bluuebunny/crossref_metadata_2025_split huggingface dataset:
EPCO-19. MULTIOMIC PROFILING OF EPIGENETIC HETEROGENEITY IN GLIOBLASTOMA AT SINGLE-CELL RESOLUTION USING MULTIOMIC SINGLE-CELL CUT&TAG
Authors: Hardigan Andrew , Blandford Emily , Mohan Aditya , Moelker Eliese , Fecci Peter , Patel Anoop 
Published: November 2024

Abstract

INTRODUCTION
Recent evidence suggests epigenetic heterogeneity may be the primary driver of cell phenotype and treatment resistance in glioblastoma (GBM). Epigenetic heterogeneity is defined by alterations of chromatin histone modifications with activating marks such as H3K27 acetylation and repressive marks such as H3K27 trimethylation. Combinations of these histone marks govern the activity of functional genomic elements such as enhancers or promoters. Single-cell (“sc-”) technologies have only recently been adapted to the study of epigenetic cell states and significant technical challenges have limited their widespread adoption, especially in complex tissues. Here, we report the development of an optimized method for performing tissue processing and simultaneous single-cell epigenetic and transcriptomic analyses in flash frozen GBM samples.

METHODS
Building on our prior work, we developed a new protocol, multiomic single-cell cleavage-under-targets and tagmentation, or “mscCUT&TAG”. We optimized nuclei isolation, cleanup and permeabilization from flash frozen GBM and reaction conditions for the scCUT&TAG portion of the assay. Critically, these changes maintained high-quality, intact nuclei in suspension while limiting well-described technical issues such as nuclei clumping and sample loss. Furthermore, we incorporated the ability to multiplex both patient samples and histone marks.

RESULTS
We applied 10X scRNA-seq, 10X Multiome (RNA+ATAC-seq), and mscCUT&TAG for five histone marks to the same GBM sample, enabling an integrated evaluation of the tumor’s transcriptome, chromatin accessibility and epigenetic regulation at single-cell resolution. mscCUT&TAG enabled the first assessment of single-cell genome segmentation in GBM. We integrated across modalities to identify specific tumor cell populations, cell-type specific coordinated changes in epigenetics and transcriptome, and define functional regulatory elements.

CONCLUSIONS
We developed a novel multiomic protocol, mscCUT&TAG, that allows for a comprehensive analysis of epigenetic heterogeneity in GBM. We anticipate this protocol will enhance our understanding of fundamental biology and potential therapeutic avenues in GBM.
EPCO-30. TRANSCRIPTOMIC ANALYSIS IDENTIFIES REGIONAL BIOLOGY AND OUTCOME HETEROGENEITY IN MEDULLOBLASTOMA AND EPENDYMOMA SUBTYPES
Authors: Arora Sonali , Nuechterlein Nicholas , Jensen Matt , Glatzer Gregory , Sahm Felix , Mack Stephen , Taylor Michael , Holland Eric 
Published: November 2024

Abstract
Medulloblastoma, the most common malignant pediatric brain tumor, includes four distinct subtypes: Wnt, Sonic Hedgehog (Shh), Group 3, and Group 4. In contrast, ependymomas, which account for 10% of pediatric brain tumors and 4% of adult brain tumors, are categorized into three types: supratentorial (ST), posterior fossa (PF), and spinal (SP). While molecular subgrouping has significantly advanced the classification of these diseases, the extent of heterogeneity within these subgroups remains unknown. To address this, we collected bulk RNA sequencing (RNASeq) data from 888 medulloblastoma and 370 ependymoma from various institutions in North America and Europe. Our goal was to establish a comprehensive reference landscape for both medulloblastoma and ependymoma. We also included 42 forebrain and 52 hindbrain fetal samples to contrast against the tumor samples. Our analysis revealed that each disease subtype forms distinct clusters. Previously, subtyping was primarily achieved using methylation data; however, we demonstrate that bulk RNASeq data can also accurately identify disease subtypes. By examining gene expression patterns, gene fusions, and copy number aberrations, we validated existing molecular features and discovered new ones for each subtype. Additionally, we developed an innovative pipeline to identify specific biological patterns for a given region within the landscape. This resource coupled with mapping of new patients’ bulk RNASeq data onto the reference landscape, facilitating accurate disease subtype identification for new patients. This extensive dataset, available through the online tool Oncoscape, will assist the scientific community in uncovering novel therapeutic targets and advancing research in pediatric brain tumors.
EPCO-44. SPATIAL TRANSCRIPTOMICS ENABLES STATISTICAL MODELING OF SEX DIFFERENCE IN GENE CO-EXPRESSION NETWORK AND IMMUNE LANDSCAPE OF GLIOBLASTOMA
Authors: Hao Yue , Baker Angela , Reid George , Luo Jingqin , Hilbush Brian , Otero Marcos , Streck Christopher , Fink Karen L , Snipes George J , Mickey Bruce E , Sloan Andrew E , Barnholtz-Sloan Jill S , Berens Michael E 
Published: November 2024

Abstract
Glioblastoma (GBM) is characterized by inter- and intra-tumor molecular heterogeneity and demonstrated cell plasticity. Sex differences have been observed in various aspects: a male:female GBM incidence ratio is 1.6:1; standard of care treatments (debulking, radiation, and temozolomide) benefit females more than males. 14 human GBM samples from two study sites were subject to spatial profiling on the Vizgen Merscope platform to examine whether distinct biological and therapeutic niches in GBM were evident, to understand how the tumor microenvironments (TME) contributed to the heterogeneity of GBM, and to interrogate sex difference in the immune landscape of GBM. Multiple sets of spatial probes were designed to capture the cell identities (cancer stem cells, astrocytes, oligodendrocytes, microglia, macrophages, etc.) and the spatially-resolved expression patterns of genes with different functions. Two of the 14 GBM samples were also profiled on another FISH-based spatial platform (Veranome). The data from the two platforms exhibited a linear correlation. We explored the gene-gene co-expression network, and calculated the interaction probabilities between genes which were then compared for sex difference. We found that female samples showed significantly lower (two-fold) CD4+/CD8A+ T-cell count ratios than male samples. We also observed sample-specific spatial niches of different GBM cell subtypes and immune cell populations. Our large-scale spatial transcriptomics study revealed intrinsic sex-based differences in immune response to tumor and illuminated microenvironmental influences on GBM.
EPCO-45. COMPLETE CHROMOSOME-SCALE DIPLOID-PHASED ASSEMBLY OF A NORMAL HUMAN ASTROCYTE CELL LINE
Authors: Ranallo-Benavidez T Rhyker , Hao Yue , Fukushima Noelle , Mbegbu Ogechukwu , Barthel Floris P 
Published: November 2024

Abstract
Genomic studies of the malignant brain tumor glioma invariably rely on aligning sequencing reads to a reference genome such as HG38, even though no single reference can capture the diversity of an individual patient or cell line. To begin addressing these biases in genomics research, we sought to construct a chromosome-scale diploid-phased assembly of a normal human astrocyte (NHA) cell line that is frequently used as a basis for developing isogenic glioma cell models. To achieve this, we generated 45x Pacific Biosciences HiFi data (N50: 16kb), 26x Oxford Nanopore Technologies ultra-long data (N50: 80kb), and 2 billion paired-end reads of Hi-C data. The assembler verkko2 was used to integrate these data and build a phased diploid assembly. We achieved an assembly size of 6.01 Gbp, with 17 gapless chromosomes. The two haplotypes contain 214 and 182 contigs, respectively. Our assembly includes 76 fully assembled telomeres and 20 fully assembled centromeres. The contiguity of our assembly is 130 Mbp, which vastly exceeds that of HG38 (N50: 68 Mbp) and approaches that of the recent telomere-to-telomere CHM13 assembly (N50: 155 Mbp). We ran BUSCO on each haplotype of the assembly using the metazoa database to quantify the single-copy genes that are expected to be present in every animal genome. The haplotypes of our assembly contain the complete sequence of 98.6% and 99.9% of these genes respectively. Finally, we used Merqury to perform a k-mer based evaluation of the two haplotypes and found quality values of 59.09 and 59.75 respectively. This diploid genome assembly of NHA will serve as a resource for researchers that wish to develop NHA-derived glioma models. It can be used to call indels or structural variants in a haplotype-specific manner which will further elucidate genotypes underlying glioma phenotypes. Most importantly, it sets the stage for personalized genome assembly and tackling the lack of diversity in genomic studies of glioma to date.
EPCO-47. SEX-BIASED BRD4 ACTIVITY DRIVES TUMORIGENESIS IN GLIOBLASTOMA
Authors: Powell Ruby , Beaumont Thomas , Kfoury-Beaumont Najla 
Published: November 2024

Abstract
Consistent sex differences in incidence and outcome have been reported in cancers including brain tumors. Glioblastoma (GBM), the most common and aggressive primary brain tumor, occurs with higher incidence and shorter survival in males compared to females. Brd4 is essential for regulating transcriptome-wide gene expression and specifying cell identity. Using an isogenic murine GBM model, we have reported correlated transcriptome-wide sex differences in gene expression, Brd4-bound enhancer usage, and Brd4 localization to oncogenic and tumor suppressor transcription factors binding sites in male and female GBM cells, respectively. Sex-biased gene expression patterns were also evident in human glioblastoma. Inhibiting Brd4 function abrogates sex differences in these transcriptional states and concomitant differences in tumorigenic phenotype. Thus, sex-biased Brd4 activity drives sex differences in GBM rendering males and females differentially sensitive to BET inhibitors. These data indicate that Brd4 can exhibit dual functions as an oncogene or tumor suppressor, dependent upon cellular sex. To better understand how to globally target Brd4, we interrogated the role played by master transcriptional Brd4 regulators in coordinating this dual phenotype in GBM. Motif-based analysis revealed that Brd4 co-localized with oncogenes such as Bach1, and Klf5 at male-biased enhancers whereas female-biased enhancers were co-occupied with tumor suppressors, such as Smad4 and p53. Knockdown of Bach1 decreased clonogenic frequency in males and females. Conversely, knockdown of Smad4 increased proliferation and clonogenicity in males, further inducing the male tumorigenic phenotype, while no change was observed in females. These results indicate that Smad4 might be necessary but not sufficient to provide females with resistance to transformation. Ongoing epitranscriptomics investigating Brd4 re-localization and enhancer usage in knockdown cells will increase our understanding of Brd4 mechanism in GBM. Targeting sex-biased Brd4 function will identify novel combinatorial sex-specific therapeutics for brain tumors and could have immediate impact on the clinical treatment of GBM patients.
EPCO-51. A MULTI-OMIC ANALYSIS OF REGIONAL HETEROGENEITY IN GLIOBLASTOMA
Authors: Ellenbogen Yosef , Landry Alexander , Liu Jeff , Wu Yiyan , Gaiti Federico , Zadeh Gelareh 
Published: November 2024

Abstract
Glioblastoma (GBM) is the most common malignant brain tumor in adults. A hallmark of GBM is its intratumoral heterogeneity as well as infiltration into the surrounding brain. The growing understanding of the cellular diversity and cellular state diversity within GBM necessitates a need for a more granular evaluation of the molecular landscape of this disease. This study aimed to investigate this using single cell RNA sequencing combined with single-cell ATAC sequencing and spatial transcriptomics in order to delineate cellular states and enriched pathways between malignant cells in different regions of GBM. The study cohort consisted of 15 patients with primary GBM. Tumor samples were taken at the time of surgical resection using intraoperative stereotactic navigation from three anatomically distinct locations: periphery – the cortex or white matter in the peri-tumoral region that is beyond contrast-enhancing tumor, border – the contrast-enhancing border of the tumor on T1-weighted MRI, and core - the hypointense core region on T1-weighted MRI. Samples underwent combined snRNA and ATAC sequencing (10x Multiome) as well as spatial transcriptomics (10x Visium). Single-nucleus RNA + ATAC sequencing captured a total of 37,547 neoplastic cells and 18,498 non-neoplastic cells within the tumor microenvironment. The majority of non-neoplastic cells were identified within the periphery alongside a distinct group of neoplastic cells that exhibited unique a unique distribution of malignant cellular state and differentially regulated genetic pathways compared to malignant cells within the tumor border and core. Gene regulatory analysis of the malignant cells in the periphery identified a previously undefined set of enriched transcription factors that are periphery-specific regulon drivers. Overall, this analysis sheds light on the genetic and epigenetic differences between malignant GBM cells based on their location and warrant further investigation into the biology and targetable aspects of the malignant cells that exist beyond the contrast-enhancing border of the tumor.
EPCO-60. EXPRESSION OF EARLY PROGENITOR MARKERS WITHIN NF2-ASSOCIATED SPINAL EPENDYMOMA
Authors: Burket Noah , Wang Jia , Gao Hongyu , Bell Robert , Zhang Chi , Liu Yunlong , Clapp Wade , Tailor Jignesh 
Published: November 2024

Abstract
Spinal ependymomas (SP-EPNs) are intramedullary spinal tumors commonly found in patients with NF2-related schwannomatosis. Recent work using bulkRNA sequencing has suggested that SP-EPNs express a molecular signature most similar to ependymal cells. However, this type of sequencing may miss rare stem cell populations within tumors. The aim of this study was to assess the spatial heterogeneity within NF2-associated SP-EPNs and explore whether a stem cell population exists within SP-EPNs. Spatial transcriptomics (ST) was performed on a SP-EPN sample previously resected from a patient with NF2-related schwannomatosis using the 10X Genomics Visium Cytassist Platform. Libraries were sequenced on the Illumina NovaSeq 6000. 28-bp reads including spatial barcode and UMI sequences and 50-bp probe reads were generated with Illumina NovaSeq 6000 at the Center for Medical Genomics at Indiana University School of Medicine. Space Ranger 2.1 was used to process the raw files after sequencing. Data analysis was performed with R package Seurat 4.9.9.9041 following preprocessing with spaceranger. Sctransform normalized the spatial transcriptome dataset. Clusters were identified using “FindClusters” and “FindNeighbors” functions. There was diffuse expression of astrocyte and ependymal cell markers throughout the SP-EPN sample with smaller areas expressing radial glia and stem cell markers, as well as overlap between these regions. Expression of vascular and connective tissue markers varied throughout the tumor. There was minimal to no expression of neural genes in the sample. ST on our SP-EPN sample revealed a tumor with marked cellular and molecular heterogeneity. Although the primarily expressed ependymal cell and astrocyte markers, there does appear to be small, stem cell-like populations. This suggests that NF2-associated SP-EPNs may have a developmental hierarchy with radial glia-like cells and more mature progenitors within the ependymal cell lineage. This proof-of-concept study offers important findings that provide justification for further investigation using single-cell sequencing.
EPCO-61. THE LANDSCAPE OF ALTERNATIVE SPLICING IN PEDIATRIC GLIOMA
Authors: Geng Baobao , Wang Lin 
Published: November 2024

Abstract
Recent studies have identified alternative splicing (AS) as a novel source of neoantigens for immunotherapy. Surprisingly little is known about the AS milieu in Pediatric glioma, including diffuse intrinsic pontine glioma (DIPG), pediatric high grade glioma (pHGG) and medulloblastoma. We analyzed 20 primary DIPG, 22 pHGG and 20 medulloblastoma human specimens via RNA sequencing and re-analyzed RNA-sequencing data from non-malignant human brain tissues. From these data, we reconstructed the landscape of AS in Pediatric glioma and contrasted that to observed isoforms in non-malignant brain. We identified novel splicing events in cell-surface proteins that are suitable targets for engineered T-cell therapies in DIPG, pHGG and medulloblastoma. We also identified novel splicing events derived neoantigens in DIPG, pHGG and medulloblastoma respectively, which are potential immunotherapy targets. We found DIPG specific isoforms of Platelet-derived growth factor pathway genes which are expressed exclusively by DIPG tumor cells. Our studies shed light on the effect of immunotherapy on AS and identify novel targets for emerging immunotherapies in pediatric glioma.
ANGI-13. LONG NON-CODING RNALINC03045 REGULATES GLIOBLASTOMA INVASION VIAWASF3
Authors: Deshpande Krutika , Doan Tammy , Tsung Kathleen , Liu Kristie , Han Jane , Loh Eddie , Ding Li , Yang Wentao , Neman Josh , Dou Yali , Attenello Frank 
Published: November 2024

Abstract
Glioblastoma (GBM) is an aggressive, heterogeneous tumor characterized by extensive infiltration of tumor cells within the surrounding brain parenchyma. Invasive tumor cells that evade surgical resection, chemoradiation and immune responses underlie GBM recurrence, necessitating the evaluation of factors that govern them. A recently completed unbiased lncRNA CRISPRi screen done by our lab identified LINC03045 as the top potential regulator of GBM invasion in Matrigel assays. Since enhanced LINC03045 expression was also found to be significantly associated with glioma tumor grade and patient survival (p&lt;0.0001), here we characterized potential mechanisms by which it regulates GBM invasion. Mediators of lncRNA effect were identified via differential expression analysis in LINC03045 knockdown glioma cells and TCGA glioma datasets, and assessed for tumor invasion in 2D and 3D assays using knockdown and rescue experiments. Differential expression analysis of protein-coding genes associated with reduced LINC03045 expression in vitro and in GBM TCGA RNAseq data revealed high positive correlation with WASF3 (p=0.016), a regulator of JAK-STAT signaling and cytoskeletal scaffolding. siRNA-mediated WASF3 knockdown was associated with significant decrease in invasion in 2D-Matrigel assays in U87 (16.4%, p&lt;0.001), U251(27.3%, p&lt;0.001) and patient-derived USC02 (25.7%, p&lt;0.001) glioma cells. Additionally, 3D invasion assays showed reduced invasion of WASF3KD U87 gliomaspheres (41.1%, p=0.011) into the surrounding matrix. Finally, WASF3 overexpression in USC02s demonstrated rescue of invasive capacity lost with LINC03045 knockdown (p&lt;0.01). Taken together, RNA-seq analysis of downstream effectors of LINC03045, coupled with tumor invasion studies establish downstream mediator WASF3 as a novel regulator of GBM invasive potential.
BIOM-12. ALLIANCE A071401: GENOMICALLY GUIDED PHASE II TRIAL OF ABEMACICLIB IN PATIENTS WITH GRADE 2/3 MENINGIOMAS HARBORING SOMATIC NF2 OR CDK PATHWAY ALTERATIONS
Authors: Brastianos Priscilla , Dooley Katharine , Geyer Susan , Gerstner Elizabeth , Kaufmann Timothy , Iafrate A John , Martinez-Lage Maria , Milhem Mohammed , Welch Mary , Kaley Thomas , Drappatz Jan , Chan Amy , Kumthekar Priya , Matsuoka Carlos Kamiya , Strowd Roy , Cohen Adam , Jaeckle Kurt , Robell Lindsay , Magge Rajiv , Nam Joo Yeon , Blondin Nicholas , Shaikh Nawal , Rabinowitz Ian , Thomas Alissa , Cahill Daniel , Santagata Sandro , Barker Frederick , Galanis Evanthia 
Published: November 2024

Abstract

BACKGROUND
Patients with meningiomas that have progressed after surgery and/or radiation have limited treatments. Loss of NF2 and CDKN2A/B are common in higher-grade meningiomas and promote meningioma progression. We evaluated the efficacy of abemaciclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, as part of Alliance A071401, a genomically-driven phase II trial in recurrent or progressive meningiomas.

METHODS
Eligible patients (pts) with grade 2/3 tumors and NF2 mutations or CDK pathway alterations were treated with abemaciclib 200 mg orally twice daily. Two co-primary endpoints were used: progression-free survival at 6 months (PFS6) and response rate (RR); the trial would be declared positive if either endpoint was met. Twenty-four evaluable pts provided ≥85% power to detect a PFS6 ≥41.5% (vs. null 15%; alpha=0.02), and ≥89% power to detect RR ≥20% (vs. null 2.5%; alpha=0.021).

RESULTS
Of 83 pts screened while the abemaciclib arm was open between September 15, 2021 and October 3, 2022, 36 eligible pts received abemaciclib. The observed PFS6 rate in the first 24 evaluable pts was 54% (95% CI 33-75%), thus the study met PFS6 endpoint. Of all 35 evaluable pts, median PFS was 7.6 months (95% CI 3.6-17.1 months), estimated 6-month PFS was 55% (95% CI 40-75%) and estimated 6-month OS was 88% (95% CI 77-100%). No objective responses were observed. Of the 36 pts who started treatment, two had a grade 4 adverse event at least possibly related to treatment which included ALT elevation(1), AST elevation(1) and vomiting(1). Patients with NF2 alterations (n=24) had favorable PFS6 outcomes (58%, 95% CI 37-78%) compared to those with CDK (n=4, 25%, 95% CI 1-81%) and CDK+NF2 alterations (n=7, 29% 95% CI 4-71%), (Fisher’s Exact p=0.036).

CONCLUSIONS
Abemaciclib was well-tolerated and resulted in improved PFS6, meeting the trial’s co-primary endpoint. Abemaciclib warrants further investigation for the treatment of patients with progressive meningiomas.
BIOM-28. WHOLE EXOME TUMOR MOLECULAR PROFILING FROM CEREBROSPINAL FLUID (CSF): PRELIMINARY RESULTS OF A PILOT STUDY
Authors: Tuohy Kyle , Xiu Joanne , Nicolaides Theodore , Mansouri Alireza , Aulakh Sonikpreet , Davidson Christian , Zhang Jian , Borderias Maria Perdigones , Spetzler David , Gilbert Mark , Glantz Michael 
Published: November 2024

Abstract

BACKGROUND
Tissue sampling for molecular profiling has become an integral part of the diagnosis, prognosis, and management of CNS malignancies. However, many tumors are surgically inaccessible, and even biopsy of accessible lesions conveys some risk, and the possibility of sampling error. We evaluated the accuracy and potential utility of CSF liquid biopsy for identifying mutations in patients with intracranial tumors.

METHODS
CSF samples were collected from ventricular reservoirs and stored at -80 degrees Celsius until analyzed. Cell-free nucleic acids were extracted, concentrated, and quantified. Samples over 110ng quantification were normalized to 110ng and input into library prep. Library prep was done using v3.5 of Caris Assure and whole exome sequencing in an S2 FC on a Novaseq 6000. Bioinformatic analysis was performed.

RESULTS
CSF samples were obtained from 20 patients (10 gliomas, 5 CNS lymphomas, 5 metastases), 19 of whom also had tumor specimens available. Two hundred eighty-one mutations were identified: 17 (6.6%) in tumor only, 184 (72.4%) in CSF only, and 80 (31.5%) in both. Key alterations found in CSF include: IDH, TERT, ATRX in gliomas, and MYD88, PIM1, BCL2, TERT in lymphomas. Glioma CSF with IDH1 mutations did not have PTEN mutations (with one exception, in which the CSF was collected 3 years after the tissue). Targetable mutations in metastases included PIK3CA, NF1, and BRCA.

CONCLUSION
These preliminary results suggest that CSF liquid biopsy may be useful for identifying mutations in primary and metastatic brain tumors. Serial sampling is feasible, and may disclose changes in diagnostic and/or driver mutations over time, with important therapeutic and diagnostic implications. Many mutations were found in CSF alone. CSF may capture the entire molecular landscape of the tumor, overcoming the sampling error inherent in tissue biopsies. Prospective studies are underway to further elucidate the clinical applications of this technique.