Midline Glioma Research Articles

LIN28B and Let-7 in Diffuse Midline Glioma: A Review

Diffuse midline glioma (DMG) is the most lethal of all childhood cancers. DMGs are driven by histone-tail-mutation-mediated epigenetic dysregulation and partner mutations in genes controlling proliferation and migration. One result of this epigenetic and genetic landscape is the overexpression of LIN28B RNA binding protein. In other systems, LIN28B has been shown to prevent let-7 microRNA biogenesis; however, let-7, when available, faithfully suppresses tumorigenic pathways and induces cellular maturation by preventing the translation of numerous oncogenes. Here, we review the current literature on LIN28A/B and the let-7 family and describe their role in gliomagenesis. Future research is then recommended, with a focus on the mechanisms of LIN28B overexpression and localization in DMG.

Differences in survival prognosticators between children and adults with H3K27M-mutant diffuse midline glioma.

H3K27M-mutant diffuse midline glioma (DMG) is a rare and aggressive central nervous system tumor. The biological behavior, clinicopathological characteristics, and prognostic factors of DMG have not yet been completely uncovered, especially in adult patients. This study aims to investigate the clinicopathological characteristics and identify prognostic factors of H3K27M-mutant DMG in pediatric and adult patients, respectively. A total of 171 patients with H3K27M-mutant DMG were included in the study. The clinicopathological characteristics of the patients were analyzed and stratified based on age. The Cox proportional hazard model was used to determine the independent prognostic factors in pediatric and adult subgroups. The median overall survival (OS) for the entire cohort was 9.0 months. Significant differences were found in some clinicopathological characteristics between children and adults. The median OS was also significantly different between the pediatric and adult subgroups, with 7.1 months for children and 12.3 months for adults (p < 0.001). In the overall population, the multivariate analysis identified adult patients, single lesion, concurrent chemoradiotherapy/radiotherapy, and intact ATRX expression as independent favorable prognostic factors. In the age-stratified subgroups, the prognostic factors varied between children and adults, with intact ATRX expression and single lesion being independent favorable prognostic factors in adults, while infratentorial localization was significantly associated with worse prognosis in children. The differences in clinicopathological features and prognostic factors between pediatric and adult patients with H3K27M-mutant DMG suggest the need for further clinical and molecular stratification based on age.

DIPG-46. TARGETING THE METABOLIC ERK5-PFKFB3 AXIS IN PEDIATRIC DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma is an aggressive brain tumor with a median age at diagnosis of 6.3 years and 5-year survival rate of 2.2%.The defining histone mutation H3K27M precludes docking of a protein responsible for epigenetic modification resulting in erroneously accessible chromatin and subsequent transcription of oncogenic pathways, including the RAS-MERK5-ERK5 signaling cascade. To determine which oncogenic processes are regulated by extracellular signal-regulated kinase 5 (ERK5), gene-set enrichment analysis of patient-derived datasets demonstrated gene networks involved in glycolysis to be enriched with ERK5 expression. In confirmation, loss of ERK5 via shRNA interference reduced cell proliferation and glycolysis in DIPG IV and SF8628 cells. Reintroduction of ERK5 wildtype (WT) into ERK5 knockdown lines rescued cell proliferation and glycolysis, while the addition of ERK5 kinase dead domain (KDD) only partially rescued these survival and metabolic defects. Targeted evaluation of glycolysis enzyme expression via qRT-PCR revealed a direct relationship between ERK5 and proglycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Mechanistically, ERK5 activation of PFKFB3 was mediated by activation of transcriptional factor myocyte enhancer factor 2A (MEF2A) as demonstrated by coimmunoprecipitation and luciferase promoter assays. Expression of PFKFB3 was elevated at both the mRNA and protein level in DMG patient-derived samples. Genetic knockdown of PFKFB3 via shRNA interference resulted in reduced proliferation and glycolysis in DIPG IV and SF8628 cells. Similarly, pharmacologic inhibition of PFKFB3 with small molecule inhibitor PFK-158 capitulated these results. This inhibitor demonstrated blood brain barrier penetrance in silico and extended survival of in vivo mouse models. Multitargeted drug therapy against both ERK5 and PFKFB3 produced a synergetic in vitro response with increased sensitivity of these cells to apoptosis compared to single treatment alone. In conclusion, these results support ERK5 regulation of glycolysis through the critical metabolic effector PFKFB3. Multitargeted drug therapy against this axis represents a therapeutic vulnerability in pediatric diffuse midline glioma.

TRLS-02. FEASIBILITY OF EVALUATING ABEMACICLIB NEUROPHARMACOKINETICS OF DIFFUSE MIDLINE GLIOMA USING INTRATUMORAL MICRODIALYSIS

Abstract BACKGROUND Diffuse midline gliomas (DMG) are the most aggressive brain tumors of childhood and young adults, with documented 2-year survival rates &amp;lt;10%. Treatment failure is due in part to the function of the BBB. Intratumoral microdialysis sampling is an effective tool to determine brain entry of varied agents and could help to provide a better understanding of the relationship of drug permeability to DMG treatment response. METHODS This is a non-randomized, single-center, phase 1 clinical trial. Up to seven young adult (18-39 years) patients with recurrent high-grade or diffuse midline glioma will be enrolled with the goal of 5 patients completing the trial over an anticipated 24 months. All patients will take abemaciclib pre-operatively for 4.5 days at twice daily dosing. Patients will undergo resection or biopsy, placement of a microdialysis catheter, and 48 hours of dialysate sampling coupled with timed plasma collections. If intratumoral tumor or brain dialysate sampling concentrations are &amp;gt;10nmol/L, or tumor tissue studies demonstrate CDK inhibition, then restart of abemaciclib therapy along with temozolomide will be administered for maintenance therapy and discontinued with evidence of radiologic or clinical disease progression. DISCUSSION The poor survival associated with diffuse midline gliomas underscore the need for improved means to evaluate efficacy of drug delivery to tumor and peritumoral tissue. The findings of this novel study, will provide real-time measurements of BBB function which have the potential to influence future prognostic and diagnostic decisions in such a lethal disease with limited treatment options. TRIAL REGISTRATION: Clinicaltrials.gov, NCT05413304. Registered June 10, 2022, Abemaciclib Neuropharmacokinetics of Diffuse Midline Glioma Using Intratumoral Microdialysis; https://clinicaltrials.gov/ct2/show/NCT05413304?term=22c0003&amp;draw=2&amp;rank=1

BIOL-24. USING NEEDLE WASH SPECIMEN AFTER STEREOTACTIC BIOPSY TO DEVELOP PDOX MODELS OF DIPG AND DIFFUSE MIDLINE GLIOMAS

Abstract Obtaining viable tissue remains a major barrier for lab-based studies, especially in brain tumors deemed surgically unresectable such as diffuse midline gliomas. Surgical biopsy is performed for those tumors invested in deep and vital brain structures however, biopsied specimens are usually of small volume without additional tissue for research after clinical testing. To overcome the barrier of limited surgical specimen volume, we partnered with neurosurgeons to develop a protocol which procures viable cells from discarded biopsy needle after stereotactic biopsies to develop patient-derived orthotopic xenograft (PDOX) models. After procurement of surgical specimen and prior to disposal of biopsy needle, each stereotactic biopsy needle was washed with FBS media twice. Wash solution was collected and immediately transported on ice to the research lab. Cell number was counted for in vitro culture and for orthotropic in vivo tumor implantation. To date, 29 samples of biopsy needle washes have been obtained including 12 diffuse midline gliomas of the brain stem, 8 supratentorial diffuse midline gliomas, and 3 pineal region tumors with harvested cell number from no visible cells to 5.3 million cells (median 1 million). 14 tumors have been orthotopically implanted into matched locations of immunodeficient mouse brains. Tumor formation was confirmed in 4 out of 15 (28.5%), including 2 brain stem tumors, 1 infiltrating astrocytoma, and 1 midline glioma, after implantation of cell numbers as low as 12,500. 3 models were successfully sub-transplanted to passages II and III. Histological examination confirmed the replication of key pathological features of the originating patient tumors. In conclusion, we have developed a novel protocol to procure clinical specimen by using wash solution of stereotactic brain tumor biopsy needles for surgically unresectable tumors. Our work demonstrates a new possibility for clinical sample acquisition and tumor modeling, opening new frontiers in the field of pediatric brain tumor research.

DIPG-20. DELINEATING MEDIATORS OF ONCOGENESIS IN FOXR2-EXPRESSING DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND Diffuse midline gliomas (DMGs) are a universally fatal brain tumor of childhood. While histone mutations are a critical tumor initiating event, they are insufficient to drive gliomagenesis. Histone mutations co-occur with somatic alterations in other pathways including TP53, MAPK, and MYC signaling. However, the mechanisms through which these pathways are activated have not been fully elucidated. METHODS We applied an integrative approach using transcriptomics, epigenetics, proteomics, in vitro cancer models, and in vivo mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. RESULTS We have recently found that a subset of DMGs aberrantly express FOXR2, a forkhead transcription factor. FOXR2 is both sufficient to enhance tumor formation, and necessary for FOXR2-expressing DMGs. While FOXR2 indeed enhances MYC protein stability, FOXR2 exerts oncogenesis through MYC-independent functions and specifically hijacks E26-transformation specific (ETS) transcriptional circuits and FOXR2 DNA-binding is highly enriched at ETS motifs. We have performed proteomic and phospho-proteomic analysis of FOXR2-expressing human neural stem cells to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells. CONCLUSION Taken together, this study elucidates how FOXR2 interacts with ETS transcription factors to mediate oncogenesis in FOXR2-expressing diffuse midline gliomas.

DIPG-13. NEW IMMUNOCOMPETENT GENETICALLY-ENGINEERED MOUSE MODELS OF PEDIATRIC-TYPE DIFFUSE HIGH-GRADE GLIOMAS REVEAL MECHANISMS OF TUMOR INITIATION AND PROGRESSION

Abstract Pediatric-type diffuse high-grade gliomas (pHGGs) form a group of aggressive brain tumors with very poor prognosis. Recurrent driver mutations in TP53 and histone H3-encoding genes, most commonly H3F3A, are observed in a large proportion of these tumors. H3-K27M substitutions are particularly prevalent in midline gliomas, and H3-G34R mutations occur in supratentorial tumors. To better understand the molecular mechanisms driving pHGG emergence and progression, we set out to develop immunocompetent mouse models recapitulating the oncogenic lesions observed in human tumors. Given that oligodendrocyte progenitor cells (OPCs) may be the cells of origin of some pHGGs, we investigated the effect of mutation induction in the OLIG2-expressing cell lineage, using the Olig2Cre driver. Interestingly, deletion of the P53-encoding gene, Trp53, in Trp53flox/flox;Olig2Cre/+ mice led to the development of diffuse gliomas in nearly all animals. Latency to humane endpoint was relatively long (&amp;gt;1 year), and tumors developed throughout the brain with a mild preference for infratentorial (e.g. brainstem) localization. The gliomas diffusely infiltrated the brain parenchyma, and were heterogeneously immuno-reactive for OPC, astrocytic, and neural progenitor cell (NPC) markers. Based on RNA sequencing, the tumors segregated in two groups: one enriched in expression of OPC genes, and another with a prominent NPC-like signature. Whole exome sequencing revealed frequent Ccnd1 and Kras/Hras copy number gains in advanced tumors. To model additional oncogenic lesions, we generated knock-in mice in which the H3f3aK27M or the H3f3aG34R mutations can be induced in any cell type with Cre recombinase, and extinguished with Flp recombinase. These were combined with conditional Trp53 loss-of-function alleles in various cell lineages, and their effect on tumor initiation and progression was monitored. Collectively, our studies provide new reagents for accurate disease modeling of pHGG in mice, opening opportunities to better understand tumor initiation and progression at high resolution, and for pre-clinical therapeutic testing.

HGG-28. INTERIM ANALYSIS OF THE GCC1949 PHASE 2 TRIAL USING INDOXIMOD-BASED CHEMO-IMMUNOTHERAPY FOR PATIENTS WITH PEDIATRIC BRAIN TUMORS

Abstract Brain tumors are the leading cause of cancer-related death in children. We report interim results of an ongoing multi-institutional phase 2 trial (NCT04049669) of the IDO pathway-inhibitor indoximod utilized in a chemo-immunotherapy regimen for patients 3-21 years of age with either recurrent malignant brain cancer or newly-diagnosed diffuse intrinsic pontine glioma (DIPG). Patients are treated with oral indoximod (38.4 mg/kg/day, divided BID) plus oral temozolomide (200 mg/m2/day for 5 days) in 28-day cycles. Patients for whom radiation therapy is planned as standard-of-care receive indoximod during the up-front radiation regimen. During treatment, palliative radiation, surgery, or dexamethasone are allowed as needed for clinical management. To date, 53 patients have been treated. Estimated median follow-up time was 23 months (range 0.2-35.5 months), and indoximod-based therapy was well tolerated. Estimated median overall survival (OS) was 15 months for the entire mixed population (n= 53, 95%-C.I. 11.5-23.2 months); 23.8 months for recurrent ependymoma (n=26); 13.5 months for recurrent medulloblastoma (n=12); 5.8 months for recurrent glioblastoma/diffuse midline glioma (GBM/DMG, n=9, excludes DIPG); and 9.6 months for newly-diagnosed DIPG (n=6). For the 39 response-evaluable patients to date (those with measurable disease and at least one follow-up MRI on-therapy), objective response in any single lesion by pediatric RANO criteria occurred in 20/39 patients (9/17 ependymoma, 9/11 medulloblastoma, 0/6 GBM/DMG, 2/5 DIPG). Lesion response was associated with significantly better overall survival (estimated median OS 23.2 months, n=20) compared to patients without lesion response (median OS 6.6 months, n=19) (p=0.0005). Single-cell RNA sequencing of serial peripheral blood samples allowed identification of expanded T cell clones, which was correlated with survival. Updated results from the first 53 patients will be presented. These data are replicating results of the preceding phase 1 study (NCT02502708) and provide preliminary evidence of anti-tumor activity in pediatric brain tumors.

BIOL-14. FROM NON-MALIGNANT TO MALIGNANT: GENERATING AND TARGETING DIFFUSE MIDLINE GLIOMA IN A NOVEL HUMAN PONTINE ORGANOID MODEL

Abstract Diffuse Midline Glioma (DMG) is a rare and aggressive pediatric cancer with no chance of survival, demonstrating a critical need for therapy development. Predictive preclinical models that take into account the unique developmental background and anatomical environment of DMG, are so far lacking. We developed a new regionally patterned brain organoid protocol based on a timely regulated sequence of morphogens. The patterned organoids give rise to pontine and medulla oblongata identity, as confirmed by transcriptomic profiling and immunofluorescence imaging. As the tissue matures, the complexity inherently takes shape and gives rise to pons-specific neurons and glial cells, offering the first human in vitro model for this region of the brain. Introducing H3K27M, including its partner mutations P53 and PDGFRA-D842V via electroporation forms de novo H3K27M diffuse midline glioma with a similar genetic make-up as found in patients. Indeed, contrary to common cell lines or PDX models, single cell sequencing and 3D imaging reveal that Organoid-DMG resembles WHO-defined primary patient cancer, including its diverse cell population heterogeneity and invasive nature. Utilizing barcode-based lineage tracing allows us to unravel and track the cancerous developmental evolution. Thus, we can now model early emerging populations and identify their transcriptomic signatures, which is conceptually impossible to conduct with patient-derived tumor tissue. In summary, we generated a bona fide DMG in vitro model which will give insights into tumorigenesis of DMG and ultimately contribute to pre-clinical drug- and immunotherapy development.

DIPG-30. HIF2 OVEREXPRESSION DICTATES AGGRESSIVENESS ADDITIONALLY TO TP53 MUTATIONS IN H3.3K27M PEDIATRIC HIGH-GRADE GLIOMAS THROUGH RAS-MAPK AND WNT/BCATENIN PATHWAYS

Abstract Diffuse midline (DMG) and brainstem (DIPG) high-grade gliomas (HGG) bearing H3.3K27M driver mutation are aggressive and uncurable brain tumors. We evidenced by the past HIF-2a and its transcriptional gene, EPAS1, as a marker of resistance when targeting mTor/HIF-1a pathway. To understand HIF-2α potential role in chronically active hypoxic environment in DIPG/DMG, we investigate its status in a cohort of pediatric HGGs comprising notably H3.3K27M mutant tumors and in their paired in vitro derived models. Using concomitantly the low-input chromatin immunoprecipitation sequencing method CUT&amp;RUN, RNAsequencing and metabolomics, we profiled the landscape of promoters and pathways regulated by HIF-2a in HGGs, where H3.3K27M and TP53 mutations are associated. Elucidation of HIF-2a downstream genes reveal statistically significant pathways linked to DIPGs (e.g., mTor and canonical Wnt pathways) and to DMGs (glutamatergic synapse and non-canoncial Wnt signaling). Finally, HIF-2a was evidenced as a new therapeutic target opening the path for its use concomitantly to irradiation.

IMMU-15. PHASE I TRIAL OF GD2.CART-CELLS AUGMENTED WITH A CONSTITUTIVELY ACTIVE INTERLEUKIN-7 RECEPTOR (C7R) FOR TREATMENT OF HIGH-GRADE PEDIATRIC CNS TUMORS

Abstract BACKGROUND Treatment of pediatric CNS tumors with T-cells modified with chimeric antigen receptors (CARTs) provides an innovative approach. We employ GD2-directed CART-cells (GD2.CARTs) modified to express a constitutively active interleukin-7 receptor (C7R) to increase GD2.CART survival and function independent of external cytokines, thereby augmenting activity against GD2-expressing CNS malignancies. METHODS In a Phase I study, we investigated the safety of intravenous GD2.CART therapy for pediatric patients with H3K27-altered diffuse midline glioma (DMG) or other GD2-expressing recurrent CNS tumors. We used a 3 + 3 design in which the first treatment cohort received GD2.CARTs without C7R, while subsequent cohorts received GD2.CARTs co-transduced with C7R (C7R-GD2.CARTs) at two dose levels. As a secondary endpoint we measured disease response 6 weeks after CART infusion. RESULTS 12 patients were treated without dose limiting toxicity. The first cohort (n=3: thalamic DMG, spinal DMG, recurrent AT/RT) received GD2.CARTs without C7R at 1x107 cells/m2. No toxicity was observed and all 3 patients had clinical neurological improvement lasting approximately 4 weeks before disease progression. The next two cohorts received C7R-GD2.CARTs at 1x107 cells/m2 (n=3) and 3x107 cells/m2 (n=6). Mild tumor inflammation associated neurotoxicity was observed in 7 of 9 (78%) patients, which was controlled with anakinra without need for corticosteroids. Cytokine release syndrome (CRS) was observed in 5 of 9 (56%) cases [grade 1 (n=4), grade 3 (n=1)], resolving with tocilizumab. Eight of 9 patients receiving C7R-GD2.CARTs had clinical improvement or stability for more than 8 weeks (up to 10 months at last follow-up) and received repeat cell infusions at 6 week intervals (range 2-4 cycles). Partial radiologic responses were confirmed for 2 of 7 (29%) patients with DMG. CONCLUSION Intravenous treatment with C7R-modified GD2.CARTs was well tolerated in children with CNS tumors, and the duration of clinical improvement was extended by C7R co-expression.

CNSC-01. GABAERGIC NEURON-TO-GLIOMA SYNAPSES IN DIFFUSE MIDLINE GLIOMAS

Abstract High-grade gliomas include clinically and molecularly distinct subtypes that stratify by anatomical location into diffuse midline gliomas (DMG) such as diffuse intrinsic pontine glioma (DIPG) and hemispheric high-grade gliomas. Neuronal activity drives high-grade glioma progression both through paracrine signaling and direct neuron-to-glioma synapses. Glutamatergic, AMPA receptor-dependent synapses between neurons and malignant glioma cells have been demonstrated in both pediatric and adult high-grade gliomas, but neuron-to-glioma synapses mediated by other neurotransmitters remain largely unexplored. Using whole-cell patch clamp electrophysiology, in vivo optogenetics and patient-derived glioma xenograft models, we have now identified functional, tumor-promoting GABAergic neuron-to-glioma synapses mediated by GABAA receptors in DMGs. GABAergic input has a depolarizing effect on DMG cells due to NKCC1 expression and consequently elevated intracellular chloride concentration in DMG tumor cells. As membrane depolarization increases glioma proliferation, we find that the activity of GABAergic interneurons promotes DMG proliferation in vivo. Increasing GABA signaling with the benzodiazepine lorazepam – a positive allosteric modulator of GABAA receptors commonly administered to children with DMG for nausea or anxiety - increases GABAA receptor conductance and increases glioma proliferation in orthotopic xenograft models of DMG. Conversely, levetiracetam, an anti-epileptic drug that attenuates GABAergic neuron-to-glioma synaptic currents, reduces glioma proliferation in patient-derived DMG xenografts and extends survival of mice bearing DMG xenografts. Concordant with gene expression patterns of GABAA receptor subunit genes across subtypes of glioma, depolarizing GABAergic currents were not found in hemispheric high-grade gliomas. Accordingly, neither lorazepam nor levetiracetam influenced the growth rate of hemispheric high-grade glioma patient-derived xenograft models. Retrospective real-world clinical data are consistent with these conclusions and should be replicated in future prospective clinical studies. Taken together, these findings uncover GABAergic synaptic communication between GABAergic interneurons and DMG cells, underscoring a tumor subtype-specific mechanism of brain cancer neurophysiology with important potential implications for commonly used drugs in this disease context.

DIPG-21. LINKING DETERMINATION OF CELL FATE IN THE DEVELOPING CENTRAL NERVOUS SYSTEM TO DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. Next-generation sequencing has revealed that the majority of patients with DMG harbor K27M mutations in canonical histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and an oligodendrocyte precursor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required in OPC differentiation in vivo. Co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. The Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, as well as a reduction in migration, invasion, and colony formation in vitro. An RNAseq database containing 45 human DMG cell lines showed an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DLX2high/OLIG1/2low cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2high/DLX2low expressing cell lines underwent stable transfection of an DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone H3 marks (K27me3, K27ac), K27M, and OPC signature genes will be presented along with comparison of altered OPC genes from the Dlx1/Dlx2 DKO mouse as determined by RNAseq. The manipulation of DLX2 expression in human DMG cells both in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.

IMG-02. VISUALIZATION OF TIGIT IN THE GLIOMA MICROENVIRONMENT: THE CREATION OF A PET IMAGING TRACER

Abstract Pediatric high-grade gliomas (HGGs) are aggressive tumors and are one of the drivers of pediatric brain tumor mortality. Few effective treatment options exist for pediatric HGGs beyond surgery and chemoradiation. Additionally, some HGGs, such as diffuse midline glioma, are often surgically inaccessible and respond poorly to chemotherapy leaving radiotherapy as the only treatment for this disease. While immune checkpoint inhibitors (ICIs) have seen success in treating cancers outside the CNS, ICIs for gliomas have shown limited efficacy. TIGIT is an IC receptor that is expressed on activated T-cells and has a critical role in suppressing T-cell and NK cell function. Prior work by our group has shown TIGIT to be a promising target for adult gliomas. These studies included analysis of transcriptomic data, and preclinical studies in glioma-bearing mice. We therefore assessed pediatric glioma transcriptomic datasets and identified via optimal cutoff analysis that at a specific mRNA threshold, TIGIT expression is associated with worsened survival. As TIGIT expression may be both prognostic and a biomarker for anti-TIGIT ICI therapy, we created a radiolabeled immunoPET tracer capable of binding TIGIT antigen that could be visualized via PET imaging. Referred to as “89Zr-DFO-TIGIT”, we found this tracer bound TIGIT with moderate immunoreactivity (57.7% ± 3.5%) as determined by bead-based immunoreactivity assays. As proof of feasibility, we injected glioma-bearing mice (GL261 cell line) with 89Zr-DFO-TIGIT which enabled the visualization of TIGIT in the tumor microenvironment via PET imaging. Thereafter, we performed biodistribution studies which found there to be significantly increased 89Zr-DFO-TIGIT in mouse tumor-bearing hemispheres as compared to non-tumor-bearing hemispheres. PET imaging also showed tracer-to-tumor uptake increased from day one to day eight. Our findings demonstrate the capability of 89Zr-DFO-TIGIT to visualize and quantify TIGIT in the glioma microenvironment, highlighting how this tracer may be of value in pediatric brain tumor immunotherapy studies.

IMMU-13. PRECLINICAL EFFICACY OF MULTI-ANTIGEN (HER2/EGFR806/B7-H3/IL13RA2) TARGETING QUAD-CAR T CELLS AGAINST DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG), remains a fatal disease. We have published the preclinical efficacy of HER2, EGFR806, and B7-H3 CAR-T cells and opened 3 trials (BrainChild-01, -02, and -03, respectively) that have delivered &amp;gt;350 intracranial CAR T doses to &amp;gt;70 enrolled children with recurrent/refractory CNS tumors and DIPG/DMG. While these trials have demonstrated the feasibility and tolerability of repeated, locoregional CAR-T cell dosing, responses have not been uniform in part due to tumor heterogeneity. METHODS Seattle Children’s Therapeutics generated multi-antigen (HER2, EGFR806, B7-H3, IL13ra2) quad-CAR T cells and we performed IND-enabling preclinical studies using CNS tumor models including DMG models (PBT-22, PBT-27, and PBT-29). RESULTS Quad-CAR T cells were generated by transducing T cells with a mixture of four vectors. The specificity of these CARs is conferred by scFv binding domains derived from the monoclonal antibodies Herceptin, mAb806, and MGA271 for HER2, EGFR806, and B7-H3, respectively. The IL13-zetakine, aligned with NCT00730613, carries an E13Y mutation in place of an scFv for specificity. In vitro, the quad-CAR T cells demonstrated significant in vitro cytolysis and cytokine (IL-2, IFNy, TNFa) production compared to mock (untransduced) CAR-Ts. In vivo, pilot studies in a GFP:ffLuc+Her2 U87 orthoptic xenograft mouse model showed significant survival (p&amp;lt;0.001). To validate against DMG, we are conducting a larger study of single dose intracranial quad-CAR T cells in an orthotopic xenograft PBT-29 DMG mouse model. While ongoing, at day 19 post-treatment there is already a significant bioluminescence decrease (p&amp;lt;0.001). In vivo studies will be completed and complimented by tumor spatial transcriptomics. CONCLUSION Quad-CAR T cell targeting of DMG is highly effective preclinically. A phase I trial of repeated, locoregional quad-CAR T cells for children with recurrent/refractory CNS tumors and DIPG/DMG is FDA-approved and will open in 2023.

DIPG-25. MAPPING TUMOR MICROENVIRONMENT USING EMERGING TECHNOLOGIES FOR PEDIATRIC BRAIN CANCERS

Abstract Diffuse midline glioma (DMG) is a fatal childhood brain cancer with a survival rate of less than one year from diagnosis. Pharmacological approaches as well as immunotherapy have failed to make a clinical impact. Incomplete understanding of the tumor microenvironment (TME) and tumor associated antigens have contributed to the observed poor prognosis. We hypothesize that characterization of the DMG TME will identify biology-informed targeted treatments. We thus obtained postmortem specimens from 70 patients diagnosed with brain cancers, including 50 DMGs, 20 other types and 10 non-CNS-cancer patients. Up to four anatomical brain locations were selected including the primary tumor, metastatic and adjacent healthy sites. Formalin-fixed-paraffin-embedded (FFPE) specimens were processed for constructing a tissue microarray (TMA). The TMA was stained for a number of markers (H&amp;E, H3K27M, H3K27me3, KI67) and was scored by a neuropathologist. We then used a multiplexed immunofluorescence (MxIF) technology, Cell DIVE™, to iteratively probe 33 biomarkers on a single tissue slide, focusing on immune cell type profiling and activation, and histone mutation status. Analysis of biomarker density and spatial relationships are underway. The main expressed immune markers across all patients were CD163, CD68, and CD8. CD8, a cytotoxic T-cell biomarker, was highly detected in pons, cerebellum, thalamus and the frontal lobe (tumor and healthy) of DMG patients and varied according to clinical intervention, including ONC201 treatment. Furthermore, in comparison to other tumors, DMGs exhibited a higher expression of CD3, T- cell marker, and CD4, a T-helper cell biomarker. Analysis of Iba1, a microglial marker, confirmed a higher difference in microglial activation in primary tumor compared to metastatic and adjacent healthy tissue. In contrast, CD68 was significantly increased in metastatic sites. Analysis is still ongoing. We report establishment of the most comprehensive TMA for pediatric brain tumors, which provides insights into TME comparing critically important clinical variables.

DIPG-38. PATIENT-DERIVED THREE-DIMENSIONAL AVATARS REVEAL CELL-AUTONOMOUS MECHANISMS DRIVING INVASION AND METASTATIC PROGRESSION IN DIFFUSE MIDLINE GLIOMAS

Abstract Diffuse midline gliomas H3K27-altered are pediatric tumors associated with dismal survival after diagnosis that are characterized by their ability to infiltrate the entire central nervous system. We show that the extent of tumor invasion and metastatic progression represent critical adverse prognostic factors affecting the survival of DMG patients. Yet, no genetic alterations associate to the tumor progression patterns. We therefore used glioma stem cells collected at diagnosis to create patient-specific 3D avatars. We found that their invasive behaviour faithfully predicts metastatic relapse in patients, thus proving DMG spread is mainly an autonomous ability of tumor cells. This relevant modelling strategy revealed the existence of phenotypic entities associated with distinct migration modalities and capacities. Using transcriptomics of both organoids and primary tumors, we identified the biological programs associated with variable invasiveness. Importantly, we show that Bone Morphogenetic Protein 7 (BMP7) signalling is a crucial tumour-autonomous regulator of DMG invasiveness via the control of a phenotypic switch. We further deciphered effectors downstream of BMP7 stimulation as actionable targets to reduce invasion. These findings have two possible therapeutic applications. First, glioma stem cell 3D models represent a tool for tumor phenotypic stratification to rapidly predict patients with exacerbated invasiveness and increased risk of metastatic progression. This may allow to propose whole-brain or cranio-spinal irradiation within weeks after diagnosis to prevent the appearance of distant foci. Second, targeting autocrine and short-range BMP7-related signalling may become a useful adjunct to the current therapeutics, which are mainly directed to control the disease locally.

DIPG-51. ABDOMINAL METASTASES OF DIFFUSE MIDLINE GLIOMA, H3K27-ALTERED WITH A VENTRICULOPERITONEAL SHUNT

Abstract Extraneural metastases are extremely rare in diffuse midline glioma (DMG), H3K27-altered, despite a tendency toward aggressive local growth and potential neuroaxis dissemination at progression. Ventriculoperitoneal shunt (VPS) placement for cerebrospinal fluid diversion is commonly performed in patients with thalamic tumors and increasingly considered in brainstem disease with developing hydrocephalus. Herein, we present a case of DMG seeding the abdomen presumably via the VPS. This patient was diagnosed at 22 years of age with a bithalamic tumor with extension into the brainstem and cerebellum as well as non-contiguous periventricular spread to the bilateral frontal lobes, with associated hydrocephalus. The patient underwent VPS insertion and biopsy, with pathology consistent with DMG, H3K27-altered. Whole exome sequencing revealed H3-3A and TP53 alterations as well as PDGFRA amplification. Treatment consisted of upfront craniospinal photon irradiation, with subsequent reduction in thalamic disease burden. Adjuvant therapy with an oral PDGFRA inhibitor (avapritinib) was initiated, though discontinued after two weeks due to worsening neurocognitive deficits. The patient presented three months post-completion of radiation, five months from diagnosis, and six weeks following VPS revision with rapidly progressive abdominal distension and altered mental status, with imaging demonstrating omental caking and significant malignant ascites, resulting in shunt malfunction and increased intracranial pressure. With limited therapeutic options, the family decided not to pursue further intervention and the patient died five days later. Autopsy confirmed metastatic disease throughout omentum, in addition to diffuse leptomeningeal dissemination. Comprehensive molecular profiling of diagnostic and post-mortem thalamic and omental tissue is in process to assess temporal as well as spatial genomic heterogeneity, including of metastasis-mediated pathways. In summary, abdominal dissemination of DMG, H3K27-altered is uncommon, but possible, especially in the presence of a VPS. Further research is necessary to determine biologic mechanisms and predictors of extraneural metastases in this disease.

DIPG-16. ACUTE VERSUS PROLONGED CONVECTION-ENHANCED DELIVERY OF ALISERTIB INTO H3 K27M-MUTANT MODELS OF DIFFUSE MIDLINE GLIOMA

Abstract Convection-enhanced delivery (CED) is a potentially promising strategy to administer therapeutics directly into the brainstem of children harboring H3 K27-altered diffuse midline glioma (DMG). While early-phase clinical trials have demonstrated the safety of this technique, efficacy has yet to be achieved. Here, we describe how different variations of CED may be exploited to extend survival in a DMG murine patient-derived orthotopic xenograft model. Either 1-day (8 µL/hr) or 7-day (1 µL/hr) continuous CED infusions were performed to deliver an equivalent volume of the aurora kinase inhibitor alisertib to the mouse brainstem. Bioluminescence and magnetic resonance images were acquired to monitor tumor progression, validate CED catheter positioning, and analyze infusion-related imaging changes. Both infusion regimens significantly prolonged survival versus control (median survival benefit of 6.5 and 8 days, p=0.03 and p=0.01, for 1-day and 7-day CED, respectively). Postmortem examination of brains revealed no signs of tissue necrosis, cavitary lesions, or cellular (inflammatory) infiltrate at the site of infusion. However, the tumor parenchyma surrounding the cannula tract was markedly hypocellular in treated animals. Alisertib induced a robust increase in H3 K27 trimethylation along with decreased H3 K27M and Ki-67. Our findings indicate that CED of alisertib is well tolerated and effective, in both the acute and prolonged setting, underscoring the potential of this approach in the management of DMG.

IMMU-18. TARGETING GD2 IN THALAMIC DIFFUSE MIDLINE GLIOMA WITH TRANSIENT MRNA CAR T CELLS

Abstract Chimeric antigen receptor (CAR) T cells targeting the disialoganglioside GD2 have shown promise as a therapeutic for diffuse midline glioma (DMG). However, thalamic tumors have been excluded from clinical trials due to prior studies showing neurotoxicity and fatality when using virally transduced CAR T cells. 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 and first tested against the murine thalamic DMG xenograft 7316-6349 via locoregional delivery with an indwelling 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 5x106 mRNA CAR T cells delivered intratumorally twice a week resulted in median overall survival of 9 days for GD2-treated mice compared to &amp;gt;30 days for CD19-treated controls (p&amp;lt;0.01). This toxicity could be avoided by decreasing the dose and timing of infusions to 2x106 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 -3x106 versus +20x106 p/sec/cm2/sr, p&amp;lt;0.01). To assess survival before the onset of graft-versus-host disease, we utilized the aggressive hypermutant thalamic DMG model 7316-3058, treating with 2x106 CAR T cells intratumorally once weekly. Overall survival was significantly prolonged for 7316-3058 mice treated with GD2-directed mRNA CAR T cells compared to CD19-directed controls (median OS 48 days versus 33 days, p=0.03). Notably, non-tumor bearing mice treated with GD2-directed CAR T cells developed fatal neurotoxicity within 14 days, suggesting 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.