V and G34R mutated high-grade glioma in comparison to the H3 wild-type Glioma, to explore whether these proteins have the potential to be used as immunotherapeutic targets. We have at first isolated the cell membrane fractions from a range of patient cells carrying histone 3 mutations (G34R, G34V) relative to wild type histone 3. A comparative quantitative liquid chromatographic mass-spectrometry analyses of these cell surface membrane fractions were performed to identify specific targetable factors. The results of these were analysed with the focus in identifying proteins that can be targeted for tumour specific immune modulation or immunotherapeutic intervention. Results of these analyses will be presented.
HGG-52. Combinatorial modulation of hypoxic pathways leads to anti-tumoral effects in H3.3 K27M midline gliomas
Authors: Fuchs Quentin , Lhermitte Benoit , Foppolo Sophie , Hubsch Clemence , Martin Sophie , Dontenwill Monique , Entz-Werlé Natacha
Published: June 2022

Pediatric midline high-grade gliomas (pmHGG) are aggressive and incurable tumors of the central nervous system. There is a pressing need for novel therapeutic approaches to treat them. Therefore, proactive translational studies wish to go further discovering new targetable proteins and pathways. Our objectives are then to focus on the modulation of microenvironmental extrinsic features like intra-tumor hypoxia. To do so, we looked first on expressions of hypoxia biomarkers in a pool of patient-derived preclinical models of pmHGG and tested oxygen modulations, as well as hypoxia drug targeting. We designed subsequently our work in those models H3.3 mutated to evaluate balance between HIF1 and HIF2 expressions (immunofluorescence, RTqPCR, RNAseq and metabolomics) and to evidence the impact of hypoxia targeting combined to irradiation on cell proliferation, migration and metabolism. Hypoxia is inducing mainly HIF1 expression and its upstream and downstream pathways and is stabilizing HIF2 expression. Both HIFs are part of crucial survival signaling and represent targets to combine with irradiation. The use of their specific inhibitors shows an antiproliferative effect when HIF1 is downregulated. HIF2 inhibitors are stopping HIF2 transcriptional effect letting us uncover new pathways that this hypoxic inducible factor is regulating in pmHGG (stemness, glycolytic and aminoacid metabolism and histone expression). Together with irradiation this anti-hypoxic strategy seems to be highly effective on cell arrest and migration. Those results are confirming central roles of HIFs in pmHGG and their potencies in pmHGG therapies. The therapeutic efficiency is independent from p53 abnormalities in our models.
HGG-57. Barriers in the management of pediatric high-grade gliomas in a low-resource setting during the COVID era
Authors: Manigos Kirby , Malilay Oliver Ryan , Navarro Joseph Erroll , Seng Kenny , Alcazaren Jose Carlos , Golditum Jared Paul , Baticulon Ronnie
Published: June 2022

INTRODUCTION: High-grade gliomas account for <5% of all pediatric brain tumors with a 20% 5-year overall survival even with maximal safe resection followed by concurrent radiotherapy and chemotherapy. Patients in low- and middle-income countries already face delays and barriers to the treatment they require. The current COVID pandemic has added unique challenges to the delivery of complex, multidisciplinary health services to these patients. METHODOLOGY AND RESULTS: We retrospectively reviewed the records of four patients, ages 2-18 years old, with histologically confirmed high-grade glioma managed in a tertiary government institution from 2020-2021. Three of the patients had a supratentorial tumor and one patient had multiple tumors located in both supra- and infratentorial compartments. Neurosurgical procedures performed were: gross total excision (1), subtotal excision (2), and biopsy (1). The tissue diagnoses obtained were glioblastoma (3) and high-grade astrocytoma (1). Two patients survived and are currently undergoing adjuvant radiotherapy and chemotherapy. The remaining two patients expired: one from hospital-acquired pneumonia and the other from COVID-19 infection. DISCUSSION: Decreased mobility due to lockdowns, the burden of requiring negative COVID-19 results before admission for surgery, reduced hospital capacity to comply with physical distancing measures, the postponement of elective surgery to minimize COVID-19 transmission, physician and nursing shortages due to infection or mandatory isolation of staff, cancellation of face-to-face outpatient clinics, and hesitation among patients and their families to go to the hospital for fear of exposure were found to be common causes of delays in treatment. Also, the redirection of health resources and other government and hospital policies to handle the COVID-19 pandemic resulted in an overall delay in the delivery of health services. In particular, the management of pediatric patients with cancers, especially high-grade gliomas, was significantly disrupted.
HGG-60. Structural variants shape driver combinations and outcomes in pediatric high-grade glioma
Authors: Dubois Frank , Shapira Ofer , Greenwald Noah , Zack Travis , Wala Jeremiah , Tsai Jessica , Crane Alexander , Baguette Audrey , Hadjadj Djihad , Harutyunyan Ashot , Kumar Kiran , Blattner-Johnson Mirjam , Vogelzang Jayne , Sousa Cecilia , Kang Kyung Shin , Sinai Claire , Wang Dayle , Khadka Prasidda , Malkin Hayley , Ho Patricia , O'Rourke Ryan , Zhang Shu , Gold Rose , Deng Davy , Serrano Jonathan , Snuderl Matija , Jones Chris , Wright Karen , Chi Susan , Grill Jacques , Kleinman Claudia , Goumnerova Liliana , Jabado Nada , Jones David , Kieran Mark , Ligon Keith , Beroukhim Rameen , Bandopadhayay Pratiti
Published: June 2022

Pediatric high-grade gliomas (pHGGs), encompassing hemispheric and diffuse midline gliomas (DMGs), remain a devastating disease. The last decade has revealed oncogenic drivers including single nucleotide variants (SNVs) in histones. However, the contribution of structural variants (SVs) to gliomagenesis has not been systematically explored due to limitations in early SV analysis approaches. Using SV algorithms, we recently created, we analyzed SVs in whole-genome sequences of 179 pHGGs including a novel cohort of treatment naïve samples–the largest WGS cohort assembled in adult or pediatric glioma. The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases, including a novel SV amplifying a MYC enhancer in the lncRNA CCDC26 in 12% of DMGs and revealing a more central role for MYC in these cancers than previously known. Applying de novo SV signature discovery, we identified five signatures including three (SVsig1-3) involving primarily simple SVs, and two (SVsig4-5) involving complex, clustered SVs. These SV signatures associated with genetic variants that differed from what was observed for SV signatures in other cancers, suggesting different links to underlying biology. Tumors with simple SV signatures were TP53 wild-type but were enriched with alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A, and RB1 early in tumor evolution, and with extrachromosomal amplicons that likely occurred later. All pHGGs exhibited at least one simple SV signature but complex SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with the complex SV signatures SVsig4-5 were associated with shorter overall survival independent of histone type and TP53 status. These data inform the role and impact of SVs in gliomagenesis and mechanisms that shape them.
IMG-05 Imaging characteristics of CNS NB-FOXR2 – a retrospective and MULTI-INSTITUTIONAL DESCRIPTION OF 25 CASES
Authors: Tietze Anna , Lequin Maarten H , Bison Brigitte , Ivarsson Liz , Mirsky David , Jaju Alok , Löbel Ulrike , von Hoff Katja , Mankad Kshitij
Published: June 2022

BACKGROUND: The CNS neuroblastoma with forkhead box R2 (CNS NB-FOXR2) activation is one of the new embryonal tumor entities in the WHO CNS tumor classification 2021. The data currently available suggest a comparably favourable outcome after craniospinal irradiation and chemotherapy. The entity is poorly characterized on imaging. We therefore aimed to describe imaging features in a multi-center patient cohort. MATERIAL AND METHODS: Molecularly and histologically confirmed CNS NB-FOXR2 tumors were retrospectively collected at six pediatric oncology centers. The imaging features on preoperative MRI and CT data were recorded by 8 experienced pediatric (neuro)radiologists in consensus. RESULTS: Twenty-five patients were identified (13 females). The majority of tumors were large at presentation (mean 115 ± SD 83 ml), showed mostly none or little perifocal edema, enhanced only partly, were often calcified and/or hemorrhagic, were always T2w hyperintense to gray matter, and often had cystic and/or necrotic components. The min. mean ADC values were relatively low (mean 687.8 ± SD 136.3 in 10-6mm2/s). The tumors originated never infratentorial and were never centered in the midline. Metastases were rare. CONCLUSIONS: For the first time, imaging characteristics of the recently described CNS NB-FOXR2 are described in a multi-center cohort. The results of this study may assist in diagnosing these tumors preoperatively. Collaborative pooling of cases is important to gain insight into typical imaging features of rare tumors, especially in view of the increasing molecular-driven subtyping.
IMG-09. Characterisation of a panel ofin vivo models of paediatric-type diffuse high-grade glioma (PDHGG) using magnetic resonance imaging
Authors: Boult Jessica , Carvalho Diana , Kessler Ketty , Vinci Maria , Fernandez Elisabet , Bjerke Lynn , Fofana Mariama , Molinari Valeria , Carcaboso Angel , Jones Chris , Robinson Simon
Published: June 2022

Novel therapies for paediatric-type diffuse high-grade glioma (PDHGG) are urgently required. Orthotopic models using patient-derived material are invaluable tools in preclinical drug development as they retain key genetic/epigenetic features, eg. histone H3G34 or H3K27 alterations. Their evaluation in situ is vital and requires sensitive imaging techniques such as MRI. 12 diffuse hemispheric glioma (DHG; 2 DHG-G34) and 21 diffuse midline glioma (DMG; 17 DMG-K27M) tumours have been characterised using MRI following site-specific orthotopic implantation of patient-derived cells directly from tumour material or after minimal expansion as stem cell cultures. Of the 62 models implanted; 3 DHG and 10 DMG samples were not tumourigenic and 13 DHG/3 DMG models are currently under MRI surveillance. Tumours identified on T2-weighted (T2w)-images varied from a diffuse hyperintense signal to well-defined high contrast masses. Tumour growth in 5 DMG models was too diffuse for longitudinal monitoring with T2w-MRI. Once established, diffusion-weighted, T1/T2 mapping and contrast-enhanced MRI were used to further assess tumour phenotype. Quantitative data from 15 DMG models demonstrated higher water diffusivity and T2 than 10 DHG tumours, which suggests less tightly packed tumour cells but may also reflect the closer proximity of tumours growing in the thalamus/pons/cerebellum to the ventricular system. Lack of contrast-agent enhancement in 11 DMG and 6/10 DHG models indicated an intact blood-brain barrier (BBB), with heterogeneous disruption observed in 4 DHGs; H3-G34 had no bearing on BBB integrity. Upon serial re-implantation survival was shortened in 3/4 DHG and 2/6 DMG models, while quantitative MRI parameters remained similar. Likewise, when 2 DHG and 2 DMG models grown in 2D/3D in vitro were implanted in parallel, poorer survival/improved penetrance was associated with 3D-cultured cells with no difference in imaging phenotype. The study highlights the potential of non-invasive MRI to accurately evaluate the efficacy of novel therapeutics in these PDHGG models.
IMMU-01. Combining CD28 and 4-1BB costimulationin trans enhances the anti-glioma efficacy and persistence of B7-H3 CAR T cells in immune-competent brain tumor models
Authors: Haydar Dalia , Elayan Abdul , Yi Zhongzhen , Gottschalk Stephen , DeRenzo Chris , Krenciute Giedre
Published: June 2022

We and others have demonstrated that B7-H3 CAR T-cells have potent antitumor responses in xenograft models for brain tumors; however, these models do not recapitulate the immunosuppressive tumor microenvironment (TME) in patients with high-grade glioma. To evaluate the safety and efficacy of antigen-specific CAR T-cells, we adapted the immune-competent GL261 glioma model which recapitulates human disease and host immune barriers. We generated a library of B7-H3 CARs with different transmembrane (CD8, CD28), costimulatory (CD28, 4-1BB), and activation (ζ, mutζ) domains. We then compared their cytolytic activity, expansion, and anti-tumor activity. Results show that B7-H3 CARs with CD28 transmembrane and costimulatory domains have superior efficacy compared to CARs with CD8 and 4-1BB domains. Additionally, CARs with mutated ζ activation domain have better overall persistence. However, providing costimulation signals through CD28 or 4-1BB alone does not induce superior anti-glioma efficacy of B7-H3 CAR T-cells in vivo. Thus, we next investigated whether incorporating 4-1BB signaling into CD28-based CARs using in trans design enhances the therapeutic efficacy of B7-H3 CAR T-cells. We found that in repeat stimulation assays, surface expression of 4-1BBL enhanced expansion of B7H3 CAR T-cells at least 300-folds more than T-cells with CD28 or 4-1BB costimulatory domains alone. Additionally, 4-1BBL expression significantly enhanced the sequential killing capacity compared to CD28- or 41BB-based B7-H3 CAR T-cells. High dimensional flow cytometry analysis of GL261 tumors post CAR T-cell injection revealed unique immune clusters including dendritic cells and lymphoid predominant populations in mice treated with 4-1BBL expressing CARs. Thus, expression of 4-1BBL on CD28-based CARs reshaped the TME and enhanced persistence and anti-glioma efficacy of B7-H3 CAR T-cells. Studies examining transcriptional and epigenetic programs, and TME/CAR T-cell interactions are in progress. Results will define pathways that dictate CAR T-cell performance and will identify unique mechanisms for further improvements utilizing other members of TNF-superfamily.
IMMU-05. Integrative transcriptomic analysis of pilocytic astrocytomas reveals CNS region-associated characteristics
Authors: Rozowsky Jacob S , Kranendonk Mariëtte E G , van Meeteren Antoinette Y N Schouten- , Meijer Lisethe , Hoving Eelco W , Calkoen Friso G , Wesseling Pieter , Kester Lennart A , van der Lugt Jasper
Published: June 2022

BACKGROUND: Recent insights highlight how the initiation and growth of gliomas is governed by interactions between glioma stem-like cells and stromal and immune cells in the tumor microenvironment. For pilocytic astrocytomas, the most common pediatric CNS tumor, this relationship is so far less explored. To this end, we used transcriptomic methods to investigate inter-patient heterogeneity, and the stromal and immune microenvironment of pilocytic astrocytomas. MATERIALS AND METHODS: In this study, we collected clinical data and tissue of 90 pre-treatment pilocytic astrocytomas from different CNS compartments: posterior fossa (n=57), supratentorial (n=23), and spinal (n=10). The median age at primary resection was 8 (0-16) years, and 66% (n=59) of our cohort was male. From 10 of these patients, we collected post-treatment samples after re-growth of the tumor as well. We characterized all samples by bulk RNA-sequencing and DNA methylation profiling, and selected a subset (n=10) samples for single-nucleus RNA-sequencing. RESULTS: Principal component analysis and unsupervised clustering of bulk sequencing data revealed gene expression patterns correlating to the CNS location of the tumor, consistent with prior reports. Using differential expression and functional pathway analysis, we found CNS region-associated enrichment of cell-cycle, developmental, and inflammatory-related pathways. With respect to the glioma immune microenvironment, supratentorial tumors were enriched in gene-sets related to T-cell activation and cytotoxicity, while spinal tumors had lowest expression of immune-related genes. Moreover, spinal tumors were enriched in pathways related to cell division, nucleotide synthesis, and neurodevelopment. To resolve cell-type expression programs of glioma and immune cells in the microenvironment, we collected and analyzed snRNA-seq data of 10 pilocytic astrocytomas, as well as harmonized our findings with a pre-existing dataset from Vladoiu, 2019. CONCLUSION: Our integrative transcriptomic analysis of pilocytic astrocytomas highlights CNS region-associated differences in expression programs of the glioma cells and in the immune cell composition of the tumor microenvironment.
IMMU-08. Nivolumab with or without ipilimumab in pediatric patients with high-grade CNS malignancies: efficacy, safety, biomarker, and pharmacokinetic results from Checkmate 908
Authors: Dunkel Ira J , Cohen Kenneth , Foreman Nicholas K , Hargrave Darren , Lassaletta Alvaro , André Nicolas , Hansford Jordan R , Hassall Tim , Eyrich Matthias , Gururangan Sridharan , Bartels Ute , Gajjar Amar , Howell Lisa , Warad Deepti , Pacius Misena , Tam Rachel , Wang Yu , Zhu Li , Doz François
Published: June 2022

BACKGROUND: Limited data exist regarding checkpoint inhibitor efficacy for pediatric CNS malignancies. METHODS: CheckMate 908 is an open-label, sequential-arm, phase 1b/2 study investigating nivolumab (NIVO) and NIVO + ipilimumab (IPI) in 5 cohorts of pediatric patients previously treated with standard-of-care (NCT03130959). Patients received NIVO-3mg/kg Q2W or NIVO-3mg/kg + IPI-1mg/kg Q3W (4 doses) followed by NIVO-3mg/kg Q2W. Primary endpoints included OS (newly diagnosed DIPG) and PFS (other CNS cohorts); secondary endpoints included other efficacy metrics/safety. Exploratory endpoints included pharmacokinetics/biomarker analyses. Comparisons between treatments/cohorts were not planned. RESULTS: At data cutoff (13-Jan-2021), 166 patients received NIVO (n=85) or NIVO+IPI (n=81) at median (m) ages of 10.0yrs (range, 1-21) and 11.0yrs (1-21), respectively. In newly diagnosed DIPG, mOS (80% CI) was 11.7mos (10.3-16.5) with NIVO (n=23) and 10.8mos (9.1-15.8) with NIVO+IPI (n=22). In recurrent/progressive HGG, mPFS (80% CI) was 1.7mos (1.4-2.7) with NIVO (n=16) and 1.3mos (1.2-1.5) with NIVO+IPI (n=15). In relapsed/resistant medulloblastoma, mPFS (80% CI) was 1.4mos (1.2-1.4) with NIVO (n=15) and 2.8mos (1.5-4.5) with NIVO+IPI (n=15). In relapsed/resistant ependymoma, mPFS (80% CI) was 1.4mos (1.4-2.6) with NIVO (n=12) and 4.6mos (1.4-5.4) with NIVO+IPI (n=10). In other recurrent/progressive CNS tumors, mPFS (95% CI) was 1.2mos (1.1-1.3) with NIVO (n=19) and 1.6mos (1.3-3.5) with NIVO+IPI (n=19). Median treatment duration was 2.1mos (range, 0-41.7+ [NIVO]/0-29.6+ [NIVO+IPI]). Grade 3/4 treatment-related AEs occurred in 14.1% (NIVO) and 27.2% (NIVO+IPI) of patients. NIVO and IPI first dose trough concentrations were lower in youngest and lowest-weight patients. Baseline tumor PD-L1 expression was not associated with survival. Tumor mutational burden was high in 1 patient (NIVO+IPI) with HGG (OS=11.0mos). CONCLUSIONS: NIVO±IPI demonstrated no clinical benefit in pediatric patients with high-grade CNS malignancies, consistent with available historical data. The safety profiles were manageable.
IMMU-19. Outcomes of Pediatric Patients with High-Risk CNS Tumors Treated with Multi-tumor associated antigen specific T cell (TAA-T) therapy: the ReMIND trial
Authors: Hwang Eugene , Fortiz Fernanda , Cruz Russell , Geiger Ashley , Grant Melanie , Lang Haili , Shibli Abeer , Burnett Sianna , Lazarski Chris , Tanna Jay , McCann Chase , Hoq Fahmida , Hanley Patrick , Kilburn Lindsay , Rood Brian , Packer Roger , Bollard Catherine
Published: June 2022

BACKGROUND: The ReMIND trial hypothesizes that autologous T-cells specific for three tumor-associated antigens (TAA) -WT1, PRAME, and survivin- will be safe and elicit anti-tumor immunity in pediatric patients with CNS cancer. METHODS: Patients (n=25) received autologous TAA-T for newly-diagnosed DIPG (Stratum A, up to 4x107/m2) or recurrent CNS malignancies (Stratum B, up to 8x107/m2) in this dose-escalation study (NCT03652545) and were monitored for toxicity and response. RESULTS: Autologous TAAT products were successfully manufactured from 28 patients. Using IFN-γ ELISPOT assay, 10/11 evaluable products had specificity for 1-3 TAAs. 25 patients received TAA-T (6 in Stratum A and 19 in Stratum B) and completed the 45-day safety monitoring period. Twenty-four (96%) had no dose limiting toxicities (DLT), but 1 (4%) patient with DIPG experienced a DLT related to potential immune-mediated pseudoprogression. Median overall survival for patients with DIPG (Stratum A) was 14 months (range, 6-32 months). Median progression-free survival (PFS) for Stratum B patients was 8 months (range, 2-26+ months), which exceeded their preceding median duration of disease stability of 2 months (range, 1-5 months). Plasma cytokine profiles demonstrated infusion-related immune cytokine responses. CONCLUSIONS: In summary, TAAT had a favorable toxicity profile (4%) especially compared to CAR-T therapy and may elicit anti-tumor immune responses that contribute to prolonged survival. Immunobiology studies and response assessments are ongoing for both strata. Based on these encouraging preliminary results, we have added a stratum that includes prescribed lymphodepletion pre TAA-T administration at a cell dose of 8x107/m2. Further, we plan to add an additional stratum to allow direct administration of TAA-T into the CNS via an Ommaya reservoir.
IMMU-21. Targeting the adenosinergic immune suppression pathway in high grade glioma synergizes with innate immune checkpoint blockade
Authors: Liu Yifan , Chetty Senthil Lakshmana , Cole Allison , Cruz Joselyn Cruz , Hoffmeyer Eric , Vibhakar Rajeev , Venkataraman Sujatha , Mitra Siddhartha
Published: June 2022

Immune response in the tumor microenvironment is modulated by the conversion of eATP to AMP to adenosine via the ecto-enzymes CD39 and CD73. Overexpression of CD73 in tumors leads to an increase in extracellular adenosine concentration and decrease in eATP, resulting in immune suppression. In this study, CD73 knockouts were created in adult and pediatric glioma cell lines (DIPG17, T3691, BT245) and used to quantify the effects of CD73 knockout on macrophage phagocytosis in the presence of extracellular AMP. In cell line DIPG17, CD73 knockout significantly increased phagocytosis with and without external AMP compared to the WT. In the WT condition, addition of AMP significantly reduced phagocytosis, while this decrease was not significant in the CD73-KO. The addition of anti-CD47 antibody significantly increased phagocytosis with external AMP in the WT condition. Similar results were obtained with cell line T3691. However, CD73 knockout combined with anti-CD47 treatment and external AMP showed significantly greater phagocytosis than the WT condition. This suggests that a combination of anti-CD73 and anti-CD47 treatment may be more effective in the tumor microenvironment. Lastly, in cell line BT245, CD73 knockout significantly increased phagocytosis as seen in the other cell lines. However, no significant difference was observed between WT and CD73-KO with extracellular AMP or anti-CD47. In vivo studies were conducted in orthotopic xenograft mouse models with DIPG17 CD73 knockout cells. Combination treatment with anti-CD47 antibody significantly decreased tumor burden and prolonged survival in the CD73-KO tumors compared to anti-CD47 treated DIPG13-WT tumors. We conclude that the CD73 adenosinergic pathway and the CD47-SIRPα pathway may present a target for immunotherapy in pediatric and adult gliomas.
IMMU-22. Safely targeting GD2 in thalamic diffuse midline glioma with mRNA CAR T cells
Authors: Foster Jessica , Griffin Crystal , Stern Allison , Madsen Peter , Storm Phillip , Resnick Adam
Published: June 2022

Chimeric antigen receptor (CAR) T cells targeting the disialoganglioside GD2 have shown promise as a therapeutic for diffuse midline glioma (DMG). However, prior studies raised significant concerns of neurotoxicity and fatality when using virally transduced CAR T cells against midline thalamic tumors. Building upon our prior work optimizing mRNA for use in CAR T cells (Hum Gen Ther, 2019), we hypothesized repeated dosing of transient GD2-directed mRNA CAR T cells could be employed for safe and effective treatment of thalamic DMG. GD2-directed CAR T cells were created using mRNA encoding the 14G2a single chain variable fragment paired with 41BB and CD3-zeta co-stimulatory domains and transfected into human T cells. CAR T cells were tested against the murine thalamic DMG xenograft 7316-6349 via locoregional delivery with an indwelling infusion catheter for repeated dosing. The previously reported fatal neurotoxicity observed in mice using lentiviral CAR T cells could be recapitulated with aggressive dosing. Four doses of 5 x 106 mRNA CAR T cells delivered intratumorally twice a week resulted in median overall survival of 9 days for GD2-treated mice compared to >30 days for CD19-treated controls (p<0.01). This toxicity could be avoided by decreasing the dose and timing of infusions to 2 x 106 mRNA CAR T cells delivered once weekly. Bioluminescent imaging showed regression of tumor in GD2-treated mice compared to CD19-treated controls (radiance fold change -3 x106 versus +20x106 p/sec/cm2/sr, p<0.01). Notably, non-tumor bearing mice treated with GD2-directed CAR T cells quickly developed fatal neurotoxicity within 14 days, suggesting on-target/off-tumor effect of the CAR T cells and a very narrow therapeutic window in the brain. These data highlight the utility of titratable mRNA-based CAR T cell therapy for CNS tumors and establish GD2-directed mRNA CAR T cells as a safe and effective method for treating thalamic DMG.
LGG-09. A Nationwide Service Evaluation of Safety, Radiologic and Visual Outcome Refining Bevacizumab-based Treatments in Children with Progressive Low-Grade Glioma
Authors: Green Katherine , Panagopoulou Paraskevi , D’Arco Felice , O'Hare Patricia , Bowman Richard , Walters Bronwen , Dahl Christine , Jorgensen Mette , Patel Pritesh , Slater Olga , Ahmed Rehana , Bailey Simon , Carceller Fernando , Collins Rhiannon , Corley Elizabeth , English Martin , Hayden James , Howells Lisa , Kamal Ahmed , Kilday John Paul , Lowis Stephen , Lumb Blanche , Micic Thomas , Mitra Dip , Pace Erika , Picton Susan , Pizer Barry , Shafiq Ayad , Uzunova Lena , Wilson Shaun , Wayman Harriet , Hargrave Darren , Opocher Enrico
Published: June 2022

INTRODUCTION: Bevacizumab/Irinotecan is currently 3rd-line treatment in the UK for progressive Paediatric Low-Grade Glioma (PLGG) based on limited evidence. A nationwide service evaluation was conducted to review its safety and efficacy amongst a larger cohort.

METHODS: Data from children with PLGG receiving Bevacizumab-based Treatments (BBT) from 11 UK Centres (2009-2020) were reviewed. Radiological and visual outcomes were based on standardized measurements. Clinical-radiological correlation was investigated. Time to progression from BBT stop, progression free-survival (PFS) curves and multivariate analysis of prognostic factors (p 0.05) were performed.

RESULTS: 88 children with PLGG (88% OPG, 24% NF1) had BBT for radiological (43%), visual (20%) or combined (27%) progression, after 40 months (median) from diagnosis. Amongst OPG cases, visual acuity (VA) per eye (better/worse) before BBT was logMAR 0.0-0.3 (23/7) 0.3 - 1.0 (27/20), > 1.0 (14/18) and LP/NLP (8/27), with 19/8 children respectively blind (LP/NLP) in one or both eyes. Bevacizumab 10 mg/kg every 14 days (median 24 doses) was given as 3rd line+ with Irinotecan (85%) or alongside 1st/2nd line chemo (15%) leading to remarkable radiological (88%) and visual (74%) responses (stable or improved) within 3-6 months, with limited toxicity. 12% progressed on treatment, and 8% died unrelated to BBT. After initial response 65% progressed at a median of 8 months (4 - 23) after BBT, resulting in 3-year-all-causes-PFS of 16% and 3-yr-visual-PFS of 45% from start of BBT. Visual concordance with MRI was poor (36%) but increases (47%) when better-eye determines visual outcome. Lack of NF1 and diencephalic syndrome (DS) at presentation were independent negative prognostic factors for PFS.

CONCLUSIONS: A remarkable but transient effect of BBT has been confirmed. Visual > radiological responses can be sustained after BBT. Variations in current BBT strategies justifies further research, including the potential upfront use alongside conventional first-line chemotherapy as sight-saving strategy.
LGG-11. Analysis of neurosurgical complications in pediatric supratentorial midline low-grade glioma – results from the German LGG studies
Authors: Weiß Sarah , Schulz Matthias , Kandels Daniela , Driever Pablo Hernáiz , Witt Olaf , Thomale Ulrich-Wilhelm , Gnekow Astrid , le Simon Michè
Published: June 2022

OBJECTIVE: Around 80% of all pediatric low-grade glioma (LGG) patients undergo at least one tumor surgery. Interventions in the supratentorial midline (SML) are particularly challenging due to the proximity of eloquent areas, yet associated complications are scarcely reported. We investigated the frequency of neurosurgical complications and related impairments and aimed at identifying risk factors for their appearance related to patient characteristics or the procedure. PATIENTS AND METHOD: Records were retrospectively analyzed from 321 patients with SML-LGG from the successive multicenter German LGG studies, who underwent neurosurgery at 63 hospitals between May 12th,1998 and June 27th, 2020. RESULTS: 543 operations (235 resections, 168 biopsies, 140 non-tumor interventions) were performed on 321 patients (54% male, median age 9 years, 11% NF1 positive, 43% visual pathway glioma). Surgical mortality rate was 0,93% (n=3). Applying the Drake classification postoperative surgical morbidity was observed in 259 cases (47,7%), medical morbidity in 103 cases (19%). 30-day persistence rate of newly developed neurological deficits was 44,8% (65/165 cases); neuroendocrine impairment affected 57 patients (17,8%), visual deterioration 34 (10,6%). Complications/impairments following resections were associated with patient age below 3 years at operation, tumor volume above 80 cm3, presence of hydrocephalus prior to surgery, complete resection, intervention in centers with fewer reported resections and surgery performed between 1998-2006 by univariate analysis. In contrast, the neurosurgical approach, tumor location, NF1 status as well as previous antineoplastic treatment were not associated with the frequency of complications. Regarding biopsies, open biopsies showed significantly more surgery-associated complications/impairments compared with stereotactic procedures. CONCLUSIONS: Neurosurgery-associated complications and impairments were frequent in pediatric patients with supratentorial midline LGG undergoing open surgery in the German LGG-studies. We identified six patient- and institution-associated factors that may increase the risk for surgical complications. Skills at the treating center and extent of resection should be considered appropriately prior to intervention.
LGG-27. Molecular implications of mitogen-activated protein kinase pathway inhibition by the MEK inhibitor trametinib in BRAF-fusion-driven pediatric pilocytic astrocytoma
Authors: Sigaud Romain , Stefanski Anja , Selt Florian , Hielscher Thomas , Usta Diren , Kocher Daniela , Picard Daniel , Büdenbender Isabel , Remke Marc , Pfister Stefan M , Jones David T W , Brummer Tilman , Witt Olaf , Milde Till
Published: June 2022

Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by driving alterations in the mitogen-activated protein kinase (MAPK) pathway, leading to its constitutive activation and modulating the balance between cell proliferation and oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP) factors. This makes PA susceptible to MAPK inhibitor (MAPKi) therapies, which show encouraging results in phase 1/2 clinical trials. However, little is known about the molecular implications of MAPK inhibition in PA. The DKFZ-BT66 cell line, derived from a primary KIAA:BRAF-fusion positive PA, was used as a model system. DKFZ-BT66 were treated with the MEKi trametinib for different durations in both proliferative and senescent states. Gene expression was analyzed by gene expression profiling and protein expression/phospho-regulation by data-dependent mass spectrometry followed by label-free quantitative analysis. A time course analysis based on differentially expressed genes and phosphorylated proteins was performed, followed by a single-sample gene set enrichment analysis (ssGSEA) and kinase substrate enrichment analysis, respectively. Differential gene expression analysis revealed that MEK inhibition led to the inhibition of the OIS/SASP gene programs in senescent DKFZ-BT66, with downregulation of key OIS/SASP partners such as IL1B on the protein level. This functionally translated into a de-sensitization of these cells towards the senolytic agent navitoclax. ssGSEA showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including FGFR, NTRK and TGFB pathways) were upregulated, in both proliferating and senescent DKFZ-BT66. This data indicates that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators in proliferating and senescent PA cells, identifying putative co-targets that could help increase treatment’s efficacy. Validation of these targets by post-translational modification enrichment analysis of the phospho-proteomics dataset is ongoing.
LGG-41. The clinical and molecular landscape of gliomas in adolescents and young adults
Authors: Bennett Julie , Nobre Liana , Sheth Javal , Ryall Scott , Fang Karen , Johnson Monique , Negm Logine , Chung Jiil , Komosa Martin , Nunes Nuno M , Fat Mary Jane Lim , Perry James , Sahgal Arjun , Detsky Jay , Bouffet Eric , Naz-Hazrati Lili , Dirks Peter , Ertl-Wagner Birgit , Kongkham Paul , Zadeh Gelareh , Mason Warren , Climans Seth , Cusimano Michael , Das Sunit , Gao Andrew , Tsang Derek , Nguyen Lananh , Laperriere Normand , Keith Julia , Munoz David , Tabori Uri , Hawkins Cynthia
Published: June 2022

OBJECTIVE: Gliomas in adolescents and young adults (AYA) are commonly treated with a standard chemo-radiation approach based on data from adults. The clinical impact of paediatric-type alterations in these tumours is unknown. METHODS: We compiled a multi-institutional cohort of patients diagnosed with glioma between 15-39.9 years over 20 years. Complete molecular analysis, therapeutic data and outcome was collected. For specific alterations, analysis included patients aged 0-39.9 years. RESULTS: A total of 1900 patients with 876 AYA gliomas were included. Ongoing analysis reveals genetic alterations in 95% of available tumours. IDH-mutant tumours account for only 53%, while paediatric-type mutations were found in 35% of AYA tumours with IDH-WT GBM accounting for the remaining 12%. The most common paediatric alterations in AYAs included BRAF p.V600E (11%) and FGFR alterations (6%) while BRAF fusions, H3 p.K27M and H3.3 p.G34R were rarely observed (4%, 4% and 1% respectively). BRAF fused tumours with non-canonical binding partners were enriched in AYAs. Analysis of BRAF-V600E gliomas between ages 0-40 revealed increased tendency for malignant tumours in patients >20 years suggesting malignant transformation possibly due to higher rate of secondary hits including TP53, CDKN2A and ATRX mutations. This resulted in worse overall-survival for AYA patients with BRAF-V600E glioma when compared to children under 20 years (p=0.0032). Ten-year OS of 100%, 90% and 95% was seen for BRAF fused, BRAF-V600E and FGFR-altered AYA low grade glioma respectively, compared to 14% and 25% for BRAF-V600E and FGFR-altered high grade glioma. In contrast, continuous decline was observed in the IDH-mutant gliomas with 10-year OS of 50% which declined to 29% at 15 years. CONCLUSIONS: Gliomas in AYA are enriched for paediatric-type alterations with distinct molecularly-based outcomes. As these tumours carry different outcomes than childhood glioma and may respond to targeted inhibitors, AYA gliomas would benefit from comprehensive diagnostic and therapeutic approaches.
LGG-44. Multi-omic analysis reveals integrated signalling networks in paediatric low-grade glioma
Authors: Woodward Lewis , Jones Tania A , Patel Ankit , Dokal Arran D , Stone Thomas J , Rajeeve Vinothini , Cutillas Pedro R , Jones David T W , Hargrave Darren , Jacques Thomas S , Sheer Denise
Published: June 2022

Paediatric low-grade gliomas (pLGGs) are the most common type of childhood CNS tumours. Our study included pilocytic astrocytomas (PAs; KIAA1549:BRAF), glioneuronal tumours (GNTs; BRAFV600E) and location-matched controls. We initially performed kinase substrate enrichment analysis (KSEA) to infer differential kinase activity, which allowed us to identify altered signalling networks in the two tumour types. Here we report the integration of these kinase signalling networks together with total proteomics, transcription factor enrichment analysis (TFEA) and transcriptomics (coding and non-coding). Total proteomic profiling confirmed an increase in proteins involved in cell cycle, inflammatory response and signal transduction in PAs, whilst there was an increase in proteins promoting cell growth, immune response and inflammation in GNTs. TFEA was performed using the DoRothEA database to identify master transcriptional regulators. We observed signiﬁcant activation of transcription factors (TFs) that are direct targets of MAPK signalling in both tumour types. Notable differences include the higher activation of NF-kB/STAT TFs in PAs and the increased activation of RFX1/2 in GNTs. Next, we constructed kinase-TF networks and identified multiple kinases targeting STAT3 in PAs and STAT1/3 in GNTs. Pathway analysis of RNA-Sequencing data showed enrichment of NF-kB in both tumours and repression of E2F target genes (PA) and reduced expression of MYC target genes (GNT). We developed a BRAF-OIS signature and found 23 genes commonly enriched in both tumour types, highlighting shared senescence-associated targets. MicroRNA profiling identified upregulation of microRNAs that target MAPK and NF-kB signalling networks, and many down-regulated microRNAs with tumour suppressive roles. Finally, we identified several lncRNAs known to be differentially expressed in glioma and, whilst their mechanism(s) of action are varied, they are thought to act with other well-established regulators to fine-tune cellular processes. Taken together, we present a comprehensive signalling network as a framework for studying pLGGs.
LGG-64. A Phase II Study of Pegylated Interferon in Children with Recurrent or Refractory and Radiographically or Clinically Progressive Juvenile Pilocytic Astrocytomas and Optic Pathway Gliomas (NCT02343224)
Authors: Aguilera Dolly , Mazewski Claire , Janss Anna , MacDonald Tobey , Goldman Adam , Leong Tracy , Castellino Robert Craig
Published: June 2022

Unresectable Juvenile Pilocytic Astrocytomas (JPA) and Optic Pathway gliomas (OPG) are chronic diseases that can have solid +/- cystic components. We wanted to evaluate the objective response to pegylated interferon Alpha2B in this group of patients in a prospective single arm Phase II clinical trial. Eligibility criteria: age 3 -25 years, patients with neurofibromatosis (NF) were eligible, evidence of measurable disease in MRI, no limitation in the number of prior therapies including chemotherapy and radiation. Exclusion criteria: prior pegylated interferon exposure, less than 2 years from radiation, active autoimmune disease. Subjects enrolled received pegylated interferon 1 mcg/kg/dose SQ weekly, to a max dose of 150mcg/dose in 28 day cycles for up to 2 years. The study design is a Simon two stage design. If no complete or partial responses among the first 9 patients, the study will terminate. Nine subjects enrolled: 4 females, 5 males, median age of 11years, 6 Caucasians, 3 African Americans. Two subjects with NF. Molecular findings KIAA-BRAF fusion (6), V600E mutation (1), CDK2A loss (1). Location: brain (7), brain and spine (2). We enrolled a heavily pre-treated population, patients with prior radiation ( 1), nine with prior chemotherapy, the average number of regimens 4 (range 2-6). No complete responses or partial responses were seen. Two patients with prolonged stable disease 75+months and 66+ months. At 12 and 24 month EFS 76.2% (95%CI52.1-100%). Median EFS has not been reached. The 12 and 24 months survival estimate 75% (95% CI 50.3-100%), median survival has not been reached. Side effects as expected mostly grade 1-2. No grade 4 event related to pegylated interferon were seen. This is the first report of pegylated interferon in OPG and JPA, two patients with prolonged stable disease suggesting that pegylated interferon can offer potential benefit in this population and additional studies are important.
MEDB-63. Deciphering the role of LIN28B in Group 3 medulloblastoma
Authors: Shahab Shubin , Sun Jessica , Schnepp Robert , MacDonald Tobey , Kenney Anna
Published: June 2022

BACKGROUND: Children with Group 3 medulloblastoma (MB) have a very poor long-term outcome and many do not survive beyond 5 years. Several drivers for Group 3 MB have been identified but none have resulted in targeted therapy to date. LIN28B is a stem cell factor that is upregulated in Group 3 medulloblastoma and is associated with worse survival. Here we investigate the role of the LIN28B pathway in Group 3 MB development. Pharmacologic inhibition of the LIN28B pathway is feasible and may provide a unique opportunity to target this tumor. METHODS: Using LIN28B knockdown and overexpression in G3 MB cells we test LIN28B’s effect on proliferation, self-renewal and metastasis. Similarly, we used shRNAs targeting PBK and demonstrate a similar effect on G3 MB growth. We also investigate the role of let-7 as a target of LIN28B by introducing let-7 mimetics and overexpression vectors into MB cells. Finally, we use a LIN28 inhibitor 1632 and a PBK inhibitor HITOPK032 to treat G3 MB cell lines and then assess their impact on proliferation and apoptosis. RESULTS: We find that down-regulation of LIN28B or PBK using shRNA results in significant reduction in cell proliferation. In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation. LIN28B knockdown also significantly (p< 0.01) increases survival in mice with orthotopic Group 3 tumors. The LIN28 inhibitor 1632 also leads to significant reduction in G3 MB growth through decreased cell cycle entry and increased apoptosis. In addition, HITOPK032 also demonstrates significant reduction in Group 3 MB cell proliferation at low (nanomolar to low micromolar) concentration. CONCLUSIONS: Our study establishes a critical role for the LIN28B-let-7-PBK pathway in Group3 MB and provides encouraging preliminary preclinical results for drugs that target this pathway.
MEDB-69. Clinical and molecular meta-analysis of three major medulloblastoma clinical trials (ACNS0331, SJMB03, ACNS0332) uncovers novel strategies to improve risk-stratified therapy
Authors: Smith Kyle S , Billups Catherine A , Dhanda Sandeep K , Bihannic Laure , Vasilyeva Aksana , Li Yimei , Michalski Jeff M , Olsen James M , Leary Sarah , Fouladi Maryam , Gajjar Amar , Onar Arzu , Northcott Paul A , Robinson Giles W
Published: June 2022

BACKGROUND: Given the vast molecular heterogeneity present within medulloblastoma (MB) and considerable differences in therapy, we performed a meta-analysis of three large, recently published, prospective clinical trials (ACNS0331, SJMB03, ACNS0332) comprising 898 children with newly-diagnosed MB to shape future therapy.

METHODS: Molecular subgroups, subtypes, and copy number variations were uniformly procured from DNA methylation profiles and mutations from next-generation sequencing. Patients were stratified into six clinically homogeneous groups for cross-trial comparisons: (1) ACNS0331_LDCSI - patients with non-metastatic (M0), non-residual (R0), non-anaplastic MB treated with low-dose (LD) craniospinal irradiation(CSI); (2) ACNS0331_SDCSI - patients with M0R0 non-anaplastic MB treated with standard-dose(SD) CSI; (3) SJMB03_SDCSI - patients with M0R0 non-anaplastic MB treated with SDCSI; (4) SJMB03_HDCSI - patients with metastatic (M+) MB treated with high-dose (HD) CSI; (5) ACNS0332_HDCSI - patients with M+ MB treated with HDCSI; (6) ACNS0332_HDCSI_Carbo - patients with M+ MB treated with HDCSI and carboplatin.

RESULTS: 803 (WNT=125, SHH=122, G3=189, G4=367) of 898 patients formed the cohort. No significant difference was observed between the event-free survival (EFS) from ACNS0331_SDCSI and SJMB03_SDCSI or from SJMB03_HDCSI and ACNS0332_HDCSI when analyzed as a whole or by subgroup. ACNS0331_LDCSI outcome was inferior to the combined ACNS0331_SDCSI + SJMB03_SDCSI cohorts (p<0.001) and in G3 (p=0.030). ACNS0332_HDCSI_Carbo EFS was superior to ACNS0332_HDCSI + SJMB03_HDCSI only in G3/G4_subtype III (p=0.045). Additional molecular risk factor analysis identified M0R0 G3/G4_subtype VII and SHH without high-risk features as very low risk (>90% EFS) and M0R0 G3/G4_subtype III as high risk (<40% EFS).

CONCLUSION: The comparable results observed across trials presents a welcome opportunity to reduce toxicity by eliminating excessive doses of chemotherapy (i.e. vincristine, cisplatin, and cyclophosphamide) from therapy. Furthermore, these results support molecularly driven risk classification as the means for a better, more-refined, treatment stratification.