H3 K27M-mutant Diffuse Midline Glioma Research Articles

Spinal Cord Diffuse Midline Glioma With Histone H3 K27M Mutation in a Pediatric Patient.

Background: Diffuse midline glioma (DMG) with histone H3 K27M mutation is a recently identified entity documented in the 2016 World Health Organization (WHO) Classification of Tumors of the Central Nervous System. Spinal cord DMGs with H3 K27M-mutant are commonly reported in adults. Herein, we reported a pediatric patient with spinal cord H3 K27M-mutant DMG.Case Report: A 7-year-old girl with 1-month history of neck pain and 3-week history of progressive weakness in the right hand was presented. Spinal magnetic resonance imaging showed an intramedullary lesion with slight enhancement at the C2-7 levels. With intraoperative neuroelectrophysiological monitoring, the lesion was subtotally resected. Histopathological examination revealed a DMG with histone H3 K27M mutation corresponding to WHO grade IV. Postoperatively, the neck pain was relieved, and the upper-extremity weakness remained unchanged. Oral temozolomide was administrated for 7 months, and radiotherapy was performed for 22 courses. After an 18-month follow-up, no tumor recurrence was noted.Conclusion: Spinal cord H3 K27M-mutant DMGs are extremely rare in pediatric patients. Preoperative differential diagnosis is challenging, and surgical resection with postoperative chemoradiotherapy may be an effective treatment.

HGG-35. IDENTIFICATION OF G PROTEIN-COUPLED RECEPTOR 17 (GPR17) UP-REGULATION IN PAEDIATRIC H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA AND EXAMINATION OF ITS ROLE IN DIFFUSE MIDLINE GLIOMA CELL LINES

Paediatric H3 K27M-mutant diffuse midline glioma (pDMG) is an incurable, aggressive childhood brain malignancy, that arises in a region- and age-specific nature. The underlying pathophysiology suggests dysregulation of postnatal neurodevelopmental processes causing aborted cell differentiation. The cell of origin is unclear, but data suggests an oligodendrocytic lineage (OPC), supported by the over-expression of transcription factors such as Olig1 and Olig2 in 80% of DIPG cases. In-depth bioinformatics and principal component analyses (PCA) of genes involved in brain development and pDMG support reports of OPC gene dysregulation and led to the identification of the G-protein coupled receptor 17 (GPR17) and its association with pDMG. GPR17, a rhodopsin-like orphan GPCR (unknown ligand), has the typical features of the GPCR superfamily, seven transmembrane domains (TM1-TM7), eight amphipathic helices, an extracellular N-terminal domain, and an intracellular C-terminus. GPR17 has been implicated in several normal physiological and pathological processes, such as oligodendrocyte differentiation, spinal cord injury and brain injury. GPR17 mRNA and protein expression was confirmed in all pDMG cell lines tested. Using a well-characterised agonist (MDL 299,51) and antagonist (HAMI3379) to modulate GPR17 function in pDMG cell lines resulted in phenotypic and genomic changes as well as in cell growth and migration. HAMI3379, a GPR17 specific antagonist resulted in a significant reduction in GPR17 mRNA and protein expression (p<0.006) and a significant reduction in migration (p<0.0025). When pDMG cells were pretreated with HAMI3379 in combination with known cytotoxic agents (Bleomycin, a radiation mimic, Panobinostat or Vincristine), there was a decrease in cell viability compare to cytotoxic agent alone. There are no current effective therapies for pDMG patients and the ability of blocking GPR17 function to enhance sensitivity to standard therapies is appealing and warrants further investigation.

OMIC-11. SINGLE CELL RNA SEQUENCING FROM THE CSF OF SUBJECTS WITH H3K27M+ DIPG/DMG TREATED WITH GD2 CAR T-CELLULAR THERAPY

IntroductionWe are conducting a Phase I clinical trial utilizing chimeric antigen receptor (CAR) T-cells targeting GD2 (NCT04196413) for H3K27M-mutant diffuse intrinsic pontine glioma (DIPG) and spinal cord diffuse midline glioma (DMG). Cerebrospinal fluid (CSF) is collected for correlative studies at the time of routine intracranial pressure monitoring via Ommaya catheter. Here we present single cell RNA-sequencing results from the first 3 subjects. MethodsSingle cell RNA-sequencing was performed utilizing 10X Genomics on cells isolated from CSF at various time points before and after CAR T-cell administration and on the CAR T-cell product. Output was aligned with Cell Ranger and analyzed in R. ResultsAs detailed in the Majzner et al. abstract presented at this meeting, three of four subjects treated at dose-level one exhibited clear radiographic and/or clinical benefit. We have to date completed single cell RNA-sequencing for three of these four subjects (two with benefit, one without). After filtering out low-quality signals and doublets, 89,604 cells across 3 subjects were analyzed. Of these, 4,122 cells represent cells isolated from CSF and 85,482 cells represent CAR T-cell product. Two subjects who demonstrated clear clinical and radiographic improvement exhibited fewer S100A8+S100A9+ myeloid suppressor-cells and CD25+FOXP3+ regulatory T-cells in the CSF pre-infusion compared to the subject who did not derive a therapeutic response. In one subject with DIPG who demonstrated improvement, polyclonal CAR T-cells detectable in CSF at Day +14 demonstrated enrichment of CD8A, GZMA, GNLY and PDCD1 compared to the pre-infusion CAR T-cells by trajectory analysis, suggesting differentiation toward a cytotoxic phenotype; the same subject exhibited increasing numbers of S100A8+S100A9+ myeloid cells and CX3CR1+P2RY12+ microglia over time. Further analyses will be presented as data become available. ConclusionsThe presence of immunosuppressive myeloid populations, detectable in CSF, may correlate to clinical response in CAR T cell therapy for DIPG/DMG.

Case series of diffuse extraneural metastasis in H3F3A mutant high-grade gliomas: Clinical, molecular phenotype and literature review

H3K27M and H3.3G34R/V mutations have been identified in pediatric high-grade gliomas (pHGG), though extraneural metastases are rarely reported and poorly characterized. Three pHGG patients from two institutions were identified with extraneural metastasis, harboring histone mutations. Their clinical, imaging and molecular characteristics are reported here. A 17-year old female presented with supratentorial H3.3G34R-mutant glioma with metastatic osseous lesions in the spine, pelvis, bone marrow, pleural effusion and soft tissue of pelvis. Bone marrow biopsy and soft tissue of pelvis biopsy showed neoplastic cells positive for P53. A 20-year old female was diagnosed with H3F3A H3K27M–mutant thalamic glioma. She developed diffuse sclerotic osseous lesions. Biopsy of an osseous lesion was non-diagnostic. A 17-year old female presented with a H3F3A H3K27M-mutant diffuse midline glioma with diffuse spinal cord metastasis. She further developed multifocal chest lymphadenopathy, pleural effusions, and a soft tissue mass in the abdominal wall. The latter was positive for H3K27M mutation. We present the first case series of pHGG with H3F3A mutation and diffuse extraneural dissemination, describing their clinical and molecular profile.

Single agent activity of ONC201 in non-midline H3 K27M-mutant diffuse gliomas.

e14037 Background: H3 K27M-mutant diffuse midline glioma is an invariably lethal form of brain cancer that disproportionately affects children and young adults and has no effective treatment following front-line radiation. The initial disease definition in the 2016 WHO Classification of Tumors of the Central Nervous System regarded the H3 K27M mutation as pathognomonic, though the definition was updated in 2018 restricting the diagnosis to histologically diffuse gliomas that involve midline CNS structures (cIMPACT-NOW update 2). ONC201 is an investigational anti-cancer small molecule, DRD2 antagonist and ClpP agonist that has induced durable tumor regressions by RANO-HGG criteria in a registration cohort of recurrent diffuse midline glioma, H3 K27M-mutant patients treated with single agent ONC201. Methods: We present 7 patients with H3 K27M-mutant diffuse gliomas were enrolled in ONC201 clinical studies, though excluded from the registration cohort due to involvement of non-midline CNS structures, all within the cerebral hemispheres (3 frontal, 1 temporal, 1 frontotemporal, 1 parietal, and 1 corona radiata). Results: Two of the 7 patients underwent objective responses by RANO-HGG criteria as assessed by investigator, which was associated with clinical benefit that included increased mobility and level of alertness. Conclusions: These results demonstrate that H3 K27M-mutant diffuse gliomas occur outside of midline CNS structures, and suggest that ONC201 has single agent activity in H3 K27M-mutant gliomas irrespective of CNS location.

Serial plasma and CSF cell-free tumor DNA (cf-tDNA) tracking in diffuse midline glioma patients undergoing treatment with ONC201.

2012 Background: Diffuse midline glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. There are few available means of monitoring the disease beyond serial MRI scans, making clinical decision making slow, difficult, and often reactive. Methods: We conducted a multi-site phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients enrolled on Arm D of the trial (n=24) underwent serial lumbar puncture (baseline, 2 and 6 months) for cell-free tumor DNA (cf-tDNA) analysis at time of MRI. Additionally, patients on all arms of the trial at the University of Michigan underwent serial plasma collection. CSF collection was feasible in this cohort, with no procedural complications. We collected a total of 96 plasma samples and 53 CSF samples from 29 patients, including those with H3F3A (H3.3) (n=13), HIST13HB (H3.1) (n= 4), and unknown H3 status/not biopsied (n=12) [range of 0-8 CSF samples and 0-10 plasma samples]. We performed digital droplet polymerase chain reaction (ddPCR) analysis and/or amplicon-based electronic sequencing (Oxford Nanopore) of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Results: Preliminary analysis of samples (n=58) demonstrates a correlation between changes in tumor size and H3K27M cf-tDNA VAF, when removing samples with concurrent bevacizumab. Analysis of remaining CSF and plasma samples is ongoing, including analysis of novel biomarkers of response. In multiple cases, early reduction in CSF cf-tDNA predicts long-term clinical response (&gt;1 year) to ONC201 and does not increase in cases of later-defined pseudo-progression (radiation necrosis). For example, a now 9-year old patient with thalamic H3K27M-mutant DMG underwent treatment with ONC201 after initial radiation and developed an increase in tumor size at 4 months post-radiation (124% baseline) of unclear etiology at the time. Meanwhile, her ddPCR declined from baseline 6.76% VAF to &lt;1%, which has persisted, with now near complete response (85% tumor reduction) at 30 months on treatment from diagnosis. Conclusions: In summary, we present the feasibility and utility of serial CSF/plasma monitoring of a promising experimental therapy for DMG.

Exploiting Gangliosides for the Therapy of Ewing's Sarcoma and H3K27M-Mutant Diffuse Midline Glioma.

Simple SummaryOsteosarcoma, Ewing’s sarcoma, and H3K27M-mutant diffuse midline glioma are rare but aggressive malignancies occurring mainly in children. Due to their rareness and often fatal course, drug development is challenging. Here, we repurposed the existing drugs dinutuximab and eliglustat and investigated their potential to directly target or indirectly modulate the tumor cell-specific ganglioside GD2. Our data suggest that targeting and/or modulating tumor cell-specific GD2 may offer a new therapeutic strategy for the above mentioned tumor entities.The ganglioside GD2 is an important target in childhood cancer. Nevertheless, the only therapy targeting GD2 that is approved to date is the monoclonal antibody dinutuximab, which is used in the therapy of neuroblastoma. The relevance of GD2 as a target in other tumor entities remains to be elucidated. Here, we analyzed the expression of GD2 in different pediatric tumor entities by flow cytometry and tested two approaches for targeting GD2. H3K27M-mutant diffuse midline glioma (H3K27M-mutant DMG) samples showed the highest expression of GD2 with all cells strongly positive for the antigen. Ewing’s sarcoma (ES) samples also showed high expression, but displayed intra- and intertumor heterogeneity. Osteosarcoma had low to intermediate expression with a high percentage of GD2-negative cells. Dinutuximab beta in combination with irinotecan and temozolomide was used to treat a five-year-old girl with refractory ES. Disease control lasted over 12 months until a single partially GD2-negative intracranial metastasis was detected. In order to target GD2 in H3K27M-mutant DMG, we blocked ganglioside synthesis via eliglustat, since dinutuximab cannot cross the blood–brain barrier. Eliglustat is an inhibitor of glucosylceramide synthase, and it is used for treating children with Gaucher’s disease. Eliglustat completely inhibited the proliferation of primary H3K27M-mutant DMG cells in vitro. In summary, our data provide evidence that dinutuximab might be effective in tumors with high GD2 expression. Moreover, disrupting the ganglioside metabolism in H3K27M-mutant DMG could open up a new therapeutic option for this highly fatal cancer.

THER-05. GENETICALLY STABLE POLIOVIRUS VECTOR CARRYING H3.3K27M ANTIGEN FOR TREATMENT OF DIFFUSE MIDLINE GLIOMA BY INTRAMUSCULAR INJECTION

BACKGROUNDH3 K27M-mutant diffuse midline glioma (DMG) is invariably lethal. Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses, in particular, are uniquely tropic for dendritic cells (DC) and potently activate DC, inducing Th1-dominant cytokine profiles, CD8 T cell immunity, and enhanced epitope presentation. Thus, poliovirus is ideally suited for vectored delivery of signature tumor neoantigens, e.g. the H3 K27M feature of DMG. However, poliovirus vector design is inherently limited by genetic instability and the underlying neuropathogenicity of poliovirus.METHODSWe created a genetically stable, polio:rhinovirus chimera vector devoid of neuropathogenicity and modified for stable expression of the HLA-A2 restricted H3.3 K27M antigen (RIPO (H3.3)).RESULTSRIPO(H3.3) infects, activates, and induces H3.3K27M antigen presentation in DCs in vitro. Given intramuscularly in vivo, RIPO(H3.3) recruits and activates DCs with Th1-dominant cytokine profiles, efficiently primes H3.3K27M-specific CD8 T cells, induces antigen-specific CD8 T cell migration to the tumor site, delays tumor growth, and enhances survival in murine tumor models.CONCLUSIONThis novel approach leverages the unique ability of polioviruses to activate DCs while simultaneously introducing the H3.3 K27M antigen. In this way, DCs are activated optimally in situ, while being simultaneously infected to express/present tumor antigen. RIPO(H3.3), given by intramuscular injection, will be evaluated in a clinical trial for children with H3 K27M-mutant diffuse midline glioma.

PATH-30. CLINICAL AND GENETIC CHARACTERISTICS OF HISTONE H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMAS IN ADULTS

Abstract BACKGROUND “Diffuse midline glioma, H3 K27M-mutant” is a new tumor entity established in the 2016 WHO Classification of Tumors of the CNS that comprises a set of diffuse gliomas arising in midline structures that is molecularly defined by a recurrent K27M mutation in genes encoding the histone 3 variants H3.3 or H3.1. While this tumor entity is associated with poor prognosis in children, clinical experience in adults remains limited. Given the more frequent origin in the thalamus or spinal cord in adults versus the brainstem in children, gliomas with this mutation may encompass a heterogeneous population of tumor subtypes that vary based on patient age, anatomic site of origin, and concurrent genetic alterations. METHODS The 60 patients included were 18 years or older at initial diagnosis, during the period of 2014-2019 at UCSF. Cases were identified using immunohistochemistry with a H3 K27M-mutant specific antibody and/or next-generation sequencing of histone 3 genes H3F3A, HIST1H3B and HIST1H3C. Targeted NGS was performed on tumors from 21 patients, utilizing an UCSF institutional panel or a variety of commercial sources. RESULTS Patients presented primarily in the 3rd decade of life, and 57% of tumors were located in the thalamus. Genomic profiling revealed p.K27M mutations exclusively in H3F3A and an absence of mutations in HIST1H3B or HIST1H3C, which are present in approximately one-third of pediatric diffuse midline gliomas. Additionally, these adult H3 K27M-mutant diffuse midline gliomas are universally IDH-wildtype, and have frequent mutations in TP53, PPM1D, FGFR1, NF1, and ATRX. The overall survival of this adult cohort is longer than historical averages for both H3 K27M-mutant diffuse midline glioma in children and IDH-wildtype glioblastomas in adults. CONCLUSIONS Together, these findings indicate that H3 K27M-mutant diffuse midline glioma represents a heterogeneous disease with regard to outcomes, sites of origin, and molecular pathogenesis in children versus adults.

CTNI-15. CLINICAL EFFICACY OF ONC201 IN NEWLY DIAGNOSED DIPG AND IN PREVIOUSLY IRRADIATED PEDIATRIC H3 K27M-MUTANT GLIOMAS

Abstract ONC201, an anti-cancer DRD2 antagonist and ClpP agonist, is in Phase II trials for adult H3 K27M-mutant diffuse midline gliomas. In adults, the recommended phase 2 dose (RP2D) of 625mg ONC201 once a week has been established as a biologically active dose that is well tolerated. Radiographic regressions with single agent ONC201 have been reported in adult recurrent H3 K27M-mutant glioma patients. This multi-arm, dose-escalation and dose-expansion trial determined the pediatric RP2D of ONC201 administered as an oral capsule (Arm A) or liquid formulation (Arm E) in post-radiation H3 K27M-mutant glioma (Arm A) or in newly diagnosed DIPG (Arm B) patients. Molecular assessments include intratumoral ONC201 concentrations (Arm C) and CSF H3 K27M DNA levels (Arm D). Enrollment as of April 30, 2020 is complete in Arm A (22) and Arm E (26) and continues in Arm B (18/24), Arm C (5/12), and Arm D (22/24). The RP2D of weekly 625mg ONC201 scaled by body weight was confirmed when administered as a capsule or a liquid formulation as a single agent or in combination with radiation without dose-limiting toxicity. The most frequent adverse events regardless of attribution to the drug were predominantly low grade: ONC201 capsule alone was headache (54.5%), nausea (36.4%), and fatigue (36.4%); ONC201 liquid formulation was vomiting (31.8%), headache (22.7%), VIth nerve disorder (22.7%); ONC201 capsules in combination with radiation (Arm B) was headache (47.1%), vomiting (52.9%), nausea (41.2%). Pharmacokinetic analysis in plasma of Arm A patients revealed T1/2: 8.4h; Tmax: 2.1h; Cmax: 2.3ug/mL; AUC0-tlast: 16.4ug/mL, with similar exposure across body weights. In conclusion, when scaled by body weight the ONC201 capsule or liquid formulation alone or in combination with radiation were associated with safety and pharmacokinetic profiles in pediatric H3 K27M-mutant diffuse midline glioma patients that are similar to the experience in adults.

CTNI-17. CLINICAL EFFICACY AND PREDICTIVE BIOMARKERS OF ONC201 IN H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA

Abstract Patients with diffuse midline glioma (DMG) harboring H3 K27M mutation rarely survive longer than two years and have no proven therapies following first-line radiation. ONC201, a bitopic DRD2 antagonist and allosteric ClpP agonist, has shown encouraging efficacy in early phase studies in H3 K27M-mutant DMG. In order to define response rates in H3 K27M DMG patients and to clarify the genomic, anatomic and molecular predictors of response, we performed an integrated pre-clinical and clinical analysis of ONC201 treatment. ONC201 was effective in intra-uterine electroporation (IUE)-generated H3 K27M-mutant murine glioma models with excellent CNS penetration and survival benefit. Patients with H3 K27M-mutant DMG treated with ONC201 on active clinical trials (n=50, 27 thalamic, 23 brainstem) showed an overall survival (OS) of 28.1 (range: 5.9–105) months from diagnosis (enrollment by 4/29/19, data cut-off 12/28/19), compared to historical median OS of 12 months. Median OS for non-recurrent patients has not been reached (n=16, median follow-up: 16.8 from diagnosis). For non-recurrent thalamic patients (n=8), median PFS is 20.1 (range: 9.3–27.6) months from diagnosis (median time on drug: 14.5 months). Best response for thalamic patients by RANO: 1 CR, 5 PR, 7 SD, 8 PD, 6 not reported. Decreased H3 K27M cell-free tumor DNA in plasma and CSF at 6 months correlated with long-term response. Baseline tumor gene expression profiling in patients treated with ONC201 (n=14) identified EGFR and the cortical developmental transcription factor FOXG1 as the strongest biomarkers of radiographic response to ONC201. Analysis of 541 ONC201-treated human cancer cell lines from DepMap, provided evidence for an EGFR-dependent ONC201 resistance mechanism. Analysis of 38 glioma cell lines further supports FOXG1 as a glioma-specific predictive biomarker of ONC201 response. The unprecedented survival results and radiographic responses to ONC201 in H3K27M DMG make a compelling case for later phase and combinatorial studies.

EXTH-77. ONC201 EXHIBITS PASSIVE DIFFUSION AND BROAD DISTRIBUTION IN THE CENTRAL NERVOUS SYSTEM

Abstract Imipridone ONC201 is an investigational agent in phase II clinical trials for H3 K27M-mutant diffuse midline glioma with evidence of durable objective responses and clinical symptom improvements as a single agent. The systemic pharmacokinetics of ONC201 and its ability to achieve target concentrations in supratentorial glioblastoma have been demonstrated in clinical trials; however, its clearance and distribution in midline structures and other areas of the central nervous system (CNS) haven’t been evaluated. A quantitative whole-body autoradiography study was conducted in Long-Evans rats with a single dose of [14C]-ONC201. [14C]-ONC201-related material was rapidly distributed throughout the body, with concentrations peaking at 1h for most tissues. The endocrine, metabolic/excretory, ocular and gastrointestinal tract tissues contained the highest distribution of [14C]-ONC201-derived radioactivity. Importantly, [14C]-ONC201 material was distributed evenly across brain substructures, including the meninges and midline structures of the brain; all tissues had a half-life of 1.4–7.9h, with the exception of the meninges (2397.3h). Given the uniform distribution throughout the CNS, we evaluated the permeability and efflux potential of ONC201 in bidirectional transport assays using human Caco-2 cell monolayers. ONC201 displayed high permeability in the apical to basolateral direction with apparent permeability values of 23–31×10–6 cm/s at 7–700μM. In the basolateral to apical direction, apparent permeability values were 11–24 × 10–6 cm/s. The efflux ratio values were 0.46–0.79 for ONC201, suggesting that ONC201 is not a substrate of efflux transporters. However, ONC201 exerted inhibitory potential on MDR1- (90.6%) and BCRP-mediated (81.6%) transport at 200mM. In summary, ONC201 exhibits passive diffusion without being effluxed, which may enable its rapid and wide distribution throughout the CNS. This distribution profile suggests that the compound may achieve therapeutic concentrations throughout the CNS with oral administration and that investigation of additional CNS tumors will not be hindered by drug delivery to specific anatomic structures.

H3 G34-mutant high-grade glioma.

H3F3A G34 (H3.3 G34)-mutant high-grade gliomas (HGG) are rare, and newly recognized infiltratinggliomasof the cerebral hemisphere. Here, we report the clinicopathological and molecular characteristics of four H3.3 G34-mutant gliomas in terms of its biological behavior compared to those of glioblastomas (GBMs) and H3 K27M-mutant diffuse midline gliomas (DMGs) of our hospital. The median age of the four patients with H3.3 G34 HGG was 44.5years (14-66years). Three patients had tumors in the cerebral hemisphere, whereas one patient had synchronous double tumors in the cerebral hemisphere and posterior fossa. All these tumors were high-grade glioma, but neither microvascular proliferation nor necrosis. They displayed uniform genetic and epigenetic signatures; ATRX-mutant, MGMT promoter-methylated, Olig2-negative, but IDH- and TERT promoter-wildtype. The median survival rate of H3.3 G34-mutant HGGs, IDH-was 23.5months. In conclusion, H3.3 G34-mutant gliomas were unique HGGs with uniform genetic and epigenetic abnormalities, which suggested a single phylogenic origin. The median survival of H3.3 G34-mutant HGGs was better than those of IDH-wildtype GBMs and H3 K27M-mutant DMGs, but worse than that of IDH-mutant GBM. The tumor-infiltrating area and resectability may be the crucial parameters for the prognosis of the patients.

Letter to the Editor Regarding “Posterior Calvarial Augmentation for Chiari Malformation Type 1 Refractory to Foramen Magnum Decompression”

In the revised World Health Organization 2016 classification of central nervous system tumors, “diffuse midline glioma, H3 K27M-mutant” has been added as a new diagnostic entity. However, some confusion exists concerning this diagnostic entity because H3 K27M-mutant diffuse midline glioma is diagnosed with grade IV regardless of morphologic phenotype. Furthermore, the significance of H3 K27M mutation in tumors that aren’t typical “diffuse midline glioma, H3 K27M-mutant,” such as those with an unusual location and nontypical histology, remains unclear.To elucidate further such unusual tumors, we describe here a rare case of pediatric low-grade glioma located in the tectum, which was morphologically a pilocytic astrocytoma (PA) with genetically H3 K27M mutation but no microvascular proliferation, necrosis, mitoses, or other genetic alterations, insofar as we were able to observe. At the latest follow-up, 28 months after surgery, radiotherapy, and chemotherapy, the patient was found to be free from any neurologic deficits and MRI demonstrated that the tumor was stable without tumor regrowth. This case might be identified as “diffuse midline glioma, H3 K27M-mutant”, grade IV, when applying only the current World Health Organization 2016 classification. In addition, we discuss the morphologically benign gliomas harboring the H3 K27M mutation based on the literature.We describe here a rare case and present a short literature review of circumscribed/nondiffuse gliomas, particularly in PA with H3 K27M mutation. However, the significance of H3 K27M mutation for PA remains unclear, so further studies and clinical data are needed to elucidate the biology and optimal treatment of such tumors.

Abstract 5407: Novel imipridone combination therapies targeting oncohistone H3K27M mutant diffuse midline glioma (DMG)

Abstract Malignant glioma including diffuse midline glioma (DMG) is the leading cause of cancer related death in children. Recent advances in sequencing have identified the oncohistone H3K27M mutation in ~80% of pediatric DMG, which represents a target for new therapeutics. ONC201 is a first-in-class small molecule which upregulates TNF-related apoptosis inducing ligand (TRAIL) and its proapoptotic receptor DR5, independent of p53, selecting for apoptosis/growth arrest of many different tumor types including H3K27M mutant DMG. While single agent ONC201 along with RT is currently being tested in clinical trials for newly diagnosed and refractory DMG, there is an urgent need to evaluate ONC201 and other imipridone analogs in combination with other agents. Patient derived H3K27M mutant DMG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SU-DIPG-27, SU-DIPG-29, SU-DIPG-36, and SF8628), were grown in culture and exposed to imipridones (ONC201, ONC206 and ONC212) alone, and in combination with other chemotherapeutic agents that have demonstrated pre-clinical activity against DMG (HDAC inhibitors and etoposide). Response to treatment was determined by Cell Titer-Glo cell viability assays as well as Western Immunoblotting for evidence of apoptosis and integrated stress response (ISR) activation. All H3K27M-mutant DMG cell lines tested to date exhibit a dose-dependent response to imipridone therapy. Mean IC50 values were significantly lower with the imipridone analogs ONC206 (0.11 µM) and ONC212 (0.03 µM) as compared to ONC201 (1.46 µM) when used as a single agent. ONC206 and ONC212 were associated with robust induction of apoptosis evidenced by increased expression of cleaved PARP, and ISR by increased expression ATF4. In the SU-DIPG-IV cell line, combination studies of ONC201 and panobinostat demonstrated a synergistic effect of both agents, as calculated by CompuSyn software. Combination indices below one were observed at concentrations of 0.003 - 0.05 µM of panobinostat with ONC201 at 0.156 - 5 µM, with the best combination index of 0.22. We similarly observed synergy between either ONC201 or ONC206 with Etoposide. Additional combination testing and synergy analysis is ongoing and will be presented. Our findings suggest that H3K27M DMG cells demonstrate increased sensitivity to imipridones ONC206 and ONC212 as single agents and combinational strategies with HDAC inhibitors and etoposide, and provide insights into novel therapies for further clinical testing. Citation Format: Robyn Borsuk, Lanlan Zhou, Rishi Lulla, Wafik El-Deiry. Novel imipridone combination therapies targeting oncohistone H3K27M mutant diffuse midline glioma (DMG) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5407.

Clinico-pathological and molecular characterization of diffuse midline gliomas: is there a prognostic significance?

H3K27M mutant diffuse midline gliomas (DMGs) are considered grade IV irrespective of histological features and have dismal prognosis. We evaluated clinico-pathologic, radiological, and molecular characteristics of DMGs across all ages. One twenty-six DMGs were identified over 10years. Immunohistochemistry was done for H3K27M, ATRX, IDH1, and p53, and Sanger sequencing performed for IDH1 and H3K27M mutation. Patient demographics and clinico-radiologic characteristics were reviewed and survival analysis performed. DMGs comprised 5.3% of all gliomas with 49.2% H3K27M mutant and 50.8% wild types. Majority (75.68%) of pediatric and 38.20% of adults were H3K27M mutant (p = 0.0001). Amongst H3K27M mutants, brainstem (46.43%) was the commonest location in pediatric and thalamus (61.76%) in adults. H3K27M mutation was mutually exclusive with IDH mutation in 93.55%, while p53, ATRX mutation were seen in 56.4% and 30.6% cases respectively. Software-based immunohistochemistry evaluation (H-scoring) showed 99.2% concordance with sequencing for H3K27M mutation. Radiologically, no significant difference in contrast enhancement was seen between mutant and wild types (p = 0.05). The difference in overall survival (OS) was not significant in mutant versus wild types, with age or location. Tumor resection independently and on correlation with H3K27M did not influence OS (p = 0.51 and p = 0.47). Adjuvant therapy impacted survival significantly in adults (p = 0.0009), however, not in pediatric cases (p = 0.06). The study highlights the differences in frequency and location of pediatric and adult DMGs. IHC (H-scoring) for H3K27M mutation is an excellent surrogate for sequencing. Prognosis remains dismal irrespective of age, location, and H3K27M status. Potential therapeutic targets need to be explored.

Single-agent ONC201 in recurrent H3 K27M-mutant diffuse midline glioma.

3615 Background: Recurrent H3 K27M-mutant diffuse midline glioma is a lethal brain tumor that predominantly affects children and young adults and has no effective therapy. ONC201 is a first-in-class orally administered, anti-cancer small molecule that selectively antagonizes the dopamine receptors DRD2/DRD3 and agonizes ClpP, a mitochondrial protease. Prior studies have indicated dysregulated dopamine receptor expression and enhanced ONC201 sensitivity among H3 K27M-mutant gliomas. Methods: Adults with midline H3 K27M-mutant glioma patients were enrolled to a dedicated Phase II clinical trial (NCT03295396), a multi-arm Phase II clinical trial (NCT02525692), and expanded access protocols under the Sponsor’s IND. Results were pooled among patients treated with ONC201 monotherapy through any of these trial with H3 K27M confirmed glioma, progressive and measurable disease by RANO, &gt; 90 days from completion of prior radiation, no evidence of leptomeningeal dissemination, midline location other than primarily pons or spinal cord, and baseline KPS &gt; 60. Using an enrollment cutoff of February 15, 2019 and data cutoff of July 31, 2019, there were 20 patients (NCT03295396, 12; NCT02525692, 7; expanded access, 1). Dosage was 625 mg weekly in 19 and once every 3 weeks in 1. Results: No DLTs or treatment discontinuations due to toxicity occurred. Midline gliomas can exhibit minimal contrast enhancement or exhibit a mixture of contrast-enhancing and non-contrast enhancing regions in the tumor. As a result, blinded independent central review (BICR) of tumor response by MRI was assessed by RANO-HGG and RANO-LGG for each patient to capture contrast-enhancing lesions by T1 post-contrast and non-contrast-enhancing assessments by T2/FLAIR, respectively, in the object response rate. The best response by RANO-HGG or RANO-LGG is 30% (95% CI, 11.9-54.3%). Duration of response by RANO-HGG is median 52.7 weeks (range 15.9-138.3). One patient with stable disease as of this data cutoff has continued on treatment beyond 12 months and recently underwent an investigator-reported PR by RANO-HGG that is pending confirmation. Conclusions: Single agent ONC201 is well tolerated and clinically active in recurrent H3 K27M-mutant diffuse midline glioma patients. Clinical trial information: NCT03295396, NCT02525692 .

The molecular characteristics of spinal cord gliomas with or without H3 K27M mutation

Due to the rare incidence of spinal cord astrocytomas, their molecular features remain unclear. Here, we characterized the landscapes of mutations in H3 K27M, isocitrate dehydrogenase 1 (IDH1) R132H, BRAF V600E, and the TERT promoter in 83 diffuse spinal cord astrocytic tumors. Among these samples, thirty-five patients had the H3 K27M mutation; this mutant could be observed in histological grade II (40%), III (40%), and IV (20%) astrocytomas. IDH1 mutations were absent in 58 of 58 cases tested. The BRAF V600E mutation (7/57) was only observed in H3-wildtype astrocytomas, and was associated with a better prognosis in all histological grade II/III astrocytomas. TERT promoter mutations were observed in both H3 K27M-mutant (4/25) and -wildtype (9/33) astrocytomas, and were associated with a poor prognosis in H3-wildtype histological grade II/III astrocytomas. In the 2016 WHO classification of CNS tumors, H3 K27M-mutant diffuse midline gliomas, including spinal cord astrocytomas, are categorized as WHO grade IV. Here, we noticed that the median overall survival of histological grade II/III H3 K27M-mutant cases (n = 28) was significantly longer than that of either the total histological grade IV cases (n = 12) or the H3 K27M-mutant histological grade IV cases (n = 7). We also directly compared H3 K27M-mutant astrocytomas to H3-wildtype astrocytomas of the same histological grade. In histological grade II astrocytomas, compared to H3-wildtype cases (n = 37), H3 K27M-mutant patients (n = 14) had showed a significantly higher Ki-67-positive rate and poorer survival rate. However, no significant differences in these parameters were observed in histological grade III and IV astrocytoma patients. In conclusion, these findings indicate that spinal cord astrocytomas are considerably different from hemispheric and brainstem astrocytomas in terms of their molecular profiles, and that the histological grade cannot be ignored when assessing the prognosis of H3 K27M-mutant spinal cord astrocytomas.

Clinical, radiologic, and genetic characteristics of histone H3 K27M-mutant diffuse midline gliomas in adults.

Background“Diffuse midline glioma (DMG), H3 K27M-mutant” is a new tumor entity established in the 2016 WHO classification of Tumors of the Central Nervous System that comprises a set of diffuse gliomas arising in midline structures and is molecularly defined by a K27M mutation in genes encoding the histone 3 variants H3.3 or H3.1. While this tumor entity is associated with poor prognosis in children, clinical experience in adults remains limited.MethodsPatient demographics, radiologic and pathologic characteristics, treatment course, progression, and patient survival were collected for 60 adult patients with DMG, H3 K27M-mutant. A subset of tumors also underwent next-generation sequencing. Analysis of progression-free survival and overall survival was conducted using Kaplan–Meier modeling, and univariate and multivariate analysis.ResultsMedian patient age was 32 years (range 18–71 years). Tumors were centered in the thalamus (n = 34), spinal cord (10), brainstem (5), cerebellum (4), or other midline sites (4), or were multifocal (3). Genomic profiling revealed p.K27M mutations exclusively in the H3F3A gene and an absence of mutations in HIST1H3B or HIST1H3C, which are present in approximately one-third of pediatric DMGs. Accompanying mutations in TP53, PPM1D, FGFR1, NF1, and ATRX were frequently found. The overall survival of this adult cohort was 27.6 months, longer than historical averages for both H3 K27M-mutant DMG in children and IDH-wildtype glioblastoma in adults.ConclusionsTogether, these findings indicate that H3 K27M-mutant DMG represents a heterogeneous disease with regard to outcomes, sites of origin, and molecular pathogenesis in adults versus children.

Midline Glioma in Adults: Clinicopathological, Genetic, and Epigenetic Analysis

The histone H3K27M-mutant diffuse midline glioma is often seen in children and has a very poor prognosis regardless of its histological grade. Although it can occur in adults, few studies on adult cases have been reported. We examined adult midline glioma cases for their histological grade, presence of H3K27M mutation, and expression of related factors—enhancer of zeste homolog 2 (EZH2), H3K27me3, p16, and methylthioadenosine phosphorylase. These tumor characteristics were also evaluated for their prognostic value in adult midline glioma. High histological grade, H3K27M-mutant, high EZH2 expression, and high H3K27me3 expression was detected in 12/23 (53%), 11/23 (48%), 9/23 (39%), and 12/23 (52%) cases, respectively. Histological grade and prognosis were significantly correlated (P <0.01). The high expression of EZH2 and the low expression of H3K27me3 correlated with histological malignancy (P = 0.019 and 0.009) and prognosis (P = 0.048 and 0.047). To broaden the scope of our analysis, a review of cases reported in the literature (2014–2019) was performed. In the 171 cases, H3K27M-mutant showed poor prognosis in the young adult group (P = 0.001), whereas H3K27 status had no effect on prognosis in the older age group (P = 0.141). Histological grade was correlated with prognosis in both young adults and older groups (P <0.001, P = 0.003, respectively). We demonstrate differences in prognostic factors for diffuse gliomas in the midline region for children and adults. Importantly, the H3K27M mutation significantly influences prognosis in children, but not necessarily in adults. Contrarily, histological grading and immunostaining are important prognostic tools in adults.