Brainstem Tumors Research Articles

Brainstem tumors in children: a monocentric series in the light of genetic and bio-molecular progress in pediatric neuro-oncology.

Brainstem tumors represent a challenge. Their management and prognosis vary according to anatomopathological findings and genetic and bio-molecular fingerprints. We present our experience with pediatric brainstem tumors. All patients admitted for a brainstem tumor at the Pediatric Neurosurgical Unit at Hôpital Femme Mère Enfant hospital between January 1997 and December 2019 were considered. Patients data were obtained through a retrospective review of the medical records; follow-up was from the last outpatient consultation. One hundred and twelve patients were included. Eighty-five patients (75.9%) had open surgery or stereotactic biopsy. Thirty-five patients were treated for hydrocephalus. Sixty-six received an adjuvant treatment. Several protocols were adopted according to the SFOP and SIOP during this time period. The overall survival rate was 45% with a median follow-up of five years (range 1-18 year). However, the survival rate was very different between the diffuse intrinsic pontine gliomas (DIPG) and the others tumor types. If we exclude the DIPG (59 patients), of which only 1 was alive at 3 years, the survival rate was 90.6% (only 5 deaths over 53 patients) with a median follow up of 5 years. Our series confirms that benign tumors of the brainstem have a good survival when treated with surgical removal ± adjuvant therapy. Diffuse pontine gliomas continue to have a dismal prognosis. Individualized treatment based on molecular fingerprints may help to select the best adjuvant therapy and hence potentially improve survival.

TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain stem tumor and the leading cause of pediatric cancer-related death. To date, these tumors remain incurable, underscoring the need for efficacious therapies. In this study, we demonstrate that the immune checkpoint TIM-3 (HAVCR2) is highly expressed in both tumor cells and microenvironmental cells, mainly microglia and macrophages, in DIPG. We show that inhibition of TIM-3 in syngeneic models of DIPG prolongs survival and produces long-term survivors free of disease that harbor immune memory. This antitumor effect is driven by the direct effect of TIM-3 inhibition in tumor cells, the coordinated action of several immune cell populations, and the secretion of chemokines/cytokines that create a proinflammatory tumor microenvironment favoring a potent antitumor immune response. This work uncovers TIM-3 as a bona fide target in DIPG and supports its clinical translation.

Brazilian Pediatric Patients with Gliomas: Treatment Characteristics and Survival Outcomes

Despite being the second most frequent tumors in children, pediatric central nervous system (CNS) tumors are rare, and there are limited epidemiological data. The current study aimed to determine the survival rates of patients diagnosed with pediatric gliomas in Brazil, accounting for the influence of age, treatment modalities, and tumor site using population-based national database. Patients diagnosed with pediatric gliomas of CNS from 1999-2020 were identified from The Fundação Oncocentro de São Paulo database. The Kaplan-Meier and the log-rank test were used for survival analysis. A total of 1296 patients were included. The most common histologic tumor type were glioblastomas (38.27%; n = 496), pilocytic astrocytoma (32.87%; n = 426) and astrocytoma grade II (20.76%; n = 269) A total of 379 (29.24%) had brainstem tumor. The 1-year, 3-year 5-year OS for pilocytic astrocytoma were 93.72%, 89.98%, and 88.97%; for grade II 80,36%, 71,89%, 68,60%; for grade III 53,72%; 31,87%, 28,33%; and for glioblastoma 52,90%, 28,76%, 25,20%, respectively. Brainstem tumors had the worse OS compared to no brainstem tumors (p = 0.001). For high-grade glioma (grade III and IV) excluding brainstem tumors (n = 570), young patients had greater median OS (0 to 3 years: 22 months; 4 to 18 years: 13 months - p = 0.005). Regarding the treatment modalities, combined treatments were associated with higher median survival compared to less intensive therapy (surgery: 11 months; surgery and chemotherapy: 16 months; surgery, radiotherapy, and chemotherapy: 20 months; p = 0.005) CONCLUSION: In our cohort, low-grade gliomas had favorable prognoses and outcomes. Patients diagnosed with glioblastomas and brainstem gliomas had the worst OS. For high-grade gliomas, undergoing treatment de-intensification in the Brazilian pediatric population is associated with worse survival.

Clinical Presentation, MRI findings and Molecular insights in Diffuse Intrinsic Pontine Gliomas (DIPGs): A Comprehensive Review.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive and the most common brainstem tumor affecting pediatric patients, with a poor prognosis and limited treatment options. Despite extensive research, the median overall survival remains low, and current treatment mainly relies on radiotherapy. Recent discoveries of recurrent somatic mutations, particularly in histone 3 variants, have led to a reclassification of pediatric diffuse gliomas. However, the relationship between genetic alterations, imaging features, and prognosis is not yet fully understood. This review provides an overview of DIPG, including its clinical presentation, typical imaging findings (with a focus on MRI techniques), diagnostic challenges, treatment modalities, and recent advancements in molecular understanding. DIPG presents with characteristic neurological symptoms and often manifests as an expansile intra-axial mass in the pons, usually exhibiting typical MRI findings. Diffusion-weighted imaging and magnetic resonance spectroscopy can provide additional insights. Pathologically, most DIPGs are pediatric type of diffuse high-grade gliomas, with specific histological features and occasional leptomeningeal spread. Radiotherapy remains the mainstay treatment. However, clinical trials with new agents are ongoing, trying to improve outcomes for DIPG patients.

Preoperative word-finding difficulties in children with posterior fossa tumours: a European cross-sectional study.

Posterior fossa tumour surgery in children entails a high risk for severe speech and language impairments, but few studies have investigated the effect of the tumour on language prior to surgery. The current crosslinguistic study addresses this gap. We investigated the prevalence of preoperative word-finding difficulties, examined associations with medical and demographic characteristics, and analysed lexical errors. We included 148 children aged 5-17years with a posterior fossa tumour. Word-finding ability was assessed by means of a picture-naming test, Wordrace, and difficulties in accuracy and speed were identified by cut-off values. A norm-based subanalysis evaluated performance in a Swedish subsample. We compared the demographic and medical characteristics of children with slow, inaccurate, or combined slow and inaccurate word finding to the characteristics of children without word-finding difficulties and conducted a lexical error analysis. Thirty-seven percent (n = 55) presented with slow word finding, 24% (n = 35) with inaccurate word finding, and 16% (n = 23) with both slow and inaccurate word finding. Children with posterior fossa tumours were twice as slow as children in the norming sample. Right-hemisphere and brainstem location posed a higher risk for preoperative word-finding difficulties, relative to left-hemisphere location, and difficulties were more prevalent in boys than in girls. The most frequent errors were lack of response and semantically related sideordinated words. Word-finding difficulties are frequent in children with posterior fossa tumours, especially in boys and in children with right-hemisphere and brainstem tumours. Errors resemble those observed in typical development and children with word-finding difficulties.

P11.84.A PHASE 1 TRIAL OF MARIZOMIB ALONE AND IN COMBINATION WITH PANOBINOSTAT IN CHILDREN WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract BACKGROUND DIPG is an aggressive brainstem tumor with no effective therapy beyond the temporizing effects of radiation (RT). Preclinical studies demonstrated synergistic activity of marizomib (MRZ), an irreversible CNS-penetrant, pan-proteosome inhibitor, when administered with the HDAC inhibitor, panobinostat. In a study of adults with glioblastoma, MRZ was associated with significant CNS adverse events of interest (ataxia, balance disorder, dizziness, dysarthria, fall, gait disturbance, hallucination) in the majority of patients. We performed a novel Bayesian design, dual phase 1 trial of MRZ administered as a single agent and in combination with panobinostat to children with DIPG. Primary objectives were to determine the toxicity profile, dose limiting toxicities (DLT), and maximum tolerated dose (MTD) of MRZ at doses up to 0.8 mg/m2 alone and when administered in combination with panobinostat, and to characterize the plasma pharmacokinetics of MRZ as a single agent in children with DIPG. MATERIAL AND METHODS Eligible patients were <22 years with DIPG who received prior standard RT. When available, tumor tissue was centrally reviewed for molecular diagnosis. Patients initially received MRZ alone (Course A1). If tolerated, they received the combination of MRZ and panobinostat on subsequent courses (Course B1, B2, etc.). MRZ starting dose was 0.6 mg/m2IV on Days 1 and 15 of a 28-day cycle. If no DLT was observed during Course A1, patients received MRZ in combination with panobinostat 22 mg/m2/dose p.o. every other day, 3 times/week, on a one week on, one week off schedule. Dose escalation/ de-escalation for single agent and combination was guided using the Bayesian optimal interval design. RESULTS Four patients enrolled; one withdrew prior to initiating therapy due to disease progression (PD). Median age at study entry was 7.7 (range, 4.8-8.7)yrs. Three were evaluable for MRZ toxicities, two were evaluable for the combination MRZ + panobinostat. All patients were treated at dose level 1. No DLTs or CNS AEs attributed to MRZ were observed. Adverse events were related to disease/tumor progression or considered mild (grade 1 headache, emesis, diarrhea, fatigue, lymphopenia, thrombocytopenia) with the exception of sinus tachycardia and dyspnea (grade 2) (n=1). All patients died of PD; median time from on study to death for 3 treated patients was 95 days (range, 50-112). Pre- and post-study tissues were obtained where possible for qualitative exploratory pharmacokinetic, pharmacodynamic, molecular and patient model analyses. The study was terminated early due to withdrawal of support from BMS. CONCLUSION Children with DIPG tolerated single agent MRZ at a dose of 0.6 mg/m2/dose on Days 1 and 15 and in combination with panobinostat without any DLT or CNS toxicity. Target dose levels were not evaluated as clinical development program for marizomib ended and the study was discontinued early.

A combined immunopeptidomics, proteomics, and cell surface proteomics approach to identify immunotherapy targets for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging. In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy. Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13Rα2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3). The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.

Pediatric Brain Stem Tumors: Analysis of 25 Cases

The charts of twenty-five pediatria patients with brain stem tumors diagnosed between March 1977 and December 1980 have been reviewed. Ages of patients ranged from 22 months to 17 years. The use of computed tomography (CT) was found to have been valuable in diagnosis and follow-up, as well as in the design of radiation therapy portals. Radiotherapy and combination chemotherapy with VM26 (4'-1 demethyl-epipodophyllo-toxin B-D-thenyUdene glucoside) and CCNU (1-2-chloroethyl-methyl-3-Cyclohexyl-1-nitrosourea) were the treatment modalities employed. The three-year survival rate for this group of patients was 29%.

Robot-assisted transcerebellar stereotactic approach to the posterior fossa in pediatric patients: a technical note.

During the last decade, there has been renewed interest in stereotactic approaches to diffuse intrinsic pontine gliomas (DIPGs) in children, due to the development of new concepts in molecular biology and management, and subsequent need for tissue sampling. Stereotactic frame-based and robot-assisted techniques are associated with reduced target error and have been incorporated into standard practice at our institution. Four children (age 2-7years) underwent a robot-assisted frame-based transcerebellar approach using the Leksell G frame coupled with Renishaw's neuromate® stereotactic robot. The procedures included 3 biopsies (two brainstem tumors and one cerebellar hemispheric lesion) and 1 depth electrode implantation into a low-grade tumor remnant (ganglioglioma) of the middle cerebellar peduncle causing drug-resistant epilepsy in a young girl. Targeting was based on MRI, and in one case, 18F-FET-PET was coregistered to MRI to improve sampling accuracy. The frame was applied 180° rotated compared to standard orientation, and patients were positioned prone during surgery and stereotactic preoperative CT scan. Postoperative CT scan ruled out complications and was coregistered to preoperative MRI to check the target accuracy. No complications occurred, and targeting was accurate in all cases. All tissue samplings provided proper histology; depth electrode EEG exploration was diagnostic and led subsequent resective surgery. According to our experience, the transcerebellar frame-based robotic stereotactic approach to the cerebellum and the brainstem is feasible, safe, and effective even in young children.

2-Hydroxyglutarate magnetic resonance spectroscopy in adult brainstem glioma.

Adult brainstem gliomas (BSGs) are rare tumors of the CNS that are poorly understood. Upregulation of the oncometabolite 2-hydroxyglutarate (2HG) in the tumor indicates the mutation of isocitrate dehydrogenase (IDH), which can be detected by magnetic resonance spectroscopy (MRS). Although histological examination is required for the definitive diagnosis of BSG, 2HG-optimized MRS (2HG-MRS) may be useful, considering the difficult nature of brainstem lesion biopsy. The aim of this study was to evaluate the utility of 2HG-MRS for diagnosing IDH-mutant adult BSG. Patients with a radiographically confirmed brainstem tumor underwent 3T MRS. A single voxel was set in the lesion with reference to the T2 or fluid-attenuated inversion recovery image and analyzed according to the 2HG-tailored MRS protocol (point-resolved spectroscopic sequence; echo time 35 msec). All patients underwent intraoperative navigation-guided or CT-guided stereotactic biopsy for histopathological diagnosis. The status of IDH and H3K27M mutations was confirmed by immunohistochemistry and direct DNA sequencing. In addition, the authors examined the relationship between patients' 2HG concentrations and survival time. Ten patients (7 men, 3 women; median age 33.5 years) underwent 2HG-MRS and biopsy. Four patients had an H3K27M mutation and 4 had an IDH1 mutation (1 R132H canonical IDH mutation, 2 R132S and 1 R132G noncanonical IDH mutations). Two had neither H3K27M nor IDH mutations. The H3K27M and IDH mutations were mutually exclusive. Most tumors were located in the pons. There was no significant radiological difference between mutant H3K27M and IDH on a conventional MRI sequence. A 2HG concentration ≥ 1.8 mM on MRS demonstrated 100% (95% CI 28%-100%) sensitivity and 100% (95% CI 42%-100%) specificity for IDH-mutant BSG (p = 0.0048). The median overall survival was 10 months in IDH-wild-type BSG patients (n = 6) and could not be estimated in IDH-mutant BSG patients (n = 4) due to the small number of deaths (p = 0.008). 2HG-MRS demonstrated high sensitivity and specificity for the prediction of IDH-mutant BSG. In addition, 2HG-MRS may be useful for predicting the prognosis of adult BSG patients.

SONIC HEDGEHOG (SHH) PROMOTES BLOOD-BRAIN BARRIER (BBB) INTEGRITY IN DIFFUSE MIDLINE GLIOMA OF THE PONS

Abstract Diffuse Midline Glioma of the Pons (DMG-P) is the most common brainstem tumor in children and is universally lethal in this age group. The intact Blood-Brain Barrier (BBB) in these tumors are believed to play a major role in preventing systemically administered therapeutic agents from reaching therapeutic concentrations in the tumor. The factors that contribute to the integrity of BBB in DMG-P is not well understood. Sonic Hedgehog (SHH) signaling promotes BBB integrity in the developing and adult brain and plays an important role in the formation and maintenance of DMG-P tumors. In the present study, we show that DMG-P tumor cells secrete SHH ligand. Secreted SHH present in the tumor conditioned media (TCM) tightens the BBB by increasing electrical impedance and decreased paracellular and transcellular permeability in BBB Endothelial cells (ECs). Mechanistically, SHH in TCM causes tightening of the BBB by increasing the expression of junctional proteins in BBB-ECs, likely by signaling through non- canonical pathways. SHH in the TCM also increased the expression of ATP-binding cassette (ABC) transporter proteins ABCB1(P- glycoprotein) and ABCG2 (BCRP- Breast cancer resistance protein) in the Endothelial cells. These pro-barrier functions of SHH in TCM can be reversed by pharmacological inhibition of the S-acylation of the SHH ligand using Hedgehog acyltransferase (HHAT) inhibitor RUSKI-201. Our study demonstrates a potential therapeutic strategy of altering the BBB permeability to increase the efficacy of systemic therapies in DMG-P tumors. Ongoing studies will validate these findings in orthotopic murine models of DMG-P.

Bilateral Vocal Fold Paralysis in a Patient With Congenital MYOD1 Myopathy.

Congenital bilateral vocal fold paralysis (BVFP) is a rare but significant cause of morbidity in pediatric otolaryngology. The differential diagnosis is expansive, with common etiologies including birth trauma, brainstem neoplasms, and neurologic disorders. There are few known genetic causes of the condition. This report details the first known case of BVFP secondary to a genetic deficiency in MYOD1, a master transcriptional regulator of skeletal muscle cell specification. Genetics consultation and testing may be a useful adjunct in the workup of congenital BVFP and may help guide prognostication, additional workup, counseling, and clinical decision-making.

Fifty years of DIPG: looking at the future with hope.

Diffuse intrinsic pontine glioma (DIPG) is a primary brainstem tumor of childhood that carries a dismal prognosis, with median survival of less than 1year. Because of the brain stem location and pattern of growth within the pons, Dr. Harvey Cushing, the father of modern neurosurgery, urged surgical abandonment. Such a dismal prognosis remained unchanged for decades, coupled with a lack of understanding of tumor biology and an unchanging therapeutic panorama. Beyond palliative external beam radiation therapy, no therapeutic approach has been widely accepted. In the last one to two decades, however, increased tissue availability, an improving understanding of biology, genetics, and epigenetics have led to the development of novel therapeutic targets. In parallel with this biological revolution, new methods intended to enhance drug delivery into the brain stem are contributing to a surge of exciting experimental therapeutic strategies.

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.

METB-05. NON-INVASIVE DIAGNOSIS OF BRAINSTEM GLIOMAS IN PEDIATRIC, ADOLESCENT, AND YOUNG ADULT PATIENTS THROUGH CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING

Abstract Intrinsic brainstem tumors arising in pediatric, adolescent, and young adult patients comprise a spectrum of entities, predominantly diffuse midline gliomas (DMG) and IDH mutant astrocytomas. Accurate molecular diagnosis is essential for prognostication and optimal therapy. Therapeutic considerations include inclusion (in IDH mutant) or exclusion (in H3K27M) of adjuvant Temozolomide post radiotherapy and use of molecular targeted therapy (IDH inhibitors). Surgical biopsy, however, is associated with increased risk of permanent neurological deficits and may yield insufficient or non-diagnostic tissue. We hypothesized that minimally-invasive “liquid biopsy” of cerebrospinal fluid (CSF) could represent a superior diagnostic modality in this patient population, employing molecular analysis of cell free DNA (cfDNA). We analyzed 44 CSF samples from 38 unique patients for recurrent driver mutations in brainstem gliomas, including H3K27M, IDH1, and IDH2. MSK-IMPACT, a NY state-authorized hybridization capture-based next-generation panel DNA sequencing assay, was used for analysis. Samples without a detectable driver mutation by MSK-IMPACT testing were further subjected to gene targeted testing by droplet digital PCR. In all, 10/44 (22.7%) samples had mutations detected by MSK-IMPACT using standard calling criteria and 13 of the remaining 34 samples (38.2%) had supporting evidence of a mutation based on manual review. Further testing by ddPCR was performed on a subset of cases (based on DNA availability) which confirmed 7 of the previous low-level mutations and uncovered 4 additional mutations. Overall, 27/44 (61.4%) of cases had detectable evidence of a mutation, and of the 23 patients without a known tissue diagnosis, a driver in the CSF was identified for 47.8% of patients (11/23). Minimally-invasive analysis of CSF cfDNA in patients with intrinsic brainstem tumors has a high diagnostic yield and may obviate the need for tissue biopsy in a majority of patients. Testing with high sensitivity assays is valuable to maximize the rate of detection.

DIPG-27. DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) CHALLENGE: FROM GENE EXPRESSION PROFILE TO DRUG

Abstract Diffuse intrinsic pontine glioma (DIPG) accounts for 80% of pediatric brainstem tumors and about 10-20% of all central nervous system tumors in childhood. Despite the adoption of aggressive therapeutic approaches, DIPG remains the leading cause of cancer-related mortality in childhood: prognosis is still poor with more than 90% of affected children dying within 18-24 months from the diagnosis.1 Decades of clinical attempts involving more than 200 trials evaluating different conventional cytotoxic drugs, targeted drugs and studies with radiation dose escalating and new radio sensitizing agents, have failed to improve the overall survival of children with DIPG. Since available drugs have proven no benefits hereto for DIPG, it is fundamental to gain insights into the gene expression profile of DIPG patients and speed up the discovery of novel therapeutics for this dismal malignancy. Only the compound ONC201 was discovered to reduce DIPG tumors size in DIPG H3K27M mutated and now is in phase III clinical trials. Given the complex molecular mechanisms underlying DIPG, it would be necessary to fully utilize gene expression profiles to build a fully knowledge of DIPG-related genes and pathways. Preliminarily, the gene expression profile of eight pediatric DIPG samples and two primary pontine tissue samples was downloaded and analyzed starting from the Gene Expression Omnibus (GEO) database2. The GEO data were used to evaluate the differentially expressed genes (DEGs) in the pediatric DIPG samples. Subsequently, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathways of DEGs were enriched, integrated, and analyzed to find out genes and signaling pathways involved in the biological and clinic-pathological features of DIPG. More data are being to be added to our meta-analysis.

DIPG-42. MAPPING THE DNA METHYLATION LANDSCAPE OF DIPG REVEALS NOTABLE METHYLOME VARIABILITY RESPONSIVE TO PHARMACOLOGIC MODULATION

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brainstem tumor, driven by the H3K27M mutation leading to epigenetic dysregulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation and altered gene expression. However, most investigations of the epigenetic landscape of DIPG only provide a static view of the epigenome. Such studies are not capable of adequately capturing the regulatory underpinnings of DIPG intratumoral heterogeneity and phenotypic plasticity. To address this, we performed Whole-Genome Bisulfite Sequencing (WGBS) on 23 primary patient DIPG specimens, 4 patient-derived DIPG neurosphere cell lines, and 5 normal controls. To analyze this data set, we applied a novel framework for analysis of DNA methylation variability, called informME analysis, which permits the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG exhibits a markedly disordered epigenome, with increased stochasticity localizing to key regulatory elements and genes. We demonstrate that epigenetic instability maps onto key genes regulating pluripotency and developmental identity and hypothesize that these changes might enable tumor cells to sample a diverse range of transcriptional programs and phenotypes. Given this, we evaluated the responsiveness of the DIPG epigenetic landscape to pharmacologic modulation. We found that treatment with the DNA hypomethylating agent decitabine produced profound genome-wide demethylation and reduced DNA methylation stochasticity at active enhancers, bivalent promoters, and promoters of key developmental genes. Decitabine treatment also elicited changes in gene expression, including upregulation of genes related to immune signaling. Finally, treatment with DAC sensitized DIPG cells to the effects of histone deacetylase inhibition. This study suggests the application of epigenetic therapies to constrain cellular heterogeneity and plasticity in DIPG.

DIPG-09. ATOVAQUONE RADIOSENSITISES DIFFUSE MIDLINE GLIOMAS BY INHIBITING MITOCHONDRIAL METABOLISM AND HYPOXIA

Abstract Diffuse Midline Glioma (DMG) is a uniformly fatal paediatric brainstem tumour with median survival of less than 1 year. Radiotherapy has been the only effective treatment for decades, but most DMGs recur within several months due to radioresistance. The hypoxic tumour microenvironment, a main feature of solid tumours including gliomas, is a major contributor to radioresistance and impedes the efficacy of radiotherapy. Therefore, alleviating tumour hypoxia to enhance the effectiveness of radiotherapy is a therapeutic strategy to improve survival outcomes of DMG patients. Here, our strategy is to decrease the oxygen consumption rate (OCR) of DMG cells by targeting their mitochondria, which in turn will alleviate hypoxia by sparing more oxygen and subsequently improve the radiosensitivity of DMG cells. Specifically, we performed a high-throughput screening to identify potent OCR inhibitors using a library of 1963 FDA-approved drugs. The most promising OCR inhibitor identified was atovaquone, a drug used for treatment of pneumocystis pneumonia and malaria. We found that atovaquone inhibited mitochondrial metabolism of DMG cells by specifically targeting the mitochondrial complex III. It induced the formation of mitochondrial reactive oxygen species suggesting that it increases oxidative stress. It alleviated hypoxia and decreased the expression of hypoxia-inducible factor-1a in several 3-dimensional DMG neurospheres and improved the radiosensitivity of a range of DMG cultures. To overcome the issues of poor bioavailability of commercially available atovaquone resulting in low therapeutically effective brain concentrations, we tested its efficacy against the amorphous solid dispersion (ASD) atovaquone formulation which appears to enhance the atovaquone levels in the brain. We found that both the formulations inhibited OCR and hypoxia at similar doses and improved the radiosensitivity of DMG. With these promising findings, our further work is assessing the in vivo efficacies of commercially available atovaquone and ASD atovaquone formulation using orthotopic DMG models.

SURG-05. MRI-GUIDED FOCUSED ULTRASOUND BLOOD-BRAIN BARRIER DISRUPTION INCREASES DRUG DELIVERY AND EFFICACY IN AN ORTHOTOPIC DIPG MODEL

Abstract Diffuse intrinsic pontine glioma is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB disruption (BBBD), allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promising in vitro agent in DIPG with poor BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Mice were orthotopically injected with a patient-biopsied DIPG cell line, BT-245. After two weeks of growth, MR images guided FUS targeting to the tumor location. Microbubbles (25µl/kg, IV) and FUS (Frequency: 1.515 MHz, PNP: 0.77MPa, PRF: 1Hz, PL: 10ms, treatment time: 3 minutes) were applied to half the mice. Passive cavitation data (PCD) was collected and analyzed. Panobinostat was given (10mg/kg IP) to all mice. After 1.5 hours, a subset of mice were sacrificed to determine the concentration of panobinostat in tumor and cortex areas. We showed a 3-fold drug concentration increase in the tumor region while only slightly increasing the concentration in the forebrain. The remaining mice received three weekly treatments. Half were treated with FUS/MB and panobinostat, while others received panobinostat only. MRI was taken weekly to evaluate BBBD extent and tumor growth. PCD during treatments showed ideal cavitation of microbubbles illustrating safety and consistency across all treatments. The combination of panobinostat and FUS exhibited a tumor size decrease of 71% on MRI compared to panobinostat alone. Furthermore, FUS/MB improved the mean survival from 21 to 31.5 days (p=0.0001). Our study demonstrates that FUS-mediated BBBD can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.

Diffuse intrinsic pontine gliomas: First registry effort in Mexico.

Brainstem tumors comprise 10.9% of all brain tumors, and pediatric diffuse intrinsic pontine gliomas (DIPG) have a fatal prognosis. Some countries have developed national and international register databases to characterize their populations to aid clinical and public policy decisions. This study provides information regarding the clinical characteristics of a retrospective cohort of children with DIPG in México from 2001 to 2021, and assesses the proposed prognostic factors previously described for survival outcome. Health institutions from Mexico were invited to contribute to a retrospective electronic registry of patients with DIPG based on the International DIPG Registry. Fisher's exact test was used to compare long- and short-term survivors. Overall survival was estimated using the Kaplan-Meier method. Differences between survival curves were evaluated using the log-rank test and Cox proportional hazard regression analysis. Total 110 patients were included. The median age of the patients at diagnosis was 7years. Sixty patients (54.5%) presented with symptoms in less than 6months; the most frequent symptom was ataxia (56.4%). Ninety patients received treatment (81.8%), the overall survival at 4years was 11.4%, and 16 patients (14.5%) were admitted for palliative end-of-life care. We found no significant survival differences for any of the prognostic factors. This study highlights the need to develop strategies to standardize healthcare processes and enhance the quality of care to improve clinical diagnosis in Mexico. We also observed a barrier to the acceptance of palliative end-of-life care in the family and medical teams.