bluuebunny/crossref_metadata_2025 huggingface dataset:
EPID-17. ANTIBODY REACTIVITY TO VARICELLA ZOSTER VIRUS IS ASSOCIATED WITH IMPROVED GLIOMA SURVIVAL AND IS MODIFIED BY GERMLINE HLA POLYMORPHISMS
Authors: Francis Stephen , Guerra Geno , Hansen Helen , Wiencke John , Wrensch Margaret
Published: November 2023

Antigenic reactivity towards varicella zoster virus (VZV) has shown a consistent reduction in glioma risk across multiple studies on multiple continents. To our knowledge this is the virus to be robustly associated with reduced risk of any cancer. Our recent work has shown that high VZV reactivity is also assumed with improved glioma prognosis. Using the UKBioBank we have previously shown that germline HLA polymorphism, rs9273325, significantly effects reactivity to VZV. In this study we investigate the VZV glioma survival association in the context of germline HLA polymorphisms. METHODS: We measured quantitative IgG reactivity towards VZV gE, and conducted genome wide array based genotyping in 891 adults with glioma. Associations of patient IgG levels (1st quartile vs. quartiles 2,3,4) with overall survival were estimated using Cox models adjusted for age, sex, self-reported race, surgery type, dexamethasone usage at blood draw. RESULTS: VZV seroreactivity was significantly associated with reduced mortality (HR=0.83, 95% CI: 0.70-0.99; p=0.034), as expected from our previous study. Among patients with rs9273325-GG, higher levels of VZV antibodies were associated better prognosis (HR=0.75, 0.62-0.92; p=0.005), but in the rs9273325-GA/AA strata this relationship with VZV antibodies was not observed (HR=0.94, 0.66-1.35; p=0.75). Similar results were observed in grade IV gliomas, IDHwt GBMs. CONCLUSIONS: Strong antibody response to VZV is associated with improved glioma prognosis and the effect of VZV reactivity on glioma survival is modified by a specific HLA polymorphism, a Gene x Virus interaction, that is unlikely due to chance. This study highlights the importance of accounting for HLA variation in immune related studies, including oncolytic viral therapies. Our findings also point towards a potentially MHC mediated mechanism by which VZV could be priming an immune response targeting glioma cells. Further investigations into this mechanism and VZV/shingles vaccination as a potential glioma therapeutic are warranted.
EPID-20. SEX-SPECIFIC DIFFERENCES IN MENINGIOMA SUBTYPES AND MUTATIONS – A RETROSPECTIVE ANALYSIS
Authors: Berghaus Natalie , Hielscher Thomas , Suwala Abigail , Ippen Franziska Maria , Ratliff Miriam , Herold-Mende Christel , Wick Wolfgang , von Deimling Andreas , Sahm Felix
Published: November 2023

A variety of intrinsic and extrinsic factors are known to influence and promote the development of cancer. Although the patients’ gender can influence extrinsic factors, biological sex is an important intrinsic factor. Here, it must also be considered that hormone levels change over the lives of patients. To further investigate these aspects, meningioma can serve as a prototype tumor. Differences are already obvious when analyzing the epidemiology and outcomes: Females are more frequently diagnosed with meningioma, but high-grade meningiomas and fatal outcomes are more frequently observed in males. To further resolve sex and potentially hormone-specific differences, we retrospectively examined a dataset of 798 patients (females n=541, males n=257). Additionally, we arbitrarily set a cut-off of 58 years to stratify for females between pre- and post-menopause. The male cohort was analyzed accordingly to exclude an age-dependent effect. Further, the female cohort was subdivided into “45-55 years” and “other” to analyze the impact of high hormone fluctuations within menopause. When examining the histological data, we found that women displayed the chordoid subtype more frequently than men (6.3% vs. 1.6%). In the age-dependent analyses, there may be a signal for a hormone-dependent effect, as for females this subtype was diagnosed more frequently < 58 than > 58 years of age (10.6% vs. 1.9%; males: 0% vs. 3.0%) and within the menopause (15.5% vs. 3.2%). The same pattern was noted for the TRAF7 mutation, which was detected more frequently in females (20.2% vs. 7.7%), females < 58 years (23.9% vs. 15.6%, males: 6.2% vs. 9.1%) and females within the menopause (27.9% vs. 17.6%) than in the respective other subgroups. This preliminary data warrants further research in larger, comprehensively annotated cohorts and analysis for hormone levels and receptor expression.
EPID-21. EPIDEMIOLOGY AND CLINICOPATHOLOGICAL FEATURES OF MENINGIOMA PATIENTS: FINDINGS FROM NATIONAL BRAIN CENTER HOSPITAL INDONESIA
Authors: The Yuli Felistia , Amanda Nadira Fildza , Susanto Nugroho Harry , Salam Sardiana , Prayanti Diorita , Maharani Viola , Baziad Chairunnisa , Lumempouw Silvia Francina , Miftahussurur Muhammad
Published: November 2023

BACKGROUNDS

Meningioma is considered as one of high prevalence disease, eight cases per 100.000 population, among intracranial tumours. This study aimed to assess the epidemiology and clinicopathological features of meningioma patients.

METHODS

This is a retrospective cohort study of hospitalized meningioma patients, excluding spinal meningioma, in National Brain Hospital, Jakarta, Indonesia between 2020 and 2022. Patient’s characteristics, symptoms, MRI results before surgery, and MRI results after surgery were obtained from medical records. Chi-square test, Fisher’s exact test, and logistic regression analysis performed with p-value < 0.05 was considered as significant.

RESULTS

We analysed 325 patients between 2020 and 2022, which number increases per year. Meningioma grade I was predominant than grade II and III, however grade II and III patients were slightly older than grade I patients (mean age 47.6 ± SD 13.7 years and 46.9 ± 10.3 years, respectively). The majority of patients were females than males with 4 to 1 ratio. The most common locations of brain meningioma were convexity (26.8%), followed by parasellar (11.7%), and falx (34%). Preoperative clinical symptoms in the form of muscle weakness (p=0.04), vision problem (p=0.02), hearing impairment (p=0.006), slusrred speech (p=0.003), and ptosis (0.01) were significantly related to more severe meningioma grading. However, in multivariable logistic regression only hearing impairment was significantly related with meningioma grading with odds ratio 0.313 (95% CI 0.113-0.869, p=0.02).

CONCLUSIONS

Meningioma prevalence alarming rise should get more attention. Especially as this tumor usually occurs productive age population. Clinical symptoms are associated with the severity, however further study is needed to support our finding.
EPID-23. FACILITY VOLUME STATUS IS PREDICTIVE OF SURVIVAL AND TREATMENT RECOMMENDATIONS IN PATIENTS WITH INTRACRANIAL MENINGIOMA
Authors: Brown Nolan , Chakravarti Sachiv , Kuo Cathleen , Neil Zachery , Nguyen Oanh , Mehkri Yusuf , Gendreau Julian
Published: November 2023

INTRODUCTION

There is a growing body of evidence demonstrating improved survival outcomes for patients with CNS neoplasms who are treated at high volume facilities (HVFs) as compared to low volume facilities (LVFs).

METHODS

The National Cancer Database (NCDB) was queried for adult patients who sought treatment for intracranial meningioma between 2004 and 2019 at one of 1,500 Commission on Cancer-accredited facilities. Data reported included patient sociodemographic information, patient comorbidity status, and relevant tumor characteristics. After patients were stratified by facility volume, the Mann-Whitney U and Fisher exact tests were used for bivariate analysis of both continuous and categorical variables. Multivariate Cox proportional hazard model and log-rank test were used to determine whether there was any association between facility volume and overall survival.

RESULTS

In total, 336,115 patients diagnosed with meningioma were included; 23,207 (6.9%) patients were treated at an HVF and 312,908 (93.1%) patients were treated at an LVF. On bivariate analysis, patients covered by private insurance were more likely to have been treated at an HVF (50.9% vs 36.8%, p=0.0043). Furthermore, with respect to county-based geographic trends, patients treated at HVFs were more likely to reside in an urban area (14.5% vs 11.6%, p=0.027). With respect to clinical characteristics, these patients had relatively larger tumors (4.22±10.47 cm vs. 4.11±11.03 cm, p< 0.001). On multivariate analysis, significant survival benefits were observed for patients treated at HVFs (HR = 0.977, 95% CI 0.973–0.982, p < 0.001). Patients treated at LVFs were significantly less likely to receive radiotherapy (OR = 0.988, 95% CI 0.985-0.992, p < 0.001) as compared to patients treated for intracranial meningioma at HVFs.

CONCLUSION

Our results indicate that – for patients who underwent treatment for intracranial meningioma during the 15-year study period – treatment recommendations differed and appeared to be associated with a survival disadvantage at LVFs.
EPID-25. SIX CONSECUTIVE CASES OF PRIMARY INTRACRANIAL SARCOMA IN CHILDREN IN TWO PRIVATE INSTITUTIONS FROM LIMA DURING COVID-19 PANDEMIC
Authors: Lovaton-Espadin Rolando , Alaba-Garcia Wesley
Published: November 2023

INTRODUCTION

Peruvian health system was severely impacted by COVID-19 pandemic. Public hospitals were saturated by patients with COVID-19 rapidly. Many patients had to seek medical attention for other health conditions in private institutions. This situation was also observed in pediatric patients with brain tumors. The objective of the present study was to describe clinical and neuroimaging characteristics from pediatric patients diagnosed with brain tumors in two private institutions fron Lima-Peru during COVID-19 pandemic.

METHODS

Descriptive study of cases of brain tumor in patients aged less than 18 years observed in two private institutions from Lima-Peru from March 2020 to December 2021.

RESULTS

Six consecutive cases of intracranial tumors were detected during a large part of COVID-19 pandemic's first year (October 2020 to March 2021), all of them with diagnosis of primary intracranial sarcoma . Before the pandemic, the last primary intracranial sarcoma in children operated occurred in June 2018, and was the only case that year.

CONCLUSION

The present study found an unusual frequency of primary intracranial sarcoma in children in two private institutions from Lima-Peru during COVID-19 pandemic.
EPID-27. LIVING NEAR OIL AND GAS WELLS AND MORTALITY IN CHILDREN WITH BRAIN TUMORS
Authors: Hoang Thanh , Rathod Rutu , Schraw Jeremy , Peckham-Gregory Erin , Lupo Philip , Hughes Amy , Scheurer Michael
Published: November 2023

METHODS: The heating profile of MIONPs was assessed in the test tube and mouse brain in vivo. Computed tomography scan and magnetic particle imaging were used to confirm intracranial MIONP localization after convection enhanced delivery. Cell viability assays were performed following treatment with MHT and/or radiation. MHT-induced alterations to the tumor microenvironment were assessed in a syngeneic murine glioma model, and a survival study was performed in a GBM patient-derived xenograft (PDX) model to investigate synergism between MHT and CRT.
EPID-31. BRAIN TUMORS IN UNITED STATES MILITARY VETERANS
Authors: Waite Kristin , Bihn John , Cioffi Gino , Neff Corey , Kruchko Carol , Ostrom Quinn , Swinnerton Kaitlin , Elbers Danne , Mooney Michael A , Rachlin Jacob , Stein Thor , Brophy Mary , Do Nhan , Priemer David , Perl Daniel , Ferguson Ryan , Hickman Richard , Nabors Burt , Rusiecki Jennifer , Barnholtz-Sloan Jill , Fillmore Nathanael
Published: November 2023

Veterans account for ~7% of the adult United States population, with half seeking care through the Veterans Health Administration (VHA). A comprehensive analysis of incidence and survival for brain tumors in the Veteran population has been lacking. Veteran data was obtained from VHA Medical Centers that diagnose and treat cancer via the VHA Corporate Data Warehouse. Brain tumor statistics on the overall US population were generated from the Central Brain Tumor Registry of the United States data. Cases were defined as individuals (≥18 years) with a primary brain tumor, diagnosed between 2004-2018, and identified using administrative criteria based on International Classification of Diseases for Oncology, Third Edition (ICD-O-3) topography and morphology codes. Annual age-adjusted incidence rates (AAIR) and 95% confidence intervals were estimated per 100,000 population. Kaplan-Meier survival curves were generated to evaluate overall survival outcomes among Veterans. The Veteran population was primarily white (78%), male (93%), and between 60-64 years old (18%). Individuals with a primary brain tumor in the general US population were mainly female (59%) and between 18-49 years old (28%). The overall AAIR of primary brain tumors from 2004-2018 within the VA cancer registry was 11.6. Non-malignant tumors were more common than malignant tumors (AAIR:7.19 vs 4.42). The most diagnosed tumors in Veterans were non-malignant pituitary tumors (AAIR:2.96), non-malignant meningioma (AAIR:2.62), and glioblastoma (AAIR:1.96). In general, in the Veteran population, survival outcomes became worse with age and were lowest among individuals diagnosed with glioblastoma. Differences between the Veteran and US population can be broadly attributed to the differences in the demographic composition of these groups. Prior to this study, there have been no reports on national level incidence rates and survival outcomes for Veterans. Statistics like these provide vital information that drive efforts to understand disease burden and improve outcomes for individuals with primary brain tumors.
EPCO-04. SPATIAL TRANSCRIPTOMIC ANALYSIS OF MALIGNANT PERIPHERAL NERVE SHEATH TUMORS REVEALS THERAPEUTICALLY TARGETABLE MOLECULAR SIGNATURES IN REGIONS UNDERGOING HISTOPATHOLOGIC TRANSFORMATION
Authors: Mirchia Kanish , Cho Soo-Jin , Reddy Alyssa T , Jacques Line , Pekmezci Melike , Perry Arie , Raleigh David , Vasudevan Harish
Published: November 2023

Malignant peripheral nerve sheath tumors (MPNSTs) evolve from plexiform neurofibromas (pNF) in patients with neurofibromatosis type-1 (NF-1) yet the cellular and transcriptomic mechanisms underlying this transformation remain unclear. Here, we perform spatial gene expression profiling on fifteen MPNSTs to correlate histologic observations with transcriptomic programs and identify mechanisms underlying malignant transformation. METHODS: Fifteen MPNSTs, including tumors with a histopathologically defined transition zone between adjacent low-grade and high-grade areas (n=3), were retrospectively identified. Spatial transcriptomic profiling was performed using Visium followed by downstream processing with SpaceRanger, Seurat, Harmony, and monocle. RESULTS: 50,807 unique spatial transcriptomes were distributed across 17 unique clusters, of which 12 were enriched for tumor cells and 5 for non-tumor microenvironment cells based on histologic evaluation, unsupervised cell-type assignment, marker gene expression, and cell signature gene sets. Transcriptomic clusters in low-grade areas were enriched for genes regulating mononuclear cell migration and cell-cell adhesion, while high-grade regions were enriched for Ras/Raf/MEK/ERK target genes, anti-apoptotic programs, and extracellular matrix reorganization genes. Trajectory inference beginning in low grade regions revealed spatially organized molecular signatures of malignant transformation enriched for cell cycle progression within histopathologically homogeneous regions. Moreover, the intermediate transformation zone between low-grade neurofibromatous and high-grade MPNST histopathologic regions was marked by loss of cell differentiation genes and increased cell cycle progression. CONCLUSIONS: Spatial transcriptomic analysis elucidates the cellular and gene expression patterns underlying progression from pNF to MPNST. Distinct cell populations exist across low-grade, transformation zone, and high-grade regions in MPNSTs highlighted by alterations in Ras/Raf/MEK/ERK signaling, cell differentiation and cell cycle programs, shedding light on targets for combinatorial molecular therapy to decrease the risk of malignant transformation and serving as a biomarker of aggressive peripheral nerve tumors to complement conventional histopathology.
EPCO-05. SPATIOTEMPORAL MODELLING OF GLIOBLASTOMA FOR DRUG PREDICTION
Authors: Manoharan Varsha Thoppey , Abdelkareem Aly , Osz Katalin , Chan Jennifer , Morrissy Sorana
Published: November 2023

Glioblastoma (GBM) is a lethal primary brain malignancy characterized by inevitable relapse. Understanding intra-tumoral heterogeneity and the microenvironmental influence on tumor progression is key to developing effective therapies. We employ 10X Genomics’ Visium Spatial Transcriptomics platform to study murine xenograft models of multi-regional and longitudinally derived cell lines from 4 GBM patients. This resource enables the study of tumor growth at spatial resolution and allows robust species-specific distinction of tumor and its microenvironment (TME). Using a matrix factorization approach, we delineate spatial gene expression programs (GEP) specific to the tumor and TME. We observe unique niches including progenitor cell-states along the leading edge (LE) of the tumor, oligodendrocyte-like (OC) and astrocytic-like (AC) states at the tumor core along with tumor-associated endothelial states. At early time-points, the homing of brain-resident microglia (MG), macrophages (BMDM), reactive astrocytes to the tumor site are observed. The invasiveness of GBM is a major obstacle, as these cells are therapeutically resistant and seed recurrence. Targeting the LE of GBM could therefore improve patient outcomes. Interestingly in our models, the LE state exhibits higher patient diversity; upregulation of ligand/receptor genes in secreted phosphoprotein, fibronectin, macrophage migration pathways highlighting prospective targets. Further, we analyzed protein-protein interaction networks of the LE programs, which revealed that upregulation of “hub genes” within the network significantly reduces patient survival. Further investigation of such program-specific vulnerabilities will help inform on combinatorial therapeutic strategies to control disease progression and paves the way for future testing of treatment response in both xenografts and syngeneic models.
EPCO-06. THE EPIGENETIC CIRCUIT OF HOXDERNA LONG NON-CODING RNA (LNCRNA) ACTIVATES GLIOBLASTOMA-SPECIFIC TRANSCRIPTION PROGRAMS AND SUPER-ENHANCERS GENOME-WIDE, SHAPING GLIAL CELL FATE AND TRANSFORMATION
Authors: Deforzh Evgeny , Kharel Prakash , Ivanov Pavel , Karelin Anton , Uhlmann Erik , Krichevsky Anna
Published: November 2023

The origin and genesis of highly malignant and heterogeneous glioblastoma remain unknown. We have recently reported a unique enhancer-associated lncRNA (eRNA) LINC01116 (named here HOXDeRNA) that is silent in the normal brain but is commonly activated during gliomagenesis and controls gene expression in miR-10b/HOXD locus in cis by reshaping the locus topology. CRISPR activation experiments suggested that HOXDeRNA is sufficient to transform human astrocytes into glioma-like cells. Conversely, the knock-down of this lncRNA strongly impacts glioma viability. We now describe the global, genome-wide regulatory role of HOXDeRNA. Once expressed in normal cells of astroglial lineage, HOXDeRNA binds to histone methyltransferase EZH2 and removes Polycomb repression from hundreds of genes encoding glioma-specific transcription factors and other essential oncogenes across the entire genome (in trans). Among them are the core neurodevelopmental transcription factors SOX2, OLIG2, POU3F2, and SALL2. This results in the activation of glioma transcriptional programs and downstream super-enhancers enriched for binding sites of glioma master transcription factors SOX2 and OLIG2. Moreover, HOXDeRNA-induced astrocyte transformation is accompanied by the activation of multiple oncogenes, such as EGFR, PDGFR, and BRAF. We further identified a specific structure within the HOXDeRNA that mediates its genome-wide promoter binding and gene regulatory activity as a PRC2 decoy. These results have been obtained using the combination of ChIRP-Seq, ChIP-Seq, RNA-Seq, CLIP, CRISPR activation, and base editing, and intersect our analysis of normal and cancer cells with large public datasets. Thus, we describe the first lncRNA driving a cancer-specific transcriptional program globally, altering the epigenetic landscape across all 23 chromosome pairs, and resulting in the neoplastic transformation of normal cells to glioma. Overall, the results help reconstruct the sequence of events underlying the process of astrocyte transformation and suggest new RNA-targeting approaches for glioblastoma therapies.
EPCO-09. CHARACTERIZING THE GBM CELLULAR LANDSCAPE BY LARGE-SCALE SINGLE-NUCLEUS RNA-SEQUENCING
Authors: Spitzer Avishay , Nomura Masashi , Garofano Luciano , Johnson Kevin , Nehar-Belaid Djamel , Oh Young Taek , Anderson Kevin J , Najac Ryan D , Bussema Lillian , Varn Frederick , D’Angelo Fulvio , Chowdhury Tamrin , Migliozzi Simona , Park Jong Bae , Ermini Luca , Golebiewska Anna , Niclou Simone , Das Sunit , Paek Sun Ha , Moon Hyo-Eun , Mathon Bertrand , Stefano Anna-Luisa Di , Bielle Franck , Laurenge Alice , Sanson Marc , Tanaka Shota , Saito Nobuhito , Keir Steve , Ashley David , Huse Jason , Yung W K Alfred , Lasorella Anna , Verhaak Roel , Iavarone Antonio , Tirosh Itay , Suva Mario
Published: November 2023

Tumor heterogeneity is a well-known hallmark of glioblastoma (GBM). Single-cell RNA-sequencing (scRNA-seq) technologies significantly extended our understanding of GBM intra-tumor heterogeneity. While the landmark scRNA-seq studies were limited by scale, recent technological advances have enabled analyzing increasing numbers of cells. Here, leveraging a large-scale single-nucleus RNA-sequencing (snRNA-seq) dataset containing 457,442 cells (271,444 malignant and 185,995 non-malignant) from 121 IDH-wildtype GBM tumor samples and matching whole-exome/whole-genome sequencing data, we dissected the GBM malignant and tumor microenvironment (TME) compartments with unprecedented resolution and scale. We identified three novel malignant cellular states - Glial progenitor cell-like (GPC-like), Neuron-like and Cilia-like - in addition to the cellular states previously defined in GBM (NPC-like, OPC-like, AC-like and MES-like). Cross-referencing our dataset with published scRNA-seq datasets suggests that GBM states mirror the developmental hierarchy observed in the developing human brain. Functional enrichment analysis demonstrated the heterogeneous nature of pathway and metabolic activities across the spectrum of cellular states. Analysis of the matched DNA-sequencing data revealed novel associations between the occurrence of certain genetic events and the abundance of specific cellular states. Similarly, we dissected the TME and robustly defined cellular states for different TME cell types, underscoring the heterogeneity found also within the TME compartment. Leveraging the large number of samples in our dataset and controlling for intra-tumor cell state frequency exposed three baseline gene expression programs - neuronal activity, glial development and extra-cellular matrix remodeling - termed State-Controlled Profiles (SCPs), that reflect the inter-tumor heterogeneity and are not influenced by the intra-tumor cell state distribution. Integrated analysis of the intra- and inter-tumor heterogeneity components revealed striking associations between certain TME cell types and states, malignant cell states and SCPs, suggesting that an interplay between the TME and malignant cells shapes the frequency of malignant states and baseline expression profile in each tumor.
EPCO-10. AN ONCOGENIC NEURODEVELOPMENTAL PROGRAM IN H3.3 K27M DIFFUSE MIDLINE GLIOMA INVOKES ABERRANT ASCL1-SMARCA4 EPIGENOMIC ACTIVITY
Authors: Knoepfler Paul , Klein Rachel , Yee Jennifer , Lewis Nichole
Published: November 2023

The histone variant H3.3 K27M mutation is common in diffuse midline gliomas (DMG) and is associated with poor prognosis. While some insights into K27M epigenetic functions have emerged, much less is known about how K27M impacts chromatin structure and function, and the resulting transcriptomic consequences. Recently, we developed isogenic CRISPR-edited DMG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. ATAC-seq analysis of the matched sets of isogenic WT and H3.3K27M glioma cells pointed to unique K27M-associated accessible chromatin at regions corresponding to specific neurogenesis, NOTCH, and neuronal development pathways. These domains corresponded to genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell lines. Uniquely accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are important for tumorigenesis. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway including, in particular, ASCL1 and NEUROD1. CUT&RUN for ASCL1 defined novel target genes in DMG and BioID identified key epigenomic cofactors for ASCL1 including SMARCA4. Large-scale computational analysis of both our and other groups’ data including by chromHMM identified novel functional chromatin domains and potential looping events related to K27M as well. Overall, our findings indicate that H3.3K27M changes specific chromatin functions altering neurodevelopmental gene expression resulting in aberrant activity of an oncogenic epigenomic program. Encouragingly, this program appears at least partially reversible upon editing K27M back to wild-type, pointing to potential translational impact moving forward to targeting this pathway.
EPCO-12. IN VIVO PERTURB-SEQ DEFINES BIOLOGICAL DRIVERS AND THERAPEUTIC VULNERABILITIES UNDERLYING RADIOTHERAPY RESISTANCE IN GLIOBLASTOMA
Authors: Liu John , Pak Joanna , Zou Christopher , Pan Sixuan , Payne Emily , Foster Kyla , Lin Katie , Casey-Clyde Tim , Seo Kyounghee , O'Loughlin Thomas , Wu David , Lim Daniel , Vasudevan Harish , Diaz Aaron , Ozawa Tomoko , Berger Mitchel , de Groot John , Weiss William , Gilbert Luke , Raleigh David
Published: November 2023

Glioblastomas are comprised of dynamic malignant cell states and microenvironment cell types. Mechanistic and functional studies of glioblastoma resistance to therapy have been limited by a lack of in vivo approaches for multiplexed interrogation of single cells. Here we use genome-wide and targeted CRISPR interference (CRISPRi) to identify therapeutic vulnerabilities in glioblastomas in vivo by coupling convection enhanced delivery (CED) with functional genomics and single-cell transcriptomics (perturb-seq). Human (GBM43, LN18) or mouse (GL261, SB28) glioblastomas expressing CRISPRi machinery were established intracranially in mice, and unmodified mouse glioblastomas were established intracranially in immunocompetent mice expressing CRISPRi machinery in the tumor microenvironment. Perturb-seq gene prioritization was performed using genome-wide CRISPRi screens ± radiotherapy in cell cultures. Dual-sgRNA Perturb-seq lentivirus libraries were transduced in vivo using CED, and single-cell RNA sequencing with sgRNA capture was performed ± radiotherapy. RESULTS: were validated using RNA sequencing of 254,288 single nuclei from 43 pairs of primary and recurrent patient-matched human glioblastomas. Perturb-seq ± radiotherapy of 98 genes underlying glioblastoma radiotherapy responses was performed across 414,092 single cells in vivo. Genetic perturbations reprogrammed tumor and microenvironment cell states in response to radiotherapy, and high dimensional manifolds revealed genetic dependencies in metabolic and DNA damage response pathways. The DNA-dependent protein kinase (DNA-PK) component PRKDC was identified as a driver of glioblastoma radiotherapy resistance through regulation of malignant cell-intrinsic growth and stress pathways, and regulation of malignant cell-extrinsic interferon pathways that controlled cell-cell interactions with the tumor microenvironment. PRKDC inhibition with nedisertib sensitized glioblastomas to radiotherapy and extended survival in vivo, and single-cell RNA sequencing revealed hyperactivation of cell stress and cytokine signatures with combination treatment. In summary, we report in vivo perturb-seq as a platform for simultaneous discovery and functional interrogation of therapeutic vulnerabilities in glioblastoma, and show DNA-PK inhibition sensitizes glioblastomas to radiotherapy in vivo.
EPCO-16. DISRUPTING DOT1L EPIGENETIC ACTIVITY REPROGRAMS GLIOBLASTOMA STEM CELLS TOWARDS A DOPAMINERGIC NEURONAL-LIKE STATE
Authors: Assaf Samir , Bozek Danielle , Luchman H Artee , Weiss Samuel
Published: November 2023

Glioblastoma is the most common and aggressive primary adult brain tumor, remaining incurable despite current therapeutic strategies. Through a genome-wide essentiality screen, we previously identified disruptor of telomeric silencing-1-like (DOT1L) as one of the most important epigenetic regulators in glioblastoma stem cells (GSCs). Chemogenic inhibition of DOT1L epigenetic activity was found to increase the expression of neuronal markers while decreasing the expression of cancer stem cell markers at the transcriptional and protein level, suggesting GSC differentiation towards a neuronal lineage. Thus, we sought to investigate the mechanism of DOT1L in GSCs and whether disruption of DOT1L epigenetic activity promotes functional integration of GSCs into neural circuits. Here, we profiled the overall transcriptional and chromatin accessibility landscape of GSCs following DOT1L inhibition. Interestingly, we identified that DOT1L inhibition results in increased chromatin accessibility at key gene loci involved in neural differentiation, synaptic transmission, in addition to dopamine synthesis, packaging, and release, which correlated with upregulated gene expression. Furthermore, these gene loci exhibited alterations of the DOT1L epigenetic mark, H3K79me2. Transcriptional motif analysis of GSCs following DOT1L inhibition revealed dysregulated accessibility of transcription factor motifs known to be involved in regulating cellular response to neuronal signals. Cell viability assessment of GSCs treated with a DOT1L inhibitor and a panel of neurotransmitters further confirmed that DOT1L inhibition sensitizes GSCs to neuronal signals. These data suggest that disrupting the epigenetic activity of DOT1L reprograms the epigenetic landscape of GSCs to promote a functional, dopaminergic, neuronal-like state that exhibits enhanced responsiveness to neuronal signals. Future experiments aim to investigate the impact of disrupted DOT1L epigenetic activity on GSC-neuronal bidirectional communication and the mechanisms by which this influences the progression and aggressiveness of glioblastoma.
EPCO-17. 3D GENOME STRUCTURE REGULATES TRANSCRIPTION IN PEDIATRIC HIGH-GRADE GLIOMA
Authors: Huang Tina , Wang Juan , Hu Ye , Piunti Andrea , Bartom Elizabeth , Yue Feng , Saratsis Amanda
Published: November 2023

INTRODUCTION

Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are highly morbid brain tumors. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding Histone H3. To investigate whether the H3K27M mutant protein is associated with distinct chromatin structure affecting transcription regulation, we generated the first high-resolution Hi-C and ATAC-Seq maps of pHGG cell lines, and integrated these with tissue and cell genomic data.

METHODS

We generated sequencing data from patient-derived cell lines (DIPG n=6, GBM n=3, normal n=2) and frozen tissue specimens (DIPG n=1, normal brainstem n=1). Analyses included cell line RNA-Seq, ChIP-Seq (H3K27ac, H3K27me3, H3K27M) and genome-wide chromatin conformation capture (Hi-C), as well as tissue ATAC-Seq. Publicly available pediatric glioma tissue ChIP-Seq data was integrated with cell data. CRISPR knock-down of target enhancer regions was performed.

RESULTS

We identified tumor-specific enhancers and regulatory networks for known oncogenes in DIPG and GBM. In DIPG, FOX, SOX, STAT and SMAD families were among top H3K27Ac enriched motifs. Significant differences in Topologically Associating Domains (TADs) and DNA looping were observed at OLIG2 and MYCN in H3K27M mutant DIPG, relative to wild-type GBM and normal cells. Pharmacologic treatment targeting H3K27Ac (BET and Bromodomain inhibition) altered these 3D structures. Functional analysis of differentially enriched enhancers in DIPG implicated SOX2, SUZ12, and TRIM24 as top activated upstream regulators. Distinct genomic structural variations leading to enhancer hijacking and gene co-amplification were identified at A2M, JAG2, and FLRT1.

CONCLUSION

We show genome structural variations enhancer-promoter interactions that impact gene expression in pHGG in the presence and absence of the H3K27M mutation. Our results imply that tridimensional genome alterations may play a critical role in the pHGG epigenetic landscape and thereby contribute to pediatric gliomagenesis. Further studies examining the impact of the alterations is therefore underway.
EPCO-19. TUMOR-WIDE NEOANTIGEN-SPECIFIC T-CELLS INFILTRATING MUTANT IDH1 LOW-GRADE GLIOMAS AND PERSISTING IN PERIPHERAL BLOOD ALLOW FOR PERSONALIZED TCR-BASED IMMUNOTHERAPIES
Authors: Kwok Darwin , Zhang Michael , Wang Cliff , Stevers Nicholas , Borrman Tyler , Pan Zheng , Yuen Benjamin , Peng Songming , Nguyen Diana , Martin Michael , Hong Chibo , Hilz Stephanie , Phillips Joanna , Shai Anny , Bush Nancy Ann Oberheim , Hervey-Jumper Shawn , McDermott Michael , Mandl Stefanie , Okada Hideho , Costello Joseph
Published: November 2023

BACKGROUND

The low mutational burden and immunologically “cold” microenvironment of mutant IDH1 low-grade gliomas (LGG) are considerable challenges in immunotherapy for these tumor types. However, we hypothesize that LGG-targeting T-cells may exist at low frequency and with limited regional infiltration within the tumor. Through multi-region tumor sampling coupled with high-throughput T-cell receptor (TCR) profiling, we identified tumor-wide neoantigens and corresponding neoantigen-specific T-cells regionally infiltrating the tumor and persisting in peripheral blood.

METHODS

Maximally-distanced anatomical sampling of at least 10 distinct tumor regions was performed at the initial resection for three WHO Grade II diffuse astrocytoma patients for exome-based prediction of clonally and subclonally expressed neoantigens, RNAseq analysis of regional immune cell composition, and TCR beta deep sequencing. We used these predictions to generate a barcoded library of patient-specific peptide-HLA multimers loaded with predicted neoepitopes. With this library, neoantigen-specific CD8+ T-cells were captured and isolated from patients’ peripheral blood. Single-cell TCR sequencing allowed us to identify the neoantigen-reactive TCR clonotypes which were transduced subsequently into Jurkat76 cell lines for functional validation.

RESULTS

We screened patient-derived peripheral blood drawn two years after initial resection in 3 mutant IDH1 LGG patients and detected a total of 20 TCR clonotypes recognizing neoepitopes derived from truncal, tumor-wide mutations in CNTNAP1 (n=8), TP53 (n=3), and MRPL46 (n=2) as well as subclonal mutations in PRMT5 (n=1) and ZDHHC5 (n=6). Jurkat76 cells transduced with the mutant-PRMT5-specific TCR demonstrated dose-dependent neoantigen-specific immune responses when co-cultured with mutant-PRMT5 pulsed-antigen presenting cells expressing HLA-A*0201.

CONCLUSION

Our study demonstrates the existence and persistence of neoantigen-targeting T-cells within the blood and tumor of mutant IDH1 LGG patients. We identified a TCR clonotype that successfully recognizes and induces an immune response against mutant-PRMT5. These findings suggest a feasible methodology to develop personalized T-cell-based immunotherapies for patients with mutant IDH1 LGGs.
EPCO-24. DNA METHYLATION-BASED SUBCLASS PREDICTS THE EFFECT OF THE EXTENT OF RESECTION ON SURVIVAL IN IDH-WILDTYPE GLIOBLASTOMA
Authors: Dankmeijer Luuk , Westerink Lucas , Wesseling Pieter , Hamer Philip de Witt , Kouwenhoven Mathilde , Kooi Evert-Jan , Verburg Niels
Published: November 2023

BACKGROUND

DNA methylation-based classification of diffuse glioma is increasingly used in daily practice. Yet, little is known regarding the prognostic and predictive value of DNA methylation-based subclasses of IDH-wildtype glioblastoma. A recent study found a predictive value for the effect of extent or resection (EOR) on survival for DNA methylation-based subclasses using the v11.4 classifier. We set out to validate these findings in an independent cohort and to test if these results are similar using a more recent version (v12.5) of the classifier.

METHODS

This concerns a single-center retrospective cohort study of all patients with a glioblastoma, IDH-wildtype of whom DNA methylation was obtained. Patient and tumor characteristics, pre-operative Karnofsky performance status and EOR (complete, subtotal, partial, biopsy) were retrieved. DNA methylation-based classification and MGMT-promoter status were obtained using version 11.4 and 12.5 of the Heidelberg classifier. Progression-free survival (PFS) and overall survival (OS) were collected and validated using the national brain tumor registry.

RESULTS

Of 149 patients, the subtype was Mesenchymal in 57 (38%), RTK1 in 41 (28%), and RTK2 in 51 patients (34%). PFS and OS did not differ between the three subclasses, also not after pooling RTK1 and RTK2 into one Classical subclass. Complete or subtotal resection resulted in a longer OS than partial resection or biopsy in the entire cohort. This was similar for the pooled Classical subclass. In the Mesenchymal subclass, however, there was no survival advantage for complete or subtotal resection over partial resection or biopsy. These results were similar with the 11.4 and 12.5 classifier version.

CONCLUSION

In IDH-wildtype glioblastoma of the Mesenchymal subclass complete or subtotal resection does not result in a survival benefit. Our findings evoke the need for pre- or intraoperative assessment of the DNA-methylation based subclass of IDH-wildtype glioblastoma.
EPCO-26. INTRATUMORAL HETEROGENEITY OF GBM IDENTIFIED AND CHARACTERIZED BY A MULTISAMPLING APPROACH AND METHYLATION PROFILING
Authors: Singh Omkar , Abdullaev Zied , Matsumoto Yuji , Brem Steven , O'Rourke Donald M , Davatzikos Christos , Aldape Kenneth , Nasrallah MacLean P
Published: November 2023

Intratumoral heterogeneity in glioblastoma is known and is considered a key feature of GBM’s clinical aggressiveness. To more fully characterize this phenomenon, we conducted methylation profiling on the EPIC array for 79 regions from 20 glioblastomas. Using the Heidelberg classifier, each of the 20 tumors had at least 1 region with a match to a GBM subtype with >0.84 score. Among these, 6 tumors (30%) showed homogeneous-high score matches to the same GBM subclass, while the remaining 14 tumors (70%) showed heterogeneity in GBM subclass, with either lower scores, or disparate matches to different glioma subtypes. Comparing these subclass-homogeneous versus subclass-heterogeneous groups of tumors using methylCIBERSORT deconvolution, the subclass-heterogeneous cases showed a significantly higher proportions of CD8+ T cells and microglia, and significantly lower proportions of CD4+ T cells, B cells and monocytes. While low tumor purity can be a cause of lower confidence scores in the methylation classifier, we did not find that the subclass-heterogeneous tumors had lower tumor purity compared to subclass-homogeneous cases, suggesting that this heterogeneity was due to factors other than tumor purity. Within the subclass-heterogeneous tumors we identified the common intratumorally most variable probes and examined their characteristics. Compared to the overall distribution of probes on the array, these intratumoral-variable probes were significantly more likely to be intergenic and located on enhancers. Conversely, they were significantly less likely to be on gene promoters and CpG islands. Gene ontology analysis showed that genes associated with these probes were enriched for pathways involved in DNA repair. Overall, our multisampling methylation profiling analysis identifies specific biologic correlates of intratumoral heterogeneity that is not simply due to tumor purity. Methylation changes associated with intratumoral heterogeneity converge on specific genomic patterns that warrant further study.
EPCO-28. MULTIPLATFORM MOLECULAR AND FUNCTIONAL GENOMIC ANALYSES OF NF1 MUTANT GLIOBLASTOMA IDENTIFY DISTINCT CO-MUTATION PATTERNS AND DRUGGABLE DEPENDENCIES UNDERLYING SELUMETINIB RESPONSE
Authors: Mirchia Kanish , Payne Emily , Pan Sixuan , Al-Adli Nadeem , Young Jacob , Chen William , Pak Joanna , Seo Kyounghee , Ozawa Tomoko , Reddy Alyssa T , Perry Arie , Liu John , Berger Mitchel , Raleigh David , Vasudevan Harish
Published: November 2023

The tumor suppressor NF1 is recurrently mutated in sporadic IDH-wildtype glioblastoma and activates Ras/Raf/MEK signaling, motivating the use of targeted MEK inhibition. Here, we combine multiplatform molecular analysis of NF1 mutant glioblastoma human resection specimens with functional genomic CRISPRi screens to the MEK inhibitor selumetinib. METHODS: We identified 123 consecutive patients treated at the University of California San Francisco between 2016-2022 with NF1 mutant, IDH-wildtype glioblastoma. DNA methylation data was processed using the minfi package, and hierarchical clustering, principal component analysis (PCA), and survival analysis was performed in R. Human NF1 mutant GBM43 and mouse NRAS mutant SB28 GBM cells expressing CRISPRi machinery were used for genome wide selumetinib-resistance screens. Immunoblotting was performed to validate CRISPRi screen hits and selumetinib responses in cell lines. RESULTS: DNA sequencing of human glioblastomas identified 12 recurrent alterations in greater than 10% of cases including cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic regulators (ATRX, SETD2), and Ras signaling (EGFR, PDGFRA, PTPN11). Methylation data (n=55 tumors) identified three groups marked by differences in MGMT methylation (p=0.04, chi-square test) and CDKN2A/B loss (p=0.01, chi-square test). CRISPRi screens in GBM43 and SB28 cells identified driver phenotypes in cell cycle regulators (TP53, CDKN2A, CDK4), PI3K signaling (PTEN, MTOR), and Ras signaling (RASA2, PTPN11) consistent with co-occurring mutations in the human NF1 mutant glioblastoma cohort. In contrast, mediators of selumetinib response converged on regulators of Ras/Raf/MEK signaling including the Ras effector SHOC2. Biochemical analysis validated SHOC2 repression is sufficient for selumetinib sensitization. CONCLUSIONS: Using a combination of human tumor genomic analysis, functional genetic screens, and biochemical validation, we identify three epigenetic groups of NF1 mutant glioblastoma with distinct co-mutation patterns and validate compensatory Ras/Raf/MEK activation as a conserved mechanism of selumetinib resistance, suggesting combination molecular therapy as a rational therapeutic combination.
EPCO-30. UNCOVERING MECHANISMS OF RESISTANCE IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG)
Authors: Mukherji Devarshi , Haase Santiago , Welch Joshua , Lowenstein Pedro , Castro Maria
Published: November 2023

Pediatric high-grade gliomas (pHGGs) are currently the leading cause of cancer-related deaths in children, remaining incurable in most cases. A molecular subgroup of pHGGs harbor G34R mutations in the histone H3.3, although the mechanisms of the H3.3-G34R/V are not well established. In most of the patients, the tumors resist radiation and relapse after standard of care (SOC). We aim to study the molecular mechanisms mediating resistance in these tumors. Knowing why certain tumors resist treatment allows us to implement targeted therapies. We performed single cell RNA sequencing on 3 non-treated (NT) glioma bearing mice, 3 mice resistant to treatment (R), and 3 mice susceptible to treatment (S). We isolated the tumor tissue (including tumor microenvironment) and processed the cells for single cell RNA sequencing. Using R, the single-cell RNA sequencing data was converted to Seurat datasets for analysis and subsequently combined and clustered using UMAP. We used the ScType and SingleR software tools to annotate cell types. After cell annotation, Gene Set Enrichment Analysis (GSEA) was performed between R and S tumors for each cell cluster as well as NT vs Treated. GSEA showed that susceptible tumor cells showed increased immune responses when compared to resistant cells. Additionally, resistant cells showed increased neuronal phenotypes as well as suppression of the ERK 1/2 cascade pathway in comparison to susceptible cells. There were also numerous differences in immune cell subpopulations, which may help uncover mechanisms of resistance. For example, certain immune cells show signs of increased epigenetic modifications in resistant vs. susceptible glioma cells. Our results indicate that the resistance of pHGG to treatment is associated with the establishment of immunosuppressive signals from tumor cells, and the suppression of tumor growth mediated by downregulation of the MAPK/ERK cascade.
EPCO-33. H3K4 METHYLATION IS A PREREQUISITE OF CELL FATE CHANGE DURING IDH-MUTANT GLIOMAGENESIS
Authors: Koch Marilin , Deo Minh , Turcan Sevin , Hoetker Michael , Herold-Mende Christel , Wick Wolfgang
Published: November 2023

RATIONALE: Low grade gliomas (LGGs) are defined by isocitrate dehydrogenase (IDH)-mutations that mediate broad epigenetic changes. However, additional determinants of low-grade gliomagenesis remain unknown, including factors necessary for the activation of LGG gene programs. Here, we aim at identifying epigenetic prerequisites of IDH-dependent gliomagenesis by modulating key chromatin modifiers in a conditionally IDHR132H-expressing immortalized human astrocyte (IHA) model system. METHODS: Using chemical compounds, we targeted key histone modifications and DNA methylation in an inducibly IDHR132H expressing IHA cell line. Flow cytometry for the LGG-associated marker L1CAM served as a primary read-out. Compound candidates were further assessed with RNA-Seq and mass spectrometry. Phenotypic evaluation included sphere formation, clonogenicity, cell viability and invasion assays. Relevant findings were additionally assessed in established LGG cell lines TS603, NCH612 and NCH1681. RESULTS: L1CAM was significantly altered by DOT1L inhibitor EPZ00477, MLL1 inhibitor MM-102, and EZH2 inhibitor GSK-126. MM-102 treatment was associated with broad transcriptional downregulation. Intersecting data from MM-102 and GSK126 treated cells, we identified potential bivalent genes directly affected by inhibitor treatment that were enriched for Wnt signaling and developmental transcription factors implicated in gliomagenesis. Overlapping transcriptional and proteomics data, MM-102-treated cells exhibited a signature of predominantly downregulated genes associated with invasion/migration, extracellular matrix (ECM) and cholesterol metabolism. Fittingly, cells showed impaired invasion, clonal proliferation and sphere forming capacity. The negative effect on sphere formation tracked inversely with aggressiveness of established LGG cell lines. CONCLUSION: Interfering with histone H3K4 methylation through MLL1 inhibition is associated with broad downregulation of gene sets related to cell identity and ECM/migration, reflected by disruption of clonal proliferation in IDHR132H astrocytes and LGG cell lines. This points to deposition of H3K4 methylation as a prerequisite of cell fate change in LGG development. Persisting effects on established LGG cell lines warrant its investigation as a potential therapeutic target.