H3.3K27M Mutation Promotes The Migration and Invasion of Glioma Cells By Activating β-Catenin/USP1 Signaling

H3.3K27M is a newly identified molecular pathology marker in glioma and is especially correlated with the malignancy of diffuse intrinsic pontine glioma (DIPG). In recent years, accumulating research has revealed that other types of glioma also contain the H3.3K27M mutation. However, the role of H3.3K27M in high-grade adult glioma, which is the most malignant glioma, has not been investigated. In this study, we focused on exploring the expression and function of H3.3K27M in high-grade adult glioma patients. We found that H3.3K27M is partly highly expressed in high-grade glioma tissues. Then, we introduced H3.3K27M into H3.3 wild-type glioma cells, U87 cells and LN229 cells. We found that H3.3K27M did not regulate the growth of glioma in vitro and in vivo; however, the survival of mice with transplanted tumors was significantly reduced. Further investigation revealed that H3.3K27M expression mainly promoted the migration and invasion of glioma cells. Moreover, we certified that H3.3K27M overexpression enhanced the protein levels of ꞵ-catenin and p-ꞵ-catenin, the protein and mRNA levels of ubiquitin-specific protease 1 (USP1), and the protein level of enhancer of zeste homolog 2 (EZH2). Importantly, the ꞵ-catenin inhibitor XAV-939 significantly attenuated the upregulation of the aforementioned proteins. Overall, the H3.3K27M mutation is present in a certain proportion of high-grade glioma patients and facilitates a poor prognosis by promoting the metastasis of glioma by regulating the ꞵ-catenin/USP1/EZH2 pathway.

Object Detection Improves Tumour Segmentation in MR Images of Rare Brain Tumours

Tumour lesion segmentation is a key step to study and characterise cancer from MR neuroradiological images. Presently, numerous deep learning segmentation architectures have been shown to perform well on the specific tumour type they are trained on (e.g., glioblastoma in brain hemispheres). However, a high performing network heavily trained on a given tumour type may perform poorly on a rare tumour type for which no labelled cases allows training or transfer learning. Yet, because some visual similarities exist nevertheless between common and rare tumours, in the lesion and around it, one may split the problem into two steps: object detection and segmentation. For each step, trained networks on common lesions could be used on rare ones following a domain adaptation scheme without extra fine-tuning. This work proposes a resilient tumour lesion delineation strategy, based on the combination of established elementary networks that achieve detection and segmentation. Our strategy allowed us to achieve robust segmentation inference on a rare tumour located in an unseen tumour context region during training. As an example of a rare tumour, Diffuse Intrinsic Pontine Glioma (DIPG), we achieve an average dice score of 0.62 without further training or network architecture adaptation.

Suggestions for Escaping The Dark Ages for Pediatric Diffuse Intrinsic Pontine Glioma Treated with Radiotherapy: Analysis of Prognostic Factors From The National Multicenter Study

Purpose This multicenter retrospective study aimed to investigate prognostic factors for survival, encompassing clinical and radiologic features and treatments, in newly diagnosed diffuse intrinsic pontine glioma (DIPG) patients treated with radiotherapy. Methods Patients <30 years of age who underwent radiotherapy as an initial treatment for DIPG between 2000 and 2018 were included; patients who did not undergo an MRI at diagnosis and those with pathologically diagnosed grade I glioma were excluded. We examined medical records of 162 patients collected from 10 participating centers in Korea. The patients’ clinical and radiological variables, molecular and histopathologic data, and treatment response were evaluated to identify the prognosticators for DIPG and estimate survival outcomes. Results The median follow-up period was 10.8 months (interquartile range, 7.5–18.1). The 1- and 2-year overall survival (OS) rates were 53.5% and 19.0%, respectively, with a median OS of 13.1 months. Long term survival rate (≥2 years) was 16.7%, and median OS was 43.6 months. Age (<10 years), poor performance status, treatment before 2010, and post-radiotherapy necrosis were independent prognostic factors related to poor OS in the multivariate analysis. In patients with increased post-radiotherapy necrosis, the median OS was 13.3 months for bevacizumab group and 11.4 months for no-bevacizumab group (P = 0.138). Conclusion Therapeutic strategy for DIPG has remained unchanged over time, and its prognosis remains poor. Our findings suggest that appropriate efforts are needed to reduce the occurrence of post-radiotherapy necrosis. Further well-designed clinical trials are recommended to improve the poor prognosis observed in DIPG patients.

In diffuse intrinsic pontine gliomas, non-coding RNA expression is significantly varied dependent on overall survival

Diffuse intrinsic pontine glioma is a kind of pediatric brain cancer that kills 99 percent of patients within five years and for which there are no conventional chemotherapies. It is crucial for new treatments to comprehend the cancer's transcriptional activity. Using a published dataset, we compared the transcriptomes of tumors from patients who lived longer or less than six months. Among the genes whose expression changed most, we observed that numerous microRNAs and snoRNAs were present. The publication's findings are the first evidence of variable levels of non-coding RNA expression in diffuse intrinsic pontine glioma.

CTIM-27. LOCOREGIONAL B7-H3-SPECIFIC CAR T CELLS FOR CHILDREN AND YOUNG ADULTS WITH DIPG: INTERIM REPORT OF BRAINCHILD-03 ARM C

Following preclinical optimization of B7-H3-specific CAR T cells against pediatric brain tumor models, we opened BrainChild-03 (NCT04185038), a phase 1 clinical trial of repeatedly dosed, outpatient, locoregional B7-H3-specific CAR T cells for children with recurrent/refractory central nervous system (CNS) tumors or diffuse intrinsic pontine glioma (DIPG). Here, we report the interim findings from patients enrolled on Arm C, dedicated to DIPG. The primary endpoints are feasibility and safety, with secondary endpoints of disease response. We utilize second-generation CAR T cells with a 4-1BB costimulatory domain and a methotrexate-resistant human DHFR mutein (huDHFRFS; L22F,F31S), allowing for methotrexate selection. We do not deliver conditioning chemotherapy. The first three evaluable patients with DIPG all met feasibility for generating a balanced CD4:CD8 CAR T cell product, with 3.85x109 CAR T cells generated for S005, 4.29x109 for S008, and 2.45x109 for S012, allowing for greater than 6 months of biweekly dosing for each patient. All subjects were treated at Dose Level 1 (1x107 CAR T cells). S005 received 10 doses before clinical progression greater than 2 years from diagnosis, S008 has received 10 doses and continues on therapy with decreased tumor volume, and S012 has received 5 doses and continues on study with stable disease. There have been no dose limiting toxicities (DLT). 3/3 patients exhibited post infusion fever, headache, and elevated serum CRP but no evidence of cytokine release syndrome (CRS) or systemic CAR T cells. 0/3 patients required PICU admissions. In the cerebrospinal fluid (CSF), 2/3 patients have had elevations of cytokines such as CXCL10 and CCL2, as well as circulating CSF CAR T cells. Advanced serial patient CSF proteomic and transcriptomic profiling are underway. Ultimately, this report provides preliminary evidence that outpatient locoregional B7-H3 CAR T cells for children with DIPG may be feasible and tolerable.

CTNI-56. EVALUATING DRUG DISTRIBUTION IN CHILDREN WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) TREATED WITH CONVECTION ENHANCED DRUG DELIVERY

BACKGROUND There is currently no method for evaluating drug distribution and tumour coverage using the convection-enhanced drug delivery (CED) technique in Diffuse Intrinsic Pontine Glioma (DIPG). AIMS To determine an imaging protocol that can be used to assess the distribution of infusate in children with DIPG treated with CED of carboplatin and sodium valproate. METHODS 12 children with DIPG received between 4–18ml of infusate, through 2 pairs of catheters to encompass tumour volume on 2 days. Volumetric T2W and Diffusion Weighted Imaging (DWI) MRI sequences were performed before and after the first cycle of CED therapy and Apparent Diffusion Coefficient (ADC) maps were calculated. The tumour volume pre and post CED was automatically segmented (ITKSnap) on T2W and ADC on the basis of signal intensity. The ADC maps pre and post infusion were registered and subtracted (FSL) to visualize the infusate distribution. RESULTS ADC and T2W demonstrated a significant (&lt; 0.001) change in mean tumour volume post-infusion (mean ADC volume pre: 19.8ml, post 24.4ml; mean T2W volume pre 19.4ml, post 23.4ml). A significant correlation (p&lt; 0.001) was observed for the difference in tumour volume and the actual infused volume (ADC, r=0.76, T2W, r=0.70). There was a significant increase (p&lt; 0.001) in mean ADC and mean T2W signal intensity ratio post-infusion, no significant correlation with infusion volume. Finally, pixel-by-pixel subtraction of the ADC maps pre and post infusion visually demonstrated high signal intensity, presumed infusate coverage of the tumour. CONCLUSIONS ADC and T2W MR sequences could potentially be used to evaluate the volume of distribution of infusate delivered by CED, paramount to ensure tumour coverage and leading to more effective therapy evaluation. This will facilitate the use of CED in future clinical trials.

NIMG-11. VOLUMETRIC ENDPOINTS IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG): COMPARISON TO CROSS-SECTIONAL MEASURES AND CORRELATION WITH OUTCOMES

INTRODUCTION Cross-sectional tumor measures are used as endpoints in clinical trials of DIPG, but may not capture meaningful changes in disease burden. Volumetric measures may provide a more accurate assessment of tumor growth. We measured the correlation between cross-sectional and volumetric measures and compared their prognostic impact to better understand response evaluation in DIPG. METHODS Patients from the International DIPG Registry with diagnostic and post-radiation MRIs were included. Utilizing mint LesionTM software, tumors were manually contoured by an experienced pediatric neuro-radiologist to extrapolate cross-sectional product (CP) and volume measures. Correlation between CP and volume was assessed by linear regression. Landmark analyses were performed to determine differences in overall survival (OS) (via log-rank) between patients classified as progressive disease (PD) versus non-PD according to CP and volumetric measurements at one-, three-, and five-months post-radiation. Imaging consistent with pseudoprogression was designated non-PD. Hazard ratios (HR) for survival after these timepoints were calculated by Cox regression. RESULTS A total of 317 MRIs from 46 patients were analyzed. When comparing change from smallest previous tumor size, CP increase of 25% (PD by RAPNO) correlated with volume increase of 28% (R2=0.685). There was no difference in OS between patients classified as PD versus non-PD by CP at one-month, three-months, or five-months post-radiation (p &gt;0.05). However, significant differences in OS were observed between patients classified as PD versus non-PD by volume (28% increase) at one-month (2.7 vs. 12.8 months, p=0.005), three-months (1.9 vs. 10.7 months, p=0.036), and five-months post-radiation (3.7 vs. 9.1 months, p=0.023). PD by volume, but not by CP, was predictive of survival at all timepoints (HR: 5.0, 2.4, 2.4). CONCLUSIONS Volumetric assessments of PD correlated better with survival than CP at all post-radiation timepoints. Tumor volume likely represents a more accurate, prognostically-relevant measure of disease burden that deserves investigation in future DIPG trials.

EXTH-58. THERAPEUTIC HDAC INHIBITION IN HYPERMUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

Diffuse intrinsic pontine glioma (DIPG) continues to carry a dismal prognosis despite a growing understanding of its epigenetic regulation. While generally reclassified as diffuse midline glioma, H3 K27M-mutant (DMG), a subgroup of DIPGs do not harbor the classic histone mutation, with a further subset exhibiting a hypermutant phenotype. To evaluate whether hypermutant DIPG shares transcriptional vulnerabilities with H3K27M-mutant DMG, we screened a biopsy-derived treatment-naive PMS2 mutant DIPG model (PBT-24FH) for sensitivity to a panel of HDAC inhibitors (HDACi). In vitro evaluation of cell viability revealed the low nanomolar IC50 of quisinostat (50nM) and romidepsin (2nM). Dose-dependent increases in H3 acetylation and c-PARP were confirmed by western blot. Despite romidepsin’s superior potency in vitro, quisinostat demonstrated greater efficacy in an in vivo PBT-24FH flank study. 42 days following drug initiation, quisinostat-treated mice displayed dramatic tumor regression (mean volume= 33mm3, n= 7) compared to mice treated with romidepsin (mean volume= 669mm3, n= 7)(p= 0.005), or vehicle (mean volume= 990mm3, n= 6)(p&lt; 0.001). Immunohistochemistry of quisinostat-treated tumors revealed few residual tumor cells displaying a low proliferative index. To evaluate cross-resistance, romidepsin-treated mice (mean volume= 1158mm3, n= 2) were switched to quisinostat treatment and displayed swift tumor regression (mean volume after 25 days of quisinostat= 419mm3), emphasizing quisinostat’s in vivo cytotoxic effect against both large tumors and tumors previously treated by another HDACi. To evaluate quisinostat’s effect on other hypermutant tumors, we tested HCT-116, a colon cancer cell line bearing a biallelic MLH1 deletion and observed similar cytotoxicity. We also aim to repeat these studies utilizing additional pediatric hypermutant high grade glioma models. Transcriptomic and proteomic investigations are underway to identify the mechanism of action underlying quisinostat-induced cytotoxicity. Ultimately, we are the first to demonstrate in vivo efficacy of the HDACi quisinostat against hypermutant DIPG, supporting further investigation and clinical advancement.

EXTH-15. MULTI-FACETED INHIBITION OF TET PATHWAY WITH CELL-PERMEABLE 2HG AND BOBCAT 339 REDUCES PROLIFERATION AND INDUCES APOPTOSIS IN DIPG

Diffuse Intrinsic Pontine Glioma (DIPG) is one of the worst forms of pediatric brain tumors, with a 100% mortality-rate. A prominent mutation observed in them is a lysine to methionine change in histone3 which causes trapping of the repressive-chromatin-complex, leading to genome-level hypomethylation, affecting global epigenetic-regulation. Ten-Eleven Translocation (TET) proteins are alpha-ketoglutarate (α-KG) dependent dioxygenases that mediate the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), a key-step in removing DNA methylation-marks. We previously identified increased levels of 5hmC and TETs in DIPG. In IDH mutant glioma, the R132H-IDH produces an oncometabolite, 2-hydroxyglutarate (2HG), instead of α-KG. 2HG competitively inhibits TET function, resulting in a hypermethylated genome. Bobcat339, a cytosine-analog targets TETs by blocking the cytosine binding-site on TETs. We hypothesized that cell-permeable 2HG and Bobcat339 would synergize to block TET activity in DIPG, restore epigenetic-balance by increasing genomic methylation, and induce cell-death. 2HG induced apoptosis in DIPG cells at concentrations ranging from 100-300uM, as measured by cleaved-PARP western and cleaved-caspase3 immunofluorescence (2-7fold increase in CC3, depending on cell line). Cell-permeable 2HG also decreased proliferation, measured by phospho-RB western in DIPG cells (30-50% reduction in pRB band intensity, depending on cell line). Similarly, Bobcat339 suppressed proliferation at concentrations from 10-50uM, measured by BrdU incorporation (25% reduction in BrdU+ at 50uM, p= 0.02 compared to control). Bobcat339 induced apoptosis, measured by cPARP western and CC3 immunofluorescence (4-10fold more of CC3+ compared to control p&lt; 0.0004). In combination, cell-permeable 2HG (100-200uM) and Bobcat339 (10-20uM) combined to suppress cell-viability, measured by CellTiterBlue assay, producing ZIP scores of ~20 in DIPG cells (indicating high-level synergy). In combination, 2HG and Bobcat339 increased apoptosis in DIPG (3-fold increase in cleaved-PARP band intensity in JHH-DIPG1 cells treated with 20uM Bobcat339 + 100uM 2HG, compared to single-treated cells). Our results may lead to new approaches that target TET pathway in DIPG tumors.