bluuebunny/crossref_metadata_2025 huggingface dataset:
EMBR-04. BET INHIBITION TARGETS RADIOTHERAPY RESISTANCE IN H3K27ME3-DEFICIENT GROUP 3 MEDULLOBLASTOMA
Authors: Gabriel Nishanth , Balaji Kumaresh , Inkman Matthew , Jayachandran Kay , Zhang Jin , Dahiya Sonika , Goldstein Michael
Published: June 2021

Medulloblastoma has been categorized into four subgroups based on genetic, epigenetic and transcriptional profiling. However, molecular pathways determining radiotherapy response in this tumor remain elusive. Here, we investigated the role of the EZH2-dependent histone H3K27 tri-methylation in radiotherapy response in medulloblastoma. We demonstrate that 47.2% of group 3 and 4 medulloblastoma patients have H3K27me3-deficient tumors. Loss of H3K27me3 was associated with a radioresistant phenotype, high relapse rates and poor overall survival. We show that an epigenetic switch from H3K27me3 to H3K27ac occurs at specific genomic loci in H3K27me3-deficient medulloblastoma cells altering the transcriptional profile. The resulting up-regulation of EPHA2 (ephrin type-A receptor 2) stimulates an excessive activation of the pro-survival AKT signaling pathway leading to radiotherapy resistance. We show that BET inhibition targets radiation resistance in H3K27me3-deficient medulloblastoma by suppressing H3K27ac levels, blunting EPHA2 overexpression and mitigating the excessive AKT signaling. Additionally, BET inhibition sensitizes medulloblastoma cells to radiation by enhancing apoptotic response through suppression of Bcl-XL and up-regulation of Bim expression. Our work demonstrates a novel mechanism of radiation resistance in medulloblastoma and identifies an epigenetic marker predictive of radiotherapy response. Based on these findings we propose an epigenetically guided treatment approach targeting radiotherapy resistance in medulloblastoma patients.
EMBR-08. CORRELATION OF HISTOPATHOLOGY, CHROMOSOMAL MICROARRAY, AND NANOSTRING BASED 22-GENE ASSAY FOR MEDULLOBLASTOMA SUBGROUP ASSIGNMENT ON “HEAD START” 4 CLINICAL TRIAL
Authors: Dhall Girish , Patel Parth , Blue Megan , Biegel Jaclyn , Almiraz-Suarez Isabel , Hwang Eugene , Pierson Christopher , Boue Daniel , Finlay Jonathan
Published: June 2021

“Head Start” 4 (HS 4) is a prospective randomized clinical trial that tailors treatment based on medulloblastoma molecular subgroups and response to induction chemotherapy to compare the efficacy of one versus three (tandem) cycles of myeloablative therapy. Advances in RNA and DNA profiling have identified four core molecular subgroups of medulloblastoma with prognostic significance: Sonic Hedgehog (SHH) subtype, WNT subtype, Group 3, and Group 4. In HS 4 trial, we utilize a combination of histopathology and immunohistochemistry (pathology/IHC), as well as chromosomal microarray analysis (CMA) utilizing OncoScanTM (Thermo Fisher) to classify medulloblastoma samples into either SHH, WNT, or non-WNT/non-SHH (Group 3/4) subgroups at the time of diagnosis. NanoString based 22-gene assay is performed retrospectively to test concordance. We have pathology/IHC, CMA, and NanoString data on 26 infants and young children with medulloblastoma enrolled on HS 4. Pathology/IHC was able to assign samples to SHH, WNT, and non-WNT/non-SHH subgroups in all but two cases: one case was classified as Group 3, and the second as SHH by both CMA and NanoString. CMA was indeterminate in six cases, of which, pathology/IHC was able to assign all six samples aforementioned three subgroups. NanoString was indeterminate in two cases: one case was classified as SHH by CMA and pathology/IHC, and the second case was indeterminate by CMA but was assigned as non-WNT/non-SHH on pathology/IHC. There is excellent correlation between NanoString and combination of histopathology and CMA for core medulloblastoma subgrouping on HS 4. Methylation studies are ongoing.
EMBR-11. SYNERGISTIC DRUG COMBINATIONS FOR THE TREATMENT OF MYC AMPLIFIED GROUP 3 MEDULLOBLASTOMA
Authors: Zeuner Simon , Vollmer Johanna , Peterziel Heike , Sigaud Romain , Oppermann Sina , ElHarouni Dina , Hielscher Thomas , Witt Olaf , Oehme Ina , Milde Till , Ecker Jonas
Published: June 2021

Background

Medulloblastoma (MB) is a highly aggressive brain tumour in children. Patients with Group 3 MB harbouring a MYC-amplification (subtype II) show a particularly poor outcome despite high-intensity multimodal therapy. We and others have previously shown that MYC amplified Group 3 MB cells are highly susceptible towards treatment with class I histone deacetylase (HDAC) inhibitors such as entinostat. However, in clinical trials HDACi as a monotherapy show only modest efficacy in solid tumours. We propose to increase the efficacy of class I HDACi by drug combinations.

Methods

To identify synergistic drug combinations (entinostat + X) for the treatment of MYC amplified MB we performed a drug screen with a library of n=75 clinically available compounds as single agents and in combination with entinostat in n=3 MYC amplified vs. n=1 MYC-non amplified cell lines. Synergistic behaviour of the six most promising drug combinations was validated by metabolic activity assays, cell count experiments and gene expression profiling. Synergy was assessed by the Loewe additivity model using a combination of ray design and checkerboard matrix.

Results

The drug screen revealed n=20/75 drugs that were particularly effective (drug sensitivity score ≥10) in combination with entinostat treatment in all three MYC amplified cell lines. Synergy assessment of the top n=6 drugs confirmed strong synergistic activity with entinostat for n=2 drugs (navitoclax, irinotecan). The BCL-2 family inhibitor navitoclax showed the most robust synergy with entinostat in subsequent validation experiments.

Conclusion

Several drugs either clinically available or currently in clinical trials, including the BCL-2/Xl/w inhibitor navitoclax, show promising effects in a combination therapy with entinostat for the treatment of MYC amplified Group 3 MB.
EMBR-17. DE-INTENSIFICATION OF RADIOTHERAPY IN RIGOROUSLY DEFINED LOW-RISK WNT-SUBGROUP MEDULLOBLASTOMA IS ASSOCIATED WITH UNACCEPTABLY HIGH RISK OF NEURAXIAL FAILURE: RESULTS FROM THE PROSPECTIVE FOR-WNT STUDY
Authors: Gupta Tejpal , Pervez Shizan , Dasgupta Archya , Chatterjee Abhishek , Krishnatry Rahul , GodaSastri Jayant , Prasad Maya , Chinnaswamy Girish , Epari Sridhar , Sahay Ayushi , Shirsat Neelam , Shetty Prakash , Moiyadi Aliasgar , Shaikh Farnaz , Bano Nazia , Jalali Rakesh
Published: June 2021

Background

Medulloblastoma is a heterogenous disease comprising four molecular subgroups (WNT, SHH, Group 3, and Group 4) with varying outcomes. Excellent long-term survival (>90%) has prompted de-intensification of therapy in WNT-subgroup medulloblastoma globally. FOR-WNT is one such prospective study (CTRI/2017/12/010767) testing the hypothesis that focal conformal radiotherapy (RT) (54Gy/30 fractions/6-weeks) with avoidance of upfront craniospinal irradiation (CSI) followed by standard adjuvant chemotherapy significantly reduces RT-related late toxicity without unduly compromising survival in low-risk WNT-subgroup medulloblastoma (residual tumor <1.5cm2 with no evidence of metastases in children aged between 3–16 years).

Methods

Patients with low-risk WNT-subgroup medulloblastoma were enrolled after written informed consent/assent. To ensure patient safety, stopping rules were devised according to group-sequential method.

Results

Between July 2017 till Feb 2019, seven children of WNT-pathway medulloblastoma were treated with focal conformal RT followed by 6-cycles of adjuvant chemotherapy (cisplatin, cyclophosphamide, and vincristine). One child succumbed to acute renal failure during chemotherapy, while the other 6 patients completed all 6-cycles as planned. Three children were detected with neuraxial failure (supratentorial brain and/or spine) without synchronous local recurrence in the treated tumor-bed on surveillance neuro-imaging between 1.5–2 years from index diagnosis following which the study was terminated prematurely. All 3 children with relapse were treated with salvage CSI (35Gy/21 fractions) with (conformal avoidance of previously treated tumor-bed) plus boost irradiation (10.8-18Gy/6–10 fractions) of metastatic deposits resulting in complete/near complete response and are alive with controlled disease. The other 3 children have not shown any evidence of relapse for over 2-years from index diagnosis and remain on active clinico-radiological surveillance.

Conclusion

In rigorously defined low-risk WNT-subgroup medulloblastoma, avoidance of upfront CSI is associated with unacceptably high risk of neuraxial failure. A successor study (FOR-WNT 2) incorporating low-dose CSI (18Gy/10 fractions) with similar tumor-bed dose and adjuvant systemic chemotherapy is currently underway.
EMBR-19. SHH-DRIVEN MEDULLOBLASTOMA WITH CONCURRENT UNILATERAL RENAL AGENESIS
Authors: Aaroe Ashley , Sarkar Sanila , Sherani Farha , McAleer Mary , Fuller Greg , Zaky Wafik
Published: June 2021

Case Presentation

A 3-year-old female with insignificant past medical history presented with 6 weeks of headaches, emesis, and lethargy. MR imaging identified a heterogeneously enhancing right cerebellar hemispheric mass and obstructive hydrocephalus. Gross total resection was performed without complications; pathology revealed classical WHO grade 4 medulloblastoma (MB). MR imaging of the spine and cerebrospinal fluid testing were negative for disseminated disease. Treatment for standard risk medulloblastoma was initiated, comprising proton craniospinal irradiation with posterior fossa boost and concurrent vincristine, followed by adjuvant chemotherapy with vincristine, lomustine, cisplatin, and cyclophosphamide as standard of care. Next generation sequencing of the tumor tissue performed using a high-multiplex PCR-based NGS panel was negative for alteration of the SMO, PTCH1 and CTBNN1 genes. Further molecular characterization via methylation profiling demonstrated the sonic hedgehog (SHH) molecular subtype. Prior to initiation of chemotherapy, renal ultrasound was performed and identified congenitally absent right kidney; audiology evaluation was unremarkable.

Discussion

In patients with Gorlin syndrome, cases of unilateral renal agenesis in association with germline SHH-pathway mutations have been reported [1]. SHH signaling is implicated in multiple steps in the development of the urinary system [2]. Outside Gorlin syndrome, however, to the best of our knowledge unilateral renal agenesis coinciding with SHH-driven MB has not been reported. Our patient notably lacks any clinical stigmata of Gorlin syndrome (skeletal abnormalities, skin pits, macrocephaly) and does not exhibit the characteristic germline genetic abnormalities that define GS (PTCH1 mutation or 9q22.3 microdeletion). There are important treatment implications for patients with the constellation of abnormalities we describe here, particularly regarding the requisite frequent monitoring of renal function during multi-agent chemotherapy courses. Our patient tolerated chemoradiation well, and is currently on maintenance chemotherapy with favorable course to date.
EMBR-33. YAP1 FUNCTION IN SHH MEDULLOBLASTOMA PROGRESSION AND IMMUNE EVASION
Authors: Abdelfattah Nourhan , Natarajan Sivaraman , Baskin David , Yun Kyuson
Published: June 2021

Our incomplete understanding of the key players in Medulloblastoma (MB) development and progression, and their roles in modulating highly Immune desert-like microenvironment in MBs present major hurdles in successfully applying existing therapies and developing new therapies for MBs. Here, we demonstrate that Yap1 acts as a critical modulator of SHH MB (fSmoM2; GFAPcre (SG) and Ptch;p53 SHH-MB mouse models) progression and immune evasion. Yap1 genetic deletion in SG mice significantly extends survival and normalizes brain development by increasing neuronal differentiation. Both bulk and single-cell RNA sequencing analyses show that Yap1 deleted tumors contain cells with more differentiated molecular signatures similar to late CGNPs and differentiating neurons, and less stem-like cells, compared to SG tumors. Additionally, integrated analyses of ChiPseq, RNAseq, and scRNAq data suggest that Yap1 directly binds to the Super enhancer region containing Sox2 and promotes Sox2 expression in SHH MB cells. We postulate that Yap1 expression is maintained or re-activated in SHH MB cells to generate long-term self-renewing tumor cells. Consistently, Yap1-deleted SHH MB or Verteporfin (a small molecule inhibitor of Yap1) -treated Ptch;p53 MB cells lose self-renewal ability in vitro. Furthermore, we hypothesize that a molecular mechanism underlying this stemness promoting function is mediated through Sox2 expression. Intriguingly, Yap1 deletion in SG MBs is accompanied by a significant change in the immune microenvironment, when compared to age-matched SG MBs. There is a significant increase in the number of bone marrow-derived immune cells (including cytotoxic T-cells, neutrophils, and macrophages). RNAseq analysis of rescued tumors shows marked enrichment of interferon-gamma response genes and pro-inflammatory cytokines. This study highlights Yap1 as a crucial mediator of MB progression and a molecular regulator of inflammatory immune cell infiltration into SHH MBs. Consequently, our work paves the way for improving immunotherapy treatments in brain malignancies.
EPEN-03. ZFTA/C11ORF95 FUSIONS DRIVE SUPRATENTORIAL EPENDYMOMA VIA SHARED ONCOGENIC MECHANISMS
Authors: Zheng Tuyu , Ghasemi David R , Okonechnikov Konstantin , Korshunov Andrey , Maaß Kendra K , Sill Martin , Gojo Johannes , Schüller Ulrich , Milde Till , Mack Stephen C , Gilbertson Richard J , von Deimling Andreas , Jones David T W , Kool Marcel , Pfister Stefan M , Sahm Felix , Kawauchi Daisuke , Pajtler Kristian W
Published: June 2021

The majority of supratentorial ependymomas (ST-EPN) are driven by fusion genes between RELA and zinc finger translocation associated, ZFTA, previously named C11orf95. Apart from fusions with a portion of the Hippo effector YAP1, which affects a small group of infant patients, the oncogenic mechanism of remaining ST-EPNs remains unclear. Aiming at refining the molecular classification of ST-EPNs, we have analyzed methylation profiles, RNA and DNA sequencing results as well as clinical data in a cohort of 613 ST-EPNs. An unbiased approach revealed distinct methylation clusters composed of tumors with ependymal but also various other histological features containing alternative translocations that shared ZFTA as a partner gene. Tumors within these additional clusters were characterized by fusions of ZFTA to numerous fusion partners different from RELA, e.g. MAML2, MAML3, NCOA2 and SS18, implying a general role of ZFTA in tumorigenesis of ST-EPN. Indeed, the transforming capacity of newly identified fusion genes was validated using an electroporation-based in vivo gene transfer technology in mice. All fusion genes themselves were sufficient to drive malignant transformation in the developing cerebral cortex and resulting tumors faithfully recapitulated molecular characteristics of their human counterparts. We found that both, the partner gene and the zinc finger DNA binding domain of ZFTA, were essential to exert tumorigenesis. Together with two additional studies, we performed a comprehensive analysis across datasets to derive a 93 gene signature of ZFTA-RELA-driven tumors, in which the Sonic Hedgehog effector gene GLI2 was identified as a promising downstream target. Subsequent co-expression of ZFTA:RELA and a dominant negative form of Gli2 indeed hampered tumorigenesis. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating GLI2 as a critical regulator of ZFTA fusion-positive tumorigenesis as well as a potential therapeutic vulnerability in these tumors.
EPEN-04. SIOP EPENDYMOMA I: FINAL RESULTS, LONG TERM FOLLOW-UP AND MOLECULAR ANALYSIS OF THE TRIAL COHORT: A BIOMECA CONSORTIUM STUDY
Authors: A. Ritzmann Timothy , Chapman Rebecca J , Macarthur Donald , Mallucci Conor , Kilday John-Paul , Thorp Nicola , Modena Piergiorgio , Giagnacovo Marzia , Dineen Rob , Jaspan Timothy , Pajtler Kristian W , Jacques Thomas S , Paine Simon M L , Ellison David W , Bouffet Eric , Grundy Richard G
Published: June 2021

Introduction

Surgery and radiotherapy are established childhood ependymoma treatments. The efficacy of chemotherapy has been debated. We report final results of the SIOP Ependymoma I trial, with 12-year follow-up, in the context of a post-hoc analysis of more recently described biomarkers.

Aims and Methods

The trial assessed event free (EFS) and overall survival (OS) of patients aged three to 21 years with non-metastatic intracranial ependymoma, treated with a staged management strategy targeting maximum local control. The study also assessed: the response rate (RR) of subtotally resected (STR) disease to vincristine, etoposide and cyclophosphamide (VEC); and surgical operability. Children with gross total resection (GTR) received radiotherapy of 54 Gy in 30 daily fractions over six weeks, whilst those with STR received VEC before radiotherapy. We retrospectively assessed methylation and 1q status alongside hTERT, RELA, Tenascin C, H3K27me3 and pAKT expression.

Results

Between 1999 and 2007, 89 participants were enrolled, 15 were excluded with metastatic (n=4) or non-ependymoma tumours (n=11) leaving a final cohort of 74. Five- and ten-year EFS was 49.5% and 46.7%, OS was 69.3% and 60.5%. 1q gain was associated with poorer EFS (p=0.002, HR=3.00, 95%CI 1.49–6.10). hTERT expression was associated with worse five-year EFS (20.0% Vs 83.3%, p=0.014, HR=5.8). GTR was achieved in 33/74 (44.6%) and associated with improved EFS (p=0.006, HR=2.81, 95% confidence interval 1.35–5.84). There was an improvement in GTR rates in the latter half of the trial (1999-2002 32.4% versus 2003-2007 56.8%). Despite the protocol, 12 participants with STR did not receive chemotherapy. However, chemotherapy RR was 65.5% (19/29, 95%CI 45.7–82.1).

Conclusions

VEC exceeded the pre-specified RR of 45% in children over three years with STR intracranial ependymoma. However, cases of inaccurate stratification at treating centres highlights the need for rapid central review. We also confirmed associations between 1q gain, hTERT expression and outcome.
EPEN-11. TUMOR DIFFERENTIATION IMPACTS THE BIOLOGY OF RECURRENCE IN CHILDHOOD POSTERIOR FOSSA EPENDYMOMA
Authors: Donson Andrew , Ritzmann Timothy , Willard Nicholas , Griesinger Andrea , Amani Vladimir , Harris Faith , Grimaldo Enrique , Sanford Bridget , Riemondy Kent , Hankinson Todd , Grundy Richard , Foreman Nicholas
Published: June 2021

Ependymoma (EPN) of childhood is curable in only 50% of cases, with recurrences in the remainder that are refractory to treatment. In recent years significant advances have been made in understanding the molecular and cellular biology of EPN. Recent studies show that PFA subgroup EPN are comprised of multiple neoplastic subpopulations that show undifferentiated, differentiated and mesenchymal characteristics. These studies focused on tumor at presentation, with recurrent EPN being less well understood. In the present longitudinal study we examine changes in neoplastic cell heterogeneity in serial presentations of PFA EPN using deconvolution (Cibersort) of bulk RNAseq data. Analysis of a cohort of 48 PFA EPN presenting at Children’s Colorado showed survival and PFA1/PFA2 subtype assignment was associated with the proportion of individual neoplastic subpopulations as determined by deconvolution. Tumors that subsequently regrew had a significantly higher estimated proportion of undifferentiated EPN cells (UEC) at presentation, than those that were non-recurrent after 5 years follow-up. This outcome association potentially age related, as UEC proportions are significantly higher in PFA arising in children < 1 year old who have a particularly poor prognosis. Changes in PFA neoplastic subpopulations at recurrence was performed in two cohorts of patients from Children’s Colorado (n=23) and Nottingham, UK (n=15). As a whole, no subpopulation proportion was significantly changed at recurrence. However, separation of PFA into subtypes PFA1 and PFA2 revealed an increase in the proportion of the cilia-differentiated EPN cell subpopulation is more frequent event in PFA1 (15/24), and rare in PFA2 (2/11). Changes in other neoplastic subpopulations at recurrence were smaller and only seen in PFA1, both UEC and mesenchymal subpopulations being lower at recurrence. In summary, only PFA1 showed dynamic changes in neoplastic subpopulation proportions at recurrence, with potential impacts on transcriptomic based-subgroup assignment, whereas PFA2 proportions remained largely stable.
HGG-03. THE GLYCOSPHINGOLIPIDS METABOLISM IS A NOVEL TARGET IN H3K27M-MUTANT DIFFUSE MIDLINE GLIOMA
Authors: Paret Claudia , Sandhoff Roger , El Malki Khalifa , Frauenknecht Katrin B M , Wingerter Arthur , Lehmann Nadine , Vewinger Nadine , Faber Jörg
Published: June 2021

H3K27M-mutant diffuse midline glioma (H3K27M-mutant DMG) is a rare malignant brain tumour entity in children and adults with a median overall survival of around 12 months. Genomic and proteomic analysis may help to identify new target structures, however not all relevant targets are covered by these analyses. Glycosphingolipids and particularly gangliosides play a major role in brain development and have been involved in the pathology of brain tumours. The conversion of ceramide to glucosylceramide via glucosylceramide synthase (GCS) is one of the first steps in the synthesis of glycosphingolipids. Therefore, targeting GCS will inhibit their synthesis. Here we analysed the glycosphingolipids composition and tested GCS inhibitors in an in vitro model of H3K27M-mutant DMG. Methods: H3K27M-mutant DMG primary cells were established from needle biopsies of two paediatric patients and characterised by immunohistochemistry. Glycosphingolipids were analysed by thin layer chromatography, liquid chromatography-coupled tandem mass spectrometry and flow cytometry. The effect of the GCS inhibitor eliglustat on the cell proliferation was examined. Results. The primary cells maintained the features of the tumour of origin, including the H3K27M mutation. Neutral glycosphingolipids with 1 to 4 monosaccharides and the ganglioside GD2 were expressed at high concentration. Eliglustat completely abrogated the proliferation of the H3K27M-mutant DMG primary cells. Conclusions. The glycosphingolipids oncometabolism represents a novel target in H3K27M-mutant DMG. The GCS inhibitors eliglusat and miglustat are already used to treat paediatric patients with the lysosomal storage disorders Niemann Pick’s and Gaucher′s disease and miglustat can cross the blood-brain barrier. Thus, our finding may accelerate the access of H3K27M-mutant DMG patients to novel innovative clinical studies based on the inhibition of the glycosphingolipids metabolism.
HGG-14. ACT001 – A PROMISING THERAPEUTIC FOR DIFFUSE INTRINSIC PONTINE GLIOMAS
Authors: Upton Dannielle , George Sandra , Liu Jie , Cai Dongpo , Su Steven Yung-Chang , Manoharan Neevika , Foresto Steve , Hassall Tim , Tsoli Maria , Ziegler David
Published: June 2021

Diffuse Intrinsic Pontine Gliomas (DIPGs) are a subset of Diffuse Midline Gliomas (DMG) and are the most devastating of all brain tumors. There are no effective treatments and all children die of their tumor within 12-months. We performed a high-throughput drug screen with 3,570 biologically active, clinically approved compounds against a panel of DIPG cultures. Parthenolide, a compound derived from the herb T.parthenium, was found to be one of the most effective drugs tested, demonstrating significant anti-tumor activity. However, parthenolide also affected healthy cell viability and showed no in-vivo efficacy. ACT001 is a novel agent in clinical development that is a fumarate salt form of dimethylamino-micheliolide, which is semi-synthesized from parthenolide. ACT001 is blood-brain-barrier permeable and exerts an anti-tumor effect via inhibition of NF-κB and STAT3 pathways. ACT001 demonstrated potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells and inhibited colony formation in-vitro. To determine whether this activity could be replicated in-vivo, we tested ACT001 in a DIPG-orthotopic model. ACT001 was well tolerated and significantly improved survival of tumor-bearing animals, extending survival by 33% in ACT001 treated mice. We have initiated a Phase 1 paediatric trial of ACT001 for children with relapsed/refractory solid or CNS tumors, with an expansion cohort planned for patients with DIPG/DMG. Eleven patients have been enrolled, and the dose escalated from dose level-1 at 188mg/m2 bd to dose level-4 at 700mg/m2 bd. To date, no dose limiting or Grade 3/4 toxicities have been observed. At the highest dose level, clinical activity has been demonstrated in two patients, one with DIPG with a reduction in tumor burden, and another with DMG with H3K27M mutation with an objective radiographic and clinical response. These combined preclinical and clinical results suggest that ACT001 is an active novel therapy for patients with DIPG/DMG.
HGG-21. MALIGNANT SYNAPTIC PLASTICITY IN PEDIATRIC HIGH-GRADE GLIOMAS
Authors: Taylor Kathryn , Barron Tara , Hartmann Griffin , Zhang Helena , Hui Alexa , Gillespie Shawn , Monje Michelle
Published: June 2021

Pediatric high-grade gliomas (pHGG) are a devastating group of diseases that urgently require novel therapeutic options. We have previously demonstrated that pHGGs directly synapse onto neurons and the subsequent tumor cell depolarization, mediated by calcium-permeable AMPA channels, promotes their proliferation. The regulatory mechanisms governing these postsynaptic connections are unknown. Here, we investigated the role of BDNF-TrkB signaling in modulating the plasticity of the malignant synapse. BDNF ligand activation of its canonical receptor, TrkB (which is encoded for by the gene NTRK2), has been shown to be one important modulator of synaptic regulation in the normal setting. Electrophysiological recordings of glioma cell membrane properties, in response to acute neurotransmitter stimulation, demonstrate in an inward current resembling AMPA receptor (AMPAR) mediated excitatory neurotransmission. Extracellular BDNF increases the amplitude of this glutamate-induced tumor cell depolarization and this effect is abrogated in NTRK2 knockout glioma cells. Upon examining tumor cell excitability using in situ calcium imaging, we found that BDNF increases the intensity of glutamate-evoked calcium transients in GCaMP6s expressing glioma cells. Western blot analysis indicates the tumors AMPAR properties are altered downstream of BDNF induced TrkB activation in glioma. We find that BDNF-TrkB signaling promotes neuron-to-glioma synaptogenesis as measured by high-resolution confocal and electron microscopy in culture and tumor xenografts. Our analysis of published pHGG transcriptomic datasets, together with brain slice conditioned medium experiments in culture, indicate the tumor microenvironment as the chief source of BDNF ligand. Disruption of the BDNF-TrkB pathway in patient-derived orthotopic glioma xenograft models, both genetically and pharmacologically, results in an increased overall survival and reduced tumor proliferation rate. These findings suggest that gliomas leverage mechanisms of plasticity to modulate the excitatory channels involved in synaptic neurotransmission and they reveal the potential to target the regulatory components of glioma circuit dynamics as a therapeutic strategy for these lethal cancers.
HGG-30. BRAIN PENETRANT HDAC INHIBITOR RG2833 SYNERGIZES WITH LOMUSTINE AND RADIATION TO INDUCE DIPG CELL DEATH
Authors: Barnett Katherine , Novak Orlandi , Eberhart Charles , Raabe Eric
Published: June 2021

Diffuse intrinsic pontine glioma (DIPG) is driven by epigenetic dysregulation. The pan-HDAC inhibitor panobinostat showed pre-clinical efficacy against DIPG, but was limited by toxicity in clinical trials. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor with established brain penetration. RG2833 reverses temozolomide-resistance in glioblastoma through downregulation of the NFĸB pathway. As this pathway is upregulated in DIPG and may contribute to tumorigenesis, we hypothesized that RG2833 would kill DIPG cells. We found that RG2833 treatment inhibits cell growth in the 4 to 9μM range in both autopsy and biopsy-derived DIPG cell lines (MTS assay for HSJD007 p=0.0004, JHH-DIPG1 p=0.001, SF-7761 p=0.04, SU-DIPG13 p=0.01 by t-test). Western blot confirmed on target activity by increased histone 3 acetylation at IC50 doses. RG2833 induces apoptosis (cPARP Western blot, cleaved caspase 3 immunofluorescence HSJD007 p<0.003, JHH-DIPG1 p=0.0026 by t-test) and slows cell proliferation (phospho-Rb Western blot, BrdU immunofluorescence HSJD007 p=0.008, JHH-DIPG1 p=0.0002 by t-test) in multiple DIPG cell lines. RG2833 disrupts the NFĸB pathway through acetylation of p65, resulting in decreased expression of NFĸB regulated pro-survival genes (Western blot for BCL2, BCL-xL, and XIAP). In a DIPG flank tumor mouse model, treatment with RG2833 alone for 1 week suppresses flank tumor growth (p< 0.005 by t-test) and induces apoptosis (Western blot for cPARP). We next assessed whether RG2833 combines synergistically with conventional therapies. We found that RG2833 has strong synergism with both lomustine and radiation to slow DIPG cell growth in vitro (ZIP synergy scores by SynergyFinder for RG2833+lomustine in JHH-DIPG1 17.8 and HSJD007 17.7, RG2833+radiation in JHH-DIPG1 9.7 and JHH-DIPG16A 10.9). Evaluation of combination treatment for apoptotic effects in vitro and in vivo are ongoing. This data identifies selective HDAC inhibitor RG2833 as a promising therapy for DIPG that combines synergistically with conventional therapies.
HGG-42. PEDIATRIC H3K27M MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) SHOWS ROBUST RESPONSE TO IMIPRIDONE BASED COMBINATION THERAPY
Authors: Borsuk Robyn , Zhou Lanlan , Chang Wen-i , Zhang Yiqun , Prabhu Varun , Allen Joshua , Tapinos Nikos , Lulla Rishi , El-Deiry Wafik
Published: June 2021

ONC201 is a first-in-class small molecule imipridone therapy, which is known to selectively induce apoptosis of cancer cells independent of p53. This novel chemotherapeutic, as well as its analogs ONC206 and ONC212, has been shown to have potent preclinical efficacy against H3K27M mutant diffuse intrinsic pontine glioma (DIPG). We sought to identify synergy between imipridones and other FDA-approved chemotherapeutics. Seven patient-derived DIPG cell lines, six H3.3K27M mutant (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SF8628), and one H3.1K27M mutant (SU-DIPG-36) were grown in culture and exposed to first and second generation imipridones, both as monotherapies and in combination with histone de-acetylase inhibitors [HDACi], Marizomib, Etoposide, and Temozolomide. A dose dependent response was demonstrated across all cell lines, with increased potency of ONC206 and ONC212 as compared to ONC201, with half maximal inhibitory concentration (IC50) of 0.11 µM, 0.03 µM, and 1.46 µM respectively. Strong synergy is demonstrated between ONC201 and Panobinostat with best combination index (CI) of 0.01. ONC201 similarly shows strong synergy with Romidepsin with best CI of 0.02, and Marizomib with best CI of 0.18. Combination of ONC201 and Etoposide or Entinostat shows some synergy, with best CI of 0.53 and 0.71 respectively. When combined with Temozolomide, some synergy is evident, however, there is overall poor efficacy, with lack of cell death even at the highest doses of Temozolomide. Second generation imipridones show a similar pattern of strong synergy with Panobinostat, Romidepsin, and Marizomib. Immunoblotting showed evidence of apoptosis, as measured by the induction of PARP cleavage, with a combination of imipridones and Panobinostat, as well as induction of integrated stress response with a combination of imipridones and Romidepsin. These results are indicative of promising synergy between imipridones and Panobinostat, Romidepsin, or Marizomib against H3K27M mutant DIPG, combinations which should be considered for future clinical trials.
IMMU-05. IMMUNOTHERAPY WITH RESIQUIMOD REPOLARIZES TUMOR-ASSOCIATED MYELOID CELLS AND IMPROVES EVENT-FREE SURVIVAL IN A TRANSGENIC MOUSE MODEL OF SONIC-HEDGEHOG (SHH) MEDULLOBLASTOMA
Authors: Park Christopher , Thang Morrent , Hwang Duhyeong , Lim Chaemin , Dismuke Taylor , Sokolsky Marina , Vibhakar Rajeev , Donson Andrew , Gershon Timothy
Published: June 2021

Resiquimod is a synthetic small molecule agonist of Toll-like receptors 7 and 8 (TLR-7/8) that modulates innate immune cells. We found TLR-7/8 are expressed in medulloblastoma exclusively by tumor-associated myeloid cells (TAMs). We tested whether systemically administered resiquimod modulated TAMs in a genetic Sonic hedgehog (SHH) medulloblastoma model, and whether this modulation would be therapeutically beneficial. We generated mice with medulloblastoma by crossing hGFAP-Cre and SmoM2 mouse lines. The resulting hGFAP-Cre/SmoM2 (G-Smo) mice developed medulloblastoma with 100% frequency and showed a median survival of 14.5 days (n=12). Treatment with 3 doses of resiquimod at postnatal days 10, 12 and 14 reduced tumor size and increased median survival to 37 days (n=10) (p=0.003508). Cellular studies showed that resiquimod altered TAM phenotype, rapidly inducing expression of the inflammatory marker VCAM1, and more slowly increasing TAM populations. Responses to the 3-dose regimen were ultimately limited by recurrence and all mice eventually died of tumor progression. Continued resiquimod therapy with every other day dosing was less effective than the 3-dose regimen, suggesting that TAM responses to resiquimod are dynamic and change with prolonged exposure. Our data show that innate immunity, mediated by TAMs and stimulated by TLR-7/8 agonist therapy, can produce a significant anti-tumor effect in medulloblastoma. The common expression of TLR-7/8 on TAMs in patient-derived medulloblastoma samples and in the mouse model suggests that resiquimod may produce similar anti-medulloblastoma effects in humans. Further studies are needed to define the mechanism of the anti-tumor effect in detail, to determine the optimal dose regimen, and to determine if resiquimod can combine effectively with additional adjuvant therapies to produce curative effects.
IMMU-11. CLINICAL UPDATES AND CORRELATIVE FINDINGS FROM THE FIRST PATIENT WITH DIPG TREATED WITH INTRACRANIAL CAR T CELLS
Authors: Vitanza Nicholas , Wilson Ashley , Gust Juliane , Huang Wenjun , Perez Francisco , Albert Catherine , Pinto Navin , Gardner Rebecca , Orentas Rimas , Berens Michael , Jensen Michael , Park Julie
Published: June 2021

We report preliminary data for the first subject with diffuse intrinsic pontine glioma (DIPG) treated with intracranial CAR T cells. BrainChild-03 (NCT04185038) is a phase 1 trial of repetitively-dosed locoregional B7-H3-specific CAR T cells for children with recurrent/refractory central nervous system (CNS) tumors or DIPG. DIPG patients enroll on Arm C, on which B7H3CARs are delivered into the ventricular system via a CNS reservoir catheter. This study does not use lymphodepletion. Primary endpoints are feasibility and safety, with second endpoints of disease response. This 18-year-old female (BrainChild-03 S005) with radiographically-classic DIPG and biopsy-confirmed H3 K27M mutation enrolled on Arm C after progression 552 days from diagnosis following focal radiation and temozolomide, irinotecan, and bevacizumab. Apheresis and manufacturing produced 4.2x109 second-generation B7H3CARs with a methotrexate-resistant human DHFR mutein (huDHFRFS; L22F,F31S) in a single transcript in combination with the B7-H3-specific CAR and EGFRt, each separated by a T2A linker, allowing methotrexate selection and enrichment. At time of submission, she has received 10 every-other-week outpatient infusions of 1x107 B7H3CARs (first dose on October 2, 2020). She has had no DLTs, but has experienced grade 2 fever and grade 2–3 headache peaking ~12–48 hours after each infusion. Following the 8th CAR T cell infusion, she experienced increased focal weakness and dysarthria at ~72 hours with resolution after 48 hours. She has not experienced cytokine release syndrome (CRS). She has stable disease 138 days post-initial CAR T cell infusion. Frequently collected correlative studies have detected viable B7H3CARs in the CSF post-infusion via flow cytometry. CSF cytokine analysis has revealed elevations of CXCL10, GM-CSF, and G-CSF following B7H3CAR infusions, without correlation in the serum. A second evaluable subject with DIPG has also received 4 locoregional doses of 1x107 B7H3CARs without a DLT. She also has stable disease and detectable viable B7H3CARs in the CSF.
IMMU-15. QUANTIFYING INTRATHECAL DRUG DELIVERY UTILIZING PROGRAMMABLE VENTRICULOPERITONEAL SHUNTS
Authors: McThenia Sheila , Pandit-Taskar Neeta , Grkovski Milan , Donzelli Maria , Diagana Safiatu , Greenfield Jeffrey , Souweidane Mark , Kramer Kim
Published: June 2021

Background

Programmable ventriculoperitoneal (pVP) shunts are increasingly utilized for intraventricular chemotherapy, radioimmunotherapy, and/or cellular therapy. Shunt adjustments allow optimization of thecal space drug concentrations with minimization in the peritoneum. Drug delivery quantification using several types of pVP shunts has not been reported.

Methods

We performed a retrospective analysis on patients with CNS tumors and pVP shunts at Memorial Sloan Kettering Cancer Center from 2003–2020, noting shunt model. CSF flow through the pVP shunt was evaluated using In-111-DTPA scintigraphy at approximately 4 hours and 24 hours after injection. pVP shunts were calibrated pre-injection to minimize peritoneal flow and re-calibrated to baseline setting 4–5 hours following injection. Scintigraphy studies quantified ventricular-thecal and peritoneal drug activity at these 2 time points.

Results

Twenty-one CSF flow studies were administered to 15 patients, ages 1–27 years. Diagnoses included medulloblastoma (N=10), metastatic neuroblastoma (N=3), pineoblastoma (N=1), and choroid plexus carcinoma (N=1). pVP shunt models included Aesculap Miethke proGAV (N=3), Aesculap Miethke proGAV2.0 (N=3), Codman HAKIM (N=2), Codman Certas Plus (N=1), Medtronic STRATA (N= 5), and Sophysa Polaris (N= 1). All 21 studies (100%) demonstrated ventriculo-thecal drug activity. 29% (6 of 21) of the studies had no peritoneal uptake visible by imaging. 73% (16 of 21) of the studies had minimal peritoneal uptake (<12%), and 24% (5 of 21) demonstrated moderate peritoneal uptake (12–37%). pVP shunt models measuring minimal to no peritoneal uptake included: Aesculap Miethke proGAV (N=2), Aesculap Miethke proGAV2.0 (N=3), Codman HAKIM (N=2), Codman Certas Plus (N=1), Medtronic STRATA (N= 3), and Sophysa Polaris (N= 1).

Conclusions

Successful drug delivery to the ventriculo-thecal space can be accomplished using pVP shunts: 80% of studies have minimal (<12%) peritoneal drug activity. Though efficacy varies by shunt model, low numbers preclude conclusions regarding model superiority. CSF flow scintigraphy studies reliably assess drug distribution.
LGG-03. LONG-TERM FOLLOW UP OF TARGETED THERAPY IN PEDIATRIC LOW-GRADE GLIOMAS: THE DANA-FARBER/BOSTON CHILDREN’S EXPERIENCE
Authors: Tsai Jessica , Vogelzang Jayne , Sousa Cecilia , Yeo Kee Kiat , Ligon Keith , Bandopadhayay Pratiti , Cooney Tabitha
Published: June 2021

Background

Pediatric low grade gliomas (pLGGs) are the most common central nervous system (CNS) tumor in children and characterized by alterations in the MAPK pathway. Standard of care is not well defined, and treatment has evolved over the last decade to include molecular targeted therapies. The impact of targeted agents on the natural history of pLGGs remains unknown. We present a retrospective review of patients receiving targeted agents integrated with molecular profiling.

Methods

We performed an IRB-approved, retrospective chart review of pLGGs treated with off-label use of dabrafenib, vemurafenib, everolimus, and trametinib at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center from 2010 to 2020.

Results

Forty-nine patients were identified (dabrafenib n=9, everolimus n=27, trametinib n=10, and vemurafenib n=3). All patients receiving BRAF inhibitors harbored BRAF V600E mutation. Targeted agent was used as first-line therapy for 25% of patients, while for 31% of patients, targeted agent was second-line therapy. The median time from diagnosis to targeted therapy initiation was 4.76 years (0.10 – 23.77), median duration of targeted therapy was 0.79 years (0.01 – 4.87), median time to subsequent therapy post first-line targeted therapy was 0.2 years (0.01 – 3.33), and overall median follow-up for the entire cohort was 3.09 years (0.36 – 11.87). The 1-year, 3-year, and 5-year EFS from targeted therapy initiation was 58.0%, 32.2%, and 26.9%, respectively. Survival analyses by molecular subgroup and agent were performed. Reasons for cessation of targeted therapy included toxicities, progression, and/or planned end of therapy.

Conclusions

Further efforts are ongoing to perform volumetric analysis of growth rates before, during, and after treatment. While targeted molecular therapies show great promise, it will be critical to understand how these agents alter the natural history of pLGGs, particularly in the context of genomic profiling.
LGG-07. IS BRAF ALTERATION OR A HISTOLOGIC ‘QUALIFIER’ A PREDICTOR OF OUTCOME IN PEDIATRIC PILOCYTIC ASTROCYTOMA?
Authors: Cler Samuel J , Skidmore Alexander , Yahanda Alexander T , Mian Ali , Mackey Kimberly , Dahiya Sonika , Strahle Jennifer
Published: June 2021

Introduction

Pediatric pilocytic astrocytomas (PA) are the most common pediatric central nervous system tumor. Surgical resection is the primary treatment for PA with five-year survival rates up to 95%. Despite a favorable prognosis, our understanding about the prognostic value of histopathological findings, such as histopathologic qualifier* or BRAF alterations is evolving.

Methods

Patients treated for a WHO grade 1 PA at Washington University in St. Louis/St. Louis Children’s Hospital were analyzed for clinical details, including pathology diagnosis (*histopathologic qualifier refers to designations in the diagnosis such as “WHO Grade I pilocytic astrocytoma with increased proliferative index”). BRAF alterations include gene fusions and point mutations.

Results

224 patients were analyzed (51% female, mean age 9.6 years). Tumors were located in the cerebellum/fourth ventricle (50%), optic pathway/hypothalamus (15%), brainstem (12%), and cerebral cortex (11%). BRAF alterations were identified in 55/77 patients (71.4%) and additional histopathologic qualifiers were present in 27/220 patients (12.3%). 196 patients (87.5%) underwent surgical treatment and 22 (9.8%) had biopsy alone. 45 patients (22%) displayed tumor progression or recurrence after resection. The presence of a histopathologic ‘qualifier’ in the topline or BRAF alteration was not associated with tumor progression or recurrence (p=0.36, p=0.77). Ki-67 proliferative indices were not predictive of progression or recurrence (p=0.94), including when controlling for extent of resection and adjuvant therapy. BRAF alterations, specifically KIAA1549 fusions, were associated with cerebellar/fourth ventricular tumor location (p<0.001) and younger patient age (p=0.03). Extent of resection was the only predictor of outcome identified in this study; patients with gross total resection had significantly lower rates of progression and recurrence (p<0.0001).

Conclusion

BRAF alterations and histopathologic qualifiers were not associated with tumor progression or recurrence in pediatric PA, although BRAF fusions were more common in tumors located in the cerebellum/fourth ventricle and in younger patients.
LGG-13. PILOCYTIC ASTROCYTOMAS WITH NOVEL BRAF FUSIONS DEMONSTRATE MAPK PATHWAY ACTIVATION
Authors: Chatwin Hannah , Lind Katherine , DeSisto John , Sanford Bridget , Gilani Ahmed , Green Adam
Published: June 2021

Background

Pilocytic astrocytomas (PAs) are the most common pediatric low-grade glioma subtype. Oftentimes, PAs demonstrate somatic genetic alterations, the most common being the BRAF-KIAA1549 fusion, which results in constitutive activation of the MAPK pathway. Better understanding of the effects of other RAF fusions is necessary to determine the potential utility of MAPK-targeting therapies.

Methods

Three patients presented to Children’s Hospital Colorado and were ultimately diagnosed with PAs harboring previously unreported gene fusions identified as FYCO-RAF1, CCTTNNBP2-BRAF, and SLC44A1-BRAF. Utilizing immunohistochemistry, we stained novel samples and controls for ERK and pERK (phosphorylated ERK) to assess the activation of the MAPK pathway. PAs with known BRAF-KIAA1549 fusions (4 samples) and normal brain tissue (5 samples) were used as positive and negative controls, respectively. We additionally performed RNA sequencing to better understand expression changes associated with these fusions, utilizing Metascape and GSEA (Gene Set Enrichment Analysis) for analysis.

Results

Immunohistochemistry of negative control samples demonstrated less p-ERK than ERK (ratios of 0.6–0.9, mean 0.8). All samples with novel fusions demonstrated statistically significantly higher p-ERK expression compared to negative controls (ratios of 1.3–1.7, mean 1.4). These experimental samples also all fell within the p-ERK to ERK expression range of the positive control samples, which demonstrated the widest range of expression (ratios of 1.1–4.5, mean 2.2). Our molecular analysis further confirmed these results, with GSEA demonstrating positively upregulated MAPK and ERK pathways in 2 positive controls and 1 novel fusion sample. Metascape analysis emphasized overall similar gene expression between these samples, demonstrating many shared genes and functional pathways.

Conclusions

We identified 3 previously unreported RAF fusions in PA that demonstrate activation of the MAPK pathway, although not as extensively as seen in some positive control samples with BRAF-KIAA1549 fusions. MEK inhibition may be a useful therapeutic strategy in these tumors if targeted therapy is indicated.
TMOD-06. LOSS OF DICER COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE METASTATIC MEDULLOBLASTOMA
Authors: Alcantara Llaguno Sheila , Nazarenko Inga , Chen Yuntao , Sun Daochun , Rocca Gaspare La , Pedraza Alicia , Gudenas Brian , Saulnier Olivier , Burns Dennis , Bale Tejus , Northcott Paul , Taylor Michael , Ventura Andrea , Parada Luis
Published: June 2021

To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a previously characterized glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and tumors were histologically indistinguishable from human medulloblastoma (MB). The minimum genetic mutations required for MB formation were Dicer and Trp53. Nf1 was dispensable, while additional loss of Pten produced more invasive tumors and leptomeningeal metastases. The time window for initiation of tumorigenesis was until the 2nd postnatal week, coinciding with the disappearance of the external granule layer (EGL), where cerebellar granule neuron precursors (CGNPs) undergo proliferation. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the EGL, suggesting that the tumors may arise from CGNPs. However, targeting a subset of CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Analysis of tumor transcriptome and MB subtype-specific genes and markers show that Dicer tumors most resemble extremely high risk p53-mutated SHH MB. Small RNA and mRNA sequencing analyses showed downregulation of microRNAs and dysregulation of its targets such as N-Myc. These studies demonstrate a role for microRNAs in MB development and show a fully penetrant genetic mouse model of highly metastatic MB.