H3K27M Mutation Research Articles

FLASH Modulation of Immune Microenvironment in Diffuse Midline Glioma (DMG) at Single-Cell Resolution

<h3>Purpose/Objective(s)</h3> Diffuse Midline Glioma with H3K27M mutation (DMG) is a universally fatal tumor primarily treated with radiotherapy (RT) and typically unresponsive to immune-based therapies. FLASH is a novel RT technique associated with decreased toxicity and effective tumor control in glioma models as well as immune-sparing properties in other cancer models. However, the effect of FLASH on the DMG tumor immune microenvironment (TIME) has not yet been explored. <h3>Materials/Methods</h3> Using a syngeneic murine brainstem DMG, we performed single-cell RNA sequencing on CD45+ cells isolated from tumors 4 days after posterior fossa irradiation (15Gy x 1, 9MeV), utilizing either ultra-high dose rate (90Gy/sec, FLASH) or conventional (2Gy/min, CONV) RT, and compared to unirradiated tumor (SHAM). For differential gene expression analysis, we compared CONV to SHAM (CvS) and FLASH to SHAM (FvS). We also performed single cell gene set enrichment scores (ssGSEA) for evaluation of immune signatures from gene ontology (GO); ssGSEA scores were deemed significant by a p<0.05 using Mann-Whitney U test. We validated our most robust immune markers with dual immunofluorescence. <h3>Results</h3> Unsupervised clustering of 23,165 CD45+ cells revealed 16 immune clusters, including subtypes of resident microglia (MG, 78.2% of total cells), lymphocytes (6.4%), macrophages (MPs, 5.2%), monocytes (1.8%), and dendritic cells (DCs, 1.7%). MG consisted of four distinct subtypes representing a spectrum of homeostatic to activated cellular states. In the most activated MG subtype, both CONV and FLASH showed an upregulation of interferon (IFN) genes such as Isg15 (Fold Change (FC) 1.03 CvS, 0.94 FvS) and Ifit3 (FC 0.77 CvS, 0.66 FvS) when compared to SHAM. Furthermore, ssGSEA of MG found IFN scores increased in both FvS (p < 0.0001) and CvS (p < 0.0001) while TGFbR signaling score was increased in FvS (p < 0.0001) and decreased in CvS (p < 0.0001). In non-resident myeloid clusters, the most differential response was found in MPs and DCs with robust upregulation of IFN genes for CvS (Isg15 FC 2.30 MPs, 2.67 DCs; Ifit3 FC 2.02 MPs, 2.06 DCs), while this response was not seen with FvS. This was confirmed with ssGSEA with increased IFN scores in both MPs (CvS p<0.0001, p=0.04) and DCs (CvS p<0.0001, FvS p=0.69). FLASH showed an increase in anti-inflammatory MP markers such as Mrc1, Cd163, and Maf (FC 1.40, 1.11, 1.19) and an upregulated myeloid-derived suppressor cell signature in monocytes (FvS, p < 0.0001), not seen in CvS (p = 0.09). <h3>Conclusion</h3> Our work is the first to map CONV and FLASH immune alterations with single-cell resolution in the DMG TIME. FLASH induces IFN differentially across microglia and non-resident myeloid subsets. Further exploration is warranted to understand how FLASH impacts tumor growth through differential cytokine signaling, inflammatory responses and immune checkpoint expression within the DMG TIME.

Real-time drug testing of paediatric diffuse midline glioma to support clinical decision making: The Zurich DIPG/DMG centre experience

BackgroundChildren diagnosed with diffuse midline gliomas (DMG) have an extremely poor overall survival: 9–12 months from diagnosis with currently no curative treatment options. Given DMG molecular heterogeneity, surgical biopsies are needed for molecular profiling and as part of enrolment into molecular-based and precision medicine type clinical interventions. In this study, we describe the results of real time profiling and drug testing at the diffuse intrinsic pontine glioma/DMG Research Centre at University Children's Hospital Zurich. MethodBiopsies were taken using a frame based stereotactic robot system (NeuroMate®, Renishaw) at University Children's Hospital Zurich. Tissue samples were evaluated to confirm diagnosis by H3K27M and H3K27 trimethylation loss. Genomic analyses were done using a variety of platforms (INFORM, Oncomine, UCSF500 gene panel).Cell lines were developed by mechanical tissue dissociation and verified by either sequencing or immunofluorescence staining confirming H3K27M mutation and used afterwards for drug testing. ResultsTwenty-five robot-assisted primary biopsies were successfully performed. Median hospital stay was 2 days (range 1–4 days). Nine low-passage patient-derived cells were developed, whereas 8 cell lines were used to inform response to clinically relevant drugs. Genome and RNA expression were used to further guide treatment strategies with targeted agents such as dual PI3K/mTOR inhibitor paxalisib. ConclusionWe established a systematic workflow for safe, robot-assisted brainstem biopsies and in-house tissue processing, followed by real-time drug testing. This provides valuable insights into tumour prognostic and individual treatment strategies targeting relevant vulnerabilities in these tumours in a clinically meaningful time frame.

Conventional and Cross-Channel MR Radiomic Features do Not Predict Histone H3 Status in DIPG: Genomic and Clinical Evaluation of a Multi-Institutional Cohort

<h3>Purpose/Objective(s)</h3> Genomic profiling of diffuse pontine glioma (DIPG) has suggested distinct and clinically relevant molecular subgroups based on the presence and type of histone H3K27M mutation. Recent studies have demonstrated improved safety for biopsy, but sampling carries risks. We evaluated the impact of radiomic features on the molecular classification of 154 histologically confirmed and centrally reviewed DIPG. <h3>Materials/Methods</h3> Automatically segmented and manually reviewed tumor volumes were used to compute single & cross channel radiomic features from T1, T2, FLAIR, and T1+gadolinium MR sequences obtained from histone H3 WT (N=30) and H3K27M-mutant (N=124) DIPG. Radiomic feature stability was assessed when multiple pretreatment MRs were available using the concordance correlation coefficient. Random Forest, Logistic Regression, XGBoost, and Deep Learning classifiers were used to build predictive models of H3K27M status in a training dataset, after exclusion of correlated features. Model accuracy was assessed in the remaining validation dataset. <h3>Results</h3> Two hundred and twelve of 456 imaging features (46.4%) demonstrated stability (CCC, p<0.05) between pretreatment scans. Accuracy varied by method, but none surpassed the No Information Rate (82%). Model accuracy was not improved by efforts to remedy class imbalance (resampling and class weight adjustment). Several cross channel radiomic features were consistently selected by the models, indicating some predictive utility, although cross institutional scanning techniques were confounding. <h3>Conclusion</h3> While early efforts with single institutional data were encouraging, differences in MR technique and class imbalance seemed to limit the identification of stable radiomic features predictive of H3 status in this multi-institutional cohort. Biopsy remains key for determining trial eligibility and directing precision medicine.

Indolent presentation of a diffuse midline glioma, H3 K27-altered.

Diffuse midline glioma (DMG), H3 K27-altered, are aggressive central nervous system tumors which are universally fatal, with a median survival of 8-12months after diagnosis. Here, we present a patient who was incidentally found to have a lesion, concerning for tumor, within the right thalamus on brain magnetic resonance imaging at 2years of age. Twelve years later, subsequent imaging showed that the lesion had enlarged, with biopsy consistent with DMG harboring an H3 K27M mutation. This case illustrates an unusual presentation of a DMG, H3 K27-altered, with an indolent course. Such findings highlight the fact that more research is needed to understand what factors may contribute to these tumors' malignant course.

Oncohistone interactome profiling uncovers contrasting oncogenic mechanisms and identifies potential therapeutic targets in high grade glioma

Histone H3 mutations at amino acids 27 (H3K27M) and 34 (H3G34R) are recurrent drivers of pediatric-type high-grade glioma (pHGG). H3K27M mutations lead to global disruption of H3K27me3 through dominant negative PRC2 inhibition, while H3G34R mutations lead to local losses of H3K36me3 through inhibition of SETD2. However, their broader oncogenic mechanisms remain unclear. We characterized the H3.1K27M, H3.3K27M and H3.3G34R interactomes, finding that H3K27M is associated with epigenetic and transcription factor changes; in contrast H3G34R removes a break on cryptic transcription, limits DNA methyltransferase access, and alters mitochondrial metabolism. All 3 mutants had altered interactions with DNA repair proteins and H3K9 methyltransferases. H3K9me3 was reduced in H3K27M-containing nucleosomes, and cis-H3K9 methylation was required for H3K27M to exert its effect on global H3K27me3. H3K9 methyltransferase inhibition was lethal to H3.1K27M, H3.3K27M and H3.3G34R pHGG cells, underscoring the importance of H3K9 methylation for oncohistone-mutant gliomas and suggesting it as an attractive therapeutic target.

Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.

SYST-07 WINDOW-OF-OPPORTUNITY STUDY OF ONC201 IN PEDIATRIC PATIENTS WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND THALAMIC GLIOMA

BACKGROUNDH3 K27M-mutant diffuse midline glioma is a universally fatal malignancy primarily affecting children and young adults; no effective systemic therapy is available. ONC201, a first-in-class imipridone, is an oral, blood-brain barrier penetrating, selective small molecule antagonist of dopamine receptor D2/3 and agonist of the mitochondrial protease ClpP. ONC201 monotherapy demonstrated durable objective responses in adults with recurrent H3 K27M-mutant glioma. This phase 1 trial will evaluate ONC201±radiotherapy (RT) in pediatric patients with H3 K27M-mutant midline glioma DIPG. METHODSThis multicenter, open-label, dose escalation and expansion phase 1 study of ONC201 is comprised of eight arms that will evaluate the recommended phase 2 dose (RP2D) of ONC201, biomarkers, and pharmacokinetics (PK) of ONC201±RT in various treatment settings (NCT03416530). Arm G previously defined the RP2D for twice-weekly ONC201 on consecutive days. Arm H, for which enrollment is ongoing, will estimate the influence of tumor location and blood-brain barrier integrity on PK and intratumoral ONC201 exposure in biopsy-eligible pediatric tumors (DIPG or contrast-enhancing thalamic glioma). Patients eligible for Arm H will be aged 2-≤19 years, ≥2 weeks from last RT administration, and have a KPS/LPS ≥50; prior confirmation of H3 K27M mutation is not required. In Arm H, single-agent ONC201 administration will occur twice-weekly on consecutive days during each 21-day cycle at the RP2D defined in Arm G. Arm H has a planned enrollment of 27 patients (DIPG, n=15; thalamic glioma, n=12), with three patients undergoing a single biopsy at each of the following time points: 1-3 h post-first dose, 22-26 h post-second dose, 1-3 h post-first dose, 6-10 h post-second dose, and 22-26 h post-second dose. The 22-26 h post-first dose biopsy in thalamic glioma was previously collected and will not be assessed in this treatment arm. Plasma for PK analysis will be collected from all patients.

CLRM-02 SINGLE CENTER EXPERIENCE OF DOPAMINE ANTAGONIST ONC-201 FOR RECURRENT H3K27M-MUTANT GLIOBLASTOMA IN ADULTS

OBJECTIVETo report the single center experience of three adult subjects receiving ONC-201 as part of the ONC018 expanded access clinical trial [NCT03134131]. BACKGROUNDONC-201 is an oral investigational antagonist against the D2 dopamine receptor that has shown encouraging results for malignant gliomas harboring the H3K27M mutation in the H3 histone complex. Responses have been reported in pediatric subjects with such tumors. H3K27M tumors are generally located in the midline of the central nervous system which co-localize, for unknown reasons, with key dopaminergic pathways. An expanded access clinical trial (ONC018) was available to eligible patients allowing them access to this agent pending FDA review. DESIGN/METHODSOur site enrolled three subjects on the ONC018 trial. We present the demographic, clinical, and molecular characteristics for our enrolled subjects. We report the tolerability, adverse events, and outcome measures including survival, time-to-progression, response, Karnofsky Performance Status [KPS] and quality-of-life measured by the MD Anderson symptom inventory instrument [MDASI]. RESULTSThree subjects were registered at our site onto ONC018 with age range 18-44yrs, 2/3 female, residing in Norway, India and United states. Tumor locations: brainstem, corpus callosum and thalamus. Pathology: glioblastoma(3/3), MGMT methylated (2/3), IDH1 mutant (0/3), EGFR amplification (0/3) and ATRX (3/3). Median change from baseline KPS: ≤20% decrease; MDASI:2/3 experienced decrease from baseline [median 6%],consistent with improved quality of life. No clinically significant laboratory abnormalities. No serious adverse events observed in any cases. All adverse events grade I-II. CONCLUSIONSIn three subjects with H3K27M-mutant malignant glioma that received ONC201 as part of this expanded access clinical trial, we found that study drug was quite tolerable. No serious adverse events nor radiographic responses were seen. Analyses of larger study cohort and additional randomized controlled trials are necessary to provide insight into the safety and efficacy of this agent for this patient population.

Abstract 6049: Identifying shared features of low-H3K27me3 pediatric brain tumors

Abstract Brain tumors have the worse survival rates of all childhood malignancies, and a better understanding of their biology is needed for therapeutic development. Two especially devastating subtypes of pediatric brain tumors—diffuse midline gliomas, H3K27M-mutant (DMGs) and posterior fossa ependymomas, group A (PFAs) are both characterized by a hallmark of global reduction of histone 3 lysine 27 trimethylation, a mark associated with transcriptional repression. DMGs primarily harbor mutations of histone 3 (H3K27M) that inhibit the function of the H3 methyltransferase enhancer of zeste homolog 2 (EZH2)-containing polycomb repressive complex (PRC2). Most PFAs overexpress EZH inhibitory protein (EZHIP), resulting in a similar effect on PRC2 activity. Intriguingly, a small subset of DMGs found to be lacking H3K27M mutations express EZHIP, and a fraction of histological PFAs lacking EZHIP expression harbor H3K27M mutations. Given the highly similar putative driving alterations and epigenetic features in these two tumor types, we undertook a systematic evaluation of the clinical and molecular features of each tumor class to elucidate additional shared features and potential targetable vulnerabilities. We performed detailed analyses of genomic aberrations, gene expression, and epigenomic landscapes to identify key similarities and differences in tumor biology. From preliminary studies, our findings demonstrate similar H3K27me3 landscapes with convergence of residual tri-methylated loci between PFA and DMG tumors. We additionally found shared recurrent copy number gain of the long arm of chromosome 1 (1q) and identified several 1q genes whose expression correlated with survival differences in cohorts of both tumors. Together, these findings better define the commonalities and differences of these highly aggressive, low-H3K27me3 pediatric brain tumors and will provide a framework for understanding which therapeutic strategies may translate from one tumor to the other. Citation Format: Matthew Pun, Drew Pratt, Sriram Venneti. Identifying shared features of low-H3K27me3 pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6049.

Abstract 3965: Histone H3 K27M mediated regulation of cancer cell stemness and differentiation in diffuse intrinsic pontine glioma (DIPG)

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is a rare pediatric brain tumor with a median survival of 10-15 months. Histone H3 is mutated in 80% of DIPGs, dictating tumor location, onset, and outcome of this devastating disease. Therapeutic resistance remains a major obstacle to preventing tumor recurrence and is in part driven by cancer stem cells (CSCs), which exhibit self-renewal properties and tumorigenic potential. In previous studies, we have identified these proliferative and tumorigenic features in aldehyde dehydrogenase positive (ALDH+) CSCs in H3K27M mutant DIPG. Among the 19 ALDH isoforms known in humans, aldehyde dehydrogenase family 1 member A3 (ALDH1A3) is most highly expressed in malignant cells when compared to immune cells or oligodendrocytes in H3K27M DIPG biopsies, indicating an important mechanistic role in regulating stemness in DIPG. We hypothesize that ALDH-mediated cancer stemness and resistance may in part be driven by H3K27M. Indeed, ALDH1A3 expression was dramatically reduced in CRISPR-edited DIPG cells where the H3K27M mutation had been removed (H3K27M-KO). Furthermore, these cells lost their neurosphere forming potential, began to express glial fibrillary acidic protein (GFAP) and attached to the plastic, changes indicative of differentiation. Transcriptomic and GSEA pathway analyses suggested Wnt-dependent regulation of ALDH1A3, with Wnt 7b and Wnt 11 demonstrating the largest difference in the isogenic DIPGs. In addition, DNA repair, along with PI3K/AKT and Wnt signaling, were found to be upregulated upon radiation of DIPG cells, indicating future therapeutic opportunities to prevent radio resistance. We have co-targeted these cell-intrinsic vulnerabilities to eradicate CSCs in DIPG and prevent tumor recurrence, using a combination of radiotherapy, the PI3K/mTOR inhibitor GDC-0084 and the ALDH inhibitor Disulfiram. This combination has produced an exquisite synergy in cells, which we will test in clinically-relevant DIPG models. Taken together, through these studies we have gained mechanistic insight into H3K27M mediated regulation of cancer cell stemness and differentiation in DIPG, which appears to be mediated by Wnt-dependent signaling of ALDH1A3 and provides rationale for exploring new therapeutic targets. Citation Format: Monika Sharma, Brian Magnuson, Jeanette Cruz, McKenzie Kauss, Alexander Buschhaus, Mats Ljungman, Stefanie Galban. Histone H3 K27M mediated regulation of cancer cell stemness and differentiation in diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3965.

H3K27M Mutation Doesn't Mean Worse Prognosis in Old Patients.

ObjectiveDiffuse midline glioma (DMG), H3K27 altered is a new entity that has become widely recognized. However, studies concerning DMG in adult patients remain rare. We did a retrospective study covering the largest amount of patients to date to analyze the clinicopathological characteristics of diffuse glioma in midline structures of the adult.MethodsWe reviewed 108 cases of adult DMG, collected their clinical data, and pathological results including H3K27 mutation. Summarized their features and the connection with overall survival in different age groups.ResultsAmong 108 cases, 79 tumors were located at the thalamus. 38 patients had H3K27M mutation, whose average age was 35.7 years. The median overall survival of H3K27M-mutant gliomas and the 80 H3K27M wild-type gliomas were both 12 months. For young patients (age ≤ 35), The median survival time of the H3K27M-mutant was 18 months, while that of the H3K27M wild-type was 37 months. For older patients (age>35), the median survival time of the H3K27M-mutant was 16 months, while that of the H3K27M wild-type was 13 months. Other clinicopathological factors including sex, tumor location, the approach of surgery, histological grade, ATRX, and P53 were statistically irrelevant to prognosis.ConclusionThe DMG in adults mainly occurred in the thalamus. H3K27M mutations tend to happen more frequently in young adults, and this genetic alteration results in a worse outcome only in young patients (≤35). For old patients, age is the only independent prognostic factor. Patients who underwent different surgical operations including biopsy, subtotal resection, and total resection had similar prognoses.

HGG-06. Phase 2 Study of Veliparib and Local Irradiation, Followed by Maintenance Veliparib and Temozolomide, in Patients with Newly Diagnosed High-Grade Glioma without H3 K27M or BRAF Mutations: A Report from the Children's Oncology Group ACNS1721 Study

BACKGROUND: The outcome for pediatric patients with high-grade glioma (HGG) remains poor. Veliparib, a potent oral PARP1/2 inhibitor, enhances the activity of radiotherapy and DNA-damaging chemotherapy. Preclinical data indicates that veliparib crosses the blood-brain-barrier and enhances the efficacy of radiotherapy and temozolomide in IDH mutant and wild-type HGG models. ACNS1721 was a single-arm, non-randomized phase 2 clinical trial designed to determine whether treatment with veliparib and radiotherapy, followed by the poly (ADP-ribose) polymerase (PARP) inhibitor veliparib and temozolomide, improves progression-free survival (PFS) in pediatric patients with newly diagnosed HGG without H3 K27M or BRAF mutations compared to patient level data from historical cohorts with closely matching clinical and molecular features. METHODS: Following surgical resection, newly diagnosed children with non-metastatic HGG were screened by rapid central pathology review and molecular testing. Eligible patients without somatic H3 K27M or BRAF mutations were enrolled on Stratum 1 (IDH wild-type) or Stratum 2 (IDH mutant). Protocol radiochemotherapy consisted of involved field radiotherapy with concurrent veliparib at 65 mg/m2 twice daily. Adjuvant chemotherapy consisted of up to 10 cycles of veliparib 25 mg/m2 twice daily and temozolomide 135 mg/m2 once daily for 5 days every 4 weeks. RESULTS: Both strata were closed to accrual for futility after planned interim analyses. Among the 23 eligible patients who enrolled on Stratum 1 and received protocol therapy, the 1-year progression-free survival (PFS) was 0.29 (SE = 0.09) and 1-year overall survival (OS) was 0.67 (SE = 0.10). Among the 14 eligible patients who enrolled on Stratum 2 and received protocol therapy, the 1-year PFS was 0.57 (SE = 0.15) and 1-year OS was 0.90 (SE = 0.09). CONCLUSION: Rapid central pathology review and molecular testing was feasible. The protocol therapy was well tolerated but failed to improve outcome compared to clinically and molecularly matched historical control cohorts.

HGG-03. PEDIATRIC BITHALAMIC GLIOMA H3K27M NEGATIVE AND EGFR POSITIVE: A DISTINCT ENTITY WITH POTENTIAL SENSIBILITY TO EGFR INHIBITORS

INTRODUCTION: Brain tumors are the most common solid tumors of childhood and optimal therapeutic strategies for many subtypes remain unknown. Bithalamic gliomas, in contrast with unilateral, have frequent mutations in the EGFR oncogene and only rare Histone H3 mutation. These EGFR mutations are small inframe insertions within exon 20 or missense mutations within exon 7 and occur in absence of gene amplification. CASE REPORT: A previously healthy 10-years-old boy with adequate neuropsychomotor development was admitted with a 5-day-long headache. A brain magnetic resonance imaging (MRI) showed an expansive midline lesion with thalamic extension. The tumor showed no diffusion restriction or contrast enhancement. An endoscopic septostomy and biopsy were performed.The histopahotology showed a diffused astrocytoma grade II. The immunohistochemistry was negative for IDH1 and 2, BRAF mutation, P53 and H3K27M. Next generation sequencing (NGS) ruled out histone H3K27M mutation and showed EGFR mutation in exon 7. The patient experimented clinical and radiological disease progression in a few weeks, confirming the high grade glioma despite the histopahological diagnosis.He received radiotherapy (Total dose 54Gy) and after radiation he started target therapy with Osimertinib, a third generation tirosin kinase inhibitor. As a toxiticy, he experienced neutropenia Grade 2. Disease evaluation performed after 2 cycles showed stable disease. DISCUSSION: Our case report represents a distintc molecular class of pediatric-type bithalamic glioma characterized by EGFR alteration and absence of H3K27M mutation. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.

MODL-24. Focused ultrasound-mediated blood-brain barrier opening and panobinostat in a thalamic syngeneic murine DMG model is feasible and safe.

Abstract Diffuse Midline Glioma with H3K27M mutation (DMG) is an aggressive unresectable central nervous system tumor of the brainstem, midline thalamus, or spine. Prognosis is poor and systemic agents have been ineffective partially due to limited permeability of the blood brain barrier (BBB). Non-invasive low intensity focused ultrasound (FUS) can be used for BBB opening (BBBO). Our preclinical studies showed safety and feasibility of targeting and BBBO in the brainstem; however, to our knowledge, FUS-guided BBBO for thalamic DMG has yet to be reported. RNA-seq was performed on mouse syngeneic DMG cells 4423 (PDGFB+, H3.3K27M, p53−/−) and results recapitulated molecular programs seen in human tumors. 4423 cells were injected into the thalamus of male B6 (Cg)-Tyrc-2J/J mice at 1.6mm lateral and 1.8mm posterior to the bregma at a depth of 3.2mm. MRI at 14-21 days post injection confirmed thalamic tumor growth. Histological analysis with Hematoxylin and Eosin stain was consistent with thalamic DMG. Tumor implantation rate was 85% and median survival was 38 days post injection. To test safety and feasibility of BBBO for thalamic tumors, a 1.5 MHz FUS transducer was used with concurrent microbubble injection. BBBO assessed by contrast-enhanced MRI. FUS achieved BBBO targeting the thalamic tumor without increased morbidity or mortality and BBB closure was observed on day 3 post-sonication. Next we tested the tolerance of drug delivery with panobinostat post-FUS at a dose of 20mg/kg weekly. The results are preliminary at this time; however, animals tolerated the combination therapy without morbidity or mortality. Preclinical models are crucial to improve the development of new therapeutic strategies. Establishing this syngeneic thalamic DMG murine model provides the opportunity to test FUS as a non-invasive drug delivery technology for thalamic DMG compared to brainstem DMG, and to re-visit therapeutic agents previously considered ineffective due to limited penetration of the BBB.

DIPG-47. TSO500ctDNA sequencing reveals oncogenic mutations and copy number variations in the liquid biome of children with diffuse midline glioma

BACKGROUND: Molecular profiling of childhood CNS tumors is critical for diagnosis and clinical management, yet tissue access is restricted due to sensitive neuroanatomical locations. Moreover, CNS tumors including diffuse midline glioma (DMG) exhibit mutational heterogeneity and clonal evolution, which cannot be captured by upfront diagnostic biopsy alone. To address the lack of tumor visibility, and tprovide opportunity for longitudinal sampling, we validated and optimized a commercially available deep sequencing platform for analysis of circulating tumor DNA (TSO500ctDNATM). METHODS: In a proof-of-concept study, we defined the sensitivity, specificity, and clinical relevance of our novel ctDNA platform via analysis of paired tissue, CSF, and blood from children with DMG (n=10). Paired samples were assessed for concordance and sequencing results were compared to digital droplet PCR (ddPCR) detection of prognostic H3K27M mutation. RESULTS: DMG associated mutations in genes including H3-3A, H3C2, TP53, and ACVR1 were detected in ctDNA, including in CSF samples with low (<5ng) starting DNA input. Of 9 H3K27M mutations identified in tumor, 8 were present in CSF and 3 in plasma/serum, for a positive percent agreement with tumor results of 89% and 33%, respectively. Among CSF samples, H3.3K27M was detected in 6/6 cases, and H3.1K27M in 2/3 cases, with variant allele frequencies comparable to ddPCR results. CNVs including PDGFRA, KIT, and MDM4 gains were detected in CSF and paired tumor. Low frequency events including ACVR1, PIK3CA activating mutations and KRAS amplification were detected in CSF but absent from paired tumor, indicating tissue heterogeneity. Strategies to optimize ctDNA detection, including optimization of ctDNA isolation and adjustment of library QC metrics, were identified. CONCLUSION: Targeted ctDNA deep sequencing is feasible, can inform on clinically relevant tumor mutation and CNV profiling, and provides an opportunity for longitudinal monitoring of tumor genomic evolution in the liquid biome of children with CNS tumors.

HGG-20. PRMT5 promotes the formation and growth of pediatric high-grade glioma by maintaining tumor stem cell populations

BACKGROUND: Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated CNS tumors comprising two broad subtypes: diffuse midline glioma with H3K27M mutations (DMG) and cortical high-grade glioma (H3K27-wild-type (wt) PHGG). During normal development, PRMT5 promotes stem cell self-renewal through methylation of arginine residues in histone tails. We hypothesized that PRMT5 controls self-renewal essential to the proliferation of PHGG tumor initiating cells (TICs). METHODS: We identified PRMT5 as potentially oncogenic in PHGG through a screen of 4,139 shRNAs targeting 406 genes with epigenetic activity. To elucidate PRMT5’s activity, we used lentiviral shRNA delivery to knock down (KD) PRMT5 expression in four DMG and one H3K27-wt PHGG cell lines. We performed in vitro growth, cell cycle, apoptosis, limiting dilution and bulk RNA-Seq assays to determine the phenotypic effects of PRMT5 KD. To identify PRMT5’s gene targets, we performed cleavage under targets & release using nuclease (CUT&RUN) followed by qPCR and are currently performing CUT&RUN-Seq. We orthotopically implanted PRMT5 KD PHGG cells into mice and tracked survival, tumor growth and tumor histological characteristics. RESULTS: In vitro, PRMT5 KD reduced cell growth (p<0.001), slowed cell cycle progression and increased apoptosis. PRMT KD also slowed neurosphere formation, demonstrating reduced self-renewal (p<7E-9). Geneset expression analysis showed PRMT5 KD reduced expression of self-renewal genes and increased expression of differentiation genes (FDR<0.0001). In vivo, PRMT5 KD reduced tumor growth, as monitored by bioluminescence and MRI, and aggressiveness, based on Ki-67 staining (p<0.05), leading to increased survival (p<0.001). CUT&RUN-qPCR results showed PRMT5 KD led to decreased expression and H3K4me3 promoter occupancy at PAX3, and decreased expression and increased H3K27me3 occupancy at S100A6. PAX3 and S100A6 are oncogenes that preserve TIC self-renewal. CONCLUSION. In vitro experiments show that PRMT5 KD epigenetically reduces TIC self-renewal. In vitro and in vivo, PRMT5 KD reduced PHGG tumor cell growth and aggressiveness.

TBIO-09. EZHIP is not expressed within the developing mouse or human brain

The protein EZHIP, first described as part of the genetic signature of group A posterior fossa ependymoma (PFA), functions in a manner analogous to the onco-histone H3K27M, itself first described in diffuse midline glioma (DMG). These two proteins modulate activity of the Polycomb repressive complex 2 (PRC2), resulting in global loss of H3K27 trimethylation. Thus, both PFA and DMG are characterized by global loss of the H3K27me3 mark, and PRC2 modulation is critical to oncogenesis of these tumors. However, the precise mechanism(s) of EZHIP and H3K27M-induced tumorigenesis is not known. One possibility is that EZHIP is expressed within the developing brain where it acts to modulate cellular development. In turn, EZHIP overexpression or H3K27M mutation could co-opt this developmental program to drive tumorigenesis. Relatedly, whether EZHIP is expressed in the cell of origin for PFA or DMG is not known. In order to evaluate these hypotheses, we defined the landscape of EZHIP expression in both murine and human developing brain. Leveraging single cell RNA sequencing databases, we show that EZHIP is not expressed within the developing mouse brain, a finding that is consistent across separate datasets. Similarly, EZHIP is not expressed in the developing murine cerebellum, where the cell of origin for PFA is located. Furthermore, we examined bulk RNA sequencing datasets of the developing human brain. Similar to our findings in the mouse brain, EZHIP is not expressed at any time point or spatial location within the developing human brain. Taken together, these data reject the above hypothesis, that EZHIP or H3K27M co-opt an endogenous PRC2 inhibitory developmental program. Similarly, these results show that EZHIP is not expressed within the cell of origin for PFA or DMG. Further studies will seek to understand the endogenous function of EZHIP by further defining its normal expression pattern and function.

PATH-16. Noninvasive diagnosis of gliomas through CSF cfDNA sequencing in pediatric and adolescent and young adult (AYA) patients

Abstract PURPOSE: A subset of pediatric, adolescent and young adult (AYA) gliomas are located in the brainstem, eloquent locations, or present with diffuse/leptomeningeal disease, and are associated with high risk and low yield of biopsy. At the same time, accurate molecular diagnosis is necessary to direct optimal therapy. In such cases, analysis of cell free DNA (cfDNA) form cerebral spinal fluid (CSF) may represent a diagnostic alternative to biopsy. METHODS: We investigated the utility of CSF cfDNA sequencing through a stepwise approach, using clinically validated, targeted molecular assays. Testing was performed using a broad hybrid capture next generation sequencing assay (MSK-IMPACT) and subsequent targeted digital droplet PCR in a subset of cases. RESULTS: We analyzed 17 CSF samples from 17 pediatric (n=6) and AYA (n=11) glioma patients with primary or recurrent disease. Thirteen had tumors located within the brainstem, and four had leptomeningeal involvement. Somatic alterations were detected in 12/17 samples (71%). In 3/4 patients with leptomeningeal involvement, cfDNA testing revealed a BRAF fusion consistent with the diagnosis of diffuse leptomeningeal glioneuronal tumor (DLGNT). Among the 13 patients with brainstem involvement, four had somatic H3 K27M mutations, three had IDH mutations, and one had TP53 and ATRX mutations; five patients had no detectable mutations. CONCLUSION: In our analysis, we found that established glioma hotspot mutations were able to be detected within the CSF. We propose that in patients for whom tissue biopsy is high risk, not feasible, or tissue was nondiagnostic, CSF cfDNA sequencing has a substantial diagnostic yield and should be considered as a valuable novel diagnostic tool. Ongoing research is aiming to further increase the sensitivity of cfDNA testing, especially in patients with very low levels of CSF cfDNA.

HGG-26. Comprehensive Genomic Profiling of Pediatric and AYA High-Grade Gliomas Reveals a High Prevalence of ATRX and Homologous Recombination Repair (HRR) Mutations

Abstract BACKGROUND: Pediatric high-grade glioma (HGG) is a devastating heterogenous disease with a clear need for more effective treatment options. Advances in high-throughput molecular sequencing have advanced our understanding of these tumors and yet outcomes remain poor as only a very small subset of patients have demonstrated benefit from targeted therapy approaches. METHODS: We compiled genomic data from 980 pediatric (N=400) and AYA (18 to 30 yrs, N=580) HGG cases that underwent targeted, panel-based comprehensive genomic profiling (FoundationOne® CDx). RESULTS: Among pediatric patients, mutations in ATRX or HRR-related genes were among the most prevalent at 24.25% and 19.5%, respectively. Other commonly occurring mutations were seen in TP53 (58.25%), H3F3A (43%), and PIK3CA/PIK3R1 (22%). Among AYA patients, mutations in ATRX and HRR-related genes were seen in 51.72% and 17.24% of cases, respectively. The most common mutations seen in this older population were in TP53 (76.38%), IDH1/2 (50.86%), PI3KCA/PIK3R1 (20.52%), and H3F3A (18.28%). Among patients with diffuse midline glioma (harboring H3K27M mutations), ATRX and HRR mutations were reported in 21.27% and 13.57% of cases, respectively. Among pediatric patients with histone wild type HGG, mutations in ATRX and HRR-related genes occurred in 20.61% and 21.05% of cases, respectively. CONCLUSION: Alterations in ATRX and HRR-related genes are among the most prevalent mutations in pediatric and AYA HGG. Consideration should be made towards the development of subtype-specific treatment protocols using PARP and/or ATR inhibitors aimed at this subgroup of patients.

HGG-02. Epigenetic transcription regulation and 3D genome structure in pediatric high-grade glioma

INTRODUCTION: Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are highly morbid brain tumors. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding Histone H3. To investigate whether the H3K27M mutant protein is associated with distinct chromatin structure affecting transcription regulation, we generated the first high-resolution Hi-C and ATAC-Seq maps of pHGG cell lines, and integrated these with tissue and cell genomic data. METHODS: We generated sequencing data from patient-derived cell lines (DIPG n=6, GBM n=3, normal n=2) and frozen tissue specimens (DIPG n=1, normal brainstem n=1). Analyses included cell line RNA-Seq, ChIP-Seq (H3K27ac, H3K27me3, H3K27M) and genome-wide chromatin conformation capture (Hi-C), as well as tissue ATAC-Seq. Publicly available pediatric glioma tissue ChIP-Seq data was integrated with cell data. CRISPR knock-down of target enhancer regions was performed. RESULTS: We identified tumor-specific enhancers and regulatory networks for known oncogenes in DIPG and GBM. In DIPG, FOX, SOX, STAT and SMAD families were among top H3K27Ac enriched motifs. Significant differences in Topologically Associating Domains (TADs) and DNA looping were observed at OLIG2 and MYCN in H3K27M mutant DIPG, relative to wild-type GBM and normal cells. Pharmacologic treatment targeting H3K27Ac (BET and Bromodomain inhibition) altered these 3D structures. Functional analysis of differentially enriched enhancers in DIPG implicated SOX2, SUZ12, and TRIM24 as top activated upstream regulators. Distinct genomic structural variations leading to enhancer hijacking and gene co-amplification were identified at A2M, JAG2, and FLRT1. CONCLUSION: We show genome structural variations enhancer-promoter interactions that impact gene expression in pHGG in the presence and absence of the H3K27M mutation. Our results imply that tridimensional genome alterations may play a critical role in the pHGG epigenetic landscape and thereby contribute to pediatric gliomagenesis. Further studies examining the impact of the alterations are therefore underway.