bluuebunny/crossref_metadata_2025_split huggingface dataset:
ECOA-8. Lung adenocarcinoma brain metastasis prediction using tumor DNA methylation profiling
Authors: Zuccato Jeffrey , Mamatjan Yasin , Aldape Kenneth , Zadeh Gelareh 
Published: July 2021

Abstract

Introduction
The development of brain metastases from primary cancer profoundly impacts patient prognosis. Up to one quarter of lung cancers develop brain metastases and subsequent median overall survival is one year. Although clinical factors do not reliably predict brain metastasis development, DNA methylation signatures have been recently shown to predict outcomes in other cancers. It is hypothesized that DNA methylation signatures predicting brain metastasis development from lung cancer will be identified. This work may allow for treatment strategies that prevent brain metastasis development in high risk lung cancer patients.

Methods
DNA methylation profiling was undertaken on N=124 lung adenocarcinoma patients. In a randomly selected 70% training cohort, differentially methylated CpG sites between patients developing and not developing brain metastases were identified and used to build a generalized boosted regression model. Patients in the independent 30% testing cohort were assigned brain metastasis risk scores by the model.

Results
Brain metastases developed in 49/124 (39.5%) of patients and 2.3K CpG sites were significantly differentially methylated between patients developing and not developing metastases. Methylation-based brain metastasis risk scores predicted time to brain metastasis development in the testing cohort (Univariate cox: HR=3.2, 95% CI 1.1–9.4, p=0.03). A multivariate cox analysis assessing tumor size and nodal positivity together with methylation scores as covariates identified methylation scores as the only independent predictor of brain metastasis development in the testing cohort (HR=4.3, 95%CI 1.1–17, p=0.038).

Conclusions
DNA methylation signatures in lung adenocarcinomas predict brain metastasis development independently from the non-metastatic components of cancer stage. Future work developing a comprehensive nomogram utilizing methylation scores together with clinical factors to determine patient specific risk values may aid in treatment decisions and patient prognosis counselling.
NGMA-1. Quantification of IDH mutant alleles predicts outcome in diffuse gliomas
Authors: Voisin Mathew , Zadeh Gelareh 
Published: July 2021

Abstract

Background
IDH mutation is the main factor used in the prognostication of diffuse gliomas, however within IDH mutated gliomas there still remains a high variability in both tumor progression and overall survival.1 Digital droplet polymerase chain reaction (ddPCR) is one of the latest molecular amplification techniques that offers high precision in addition to the ability of absolute quantification of mutant allele copies.2

Methods
A total of 102 IDH mutant diffuse glioma tumor samples ranging from WHO grade 2 to 4 were collected. This cohort includes a total of 45 paired samples collected at two distinct surgical timepoints: initial and recurrent. All samples underwent DNA extraction. A total of 5 ng of tumor DNA from each sample was analyzed using ddPCR for the detection and quantification of IDH1 R132H mutant alleles. Sanger sequencing was performed on all samples as a gold standard.

Results
ddPCR was highly sensitive (100%) and specific (99%) for the detection of IDH mutations. Initial tumor samples with a high number of IDH mutant copies split by median demonstrated decreased overall survival (p = 0.04) and shorter progression free survival (p = 0.024). The number of IDH mutant copies was independent of WHO grade (p = 0.6) and 1p19q codeletion status (p = 0.86). Tumor pairs that had IDH mutant copies increase at recurrence were trending but not significantly related to a decrease in remaining survival (p = 0.1).

Conclusions
ddPCR is a highly sensitive and specific method of detecting IDH mutations in diffuse gliomas. The number of IDH mutant copies in tumors at initial surgery can serve as an independent prognostic factor to help guide future treatment and follow-up.
NGMA-2. Dual sgRNA-directed PD-L1 knockout in human glioblastoma cells using the CRISPR/Cas9 system
Authors: Fierro Javier , Dipasquale Jake , Aguilar Rocio , Perez Joshua , Tran An , Factoriza Chris , Dou Huanyu 
Published: July 2021

Abstract
Glioblastoma multiforme (GBM) is an astrocyte derived brain tumor. It induces an immunosuppressive microenvironment by exploiting immune checkpoints such as the PD-1/PD-L1 pathway. Targeting the PD-1/PD-L1 pathway for immunotherapy is a promising new avenue for treating GBM, but more work is needed to develop a safe and effective method for clinical applications. We identified two sgRNA sequences located on PD-L1 exon 3. The first sgRNA recognized the forward strand of human PD-L1 near the beginning of exon 3 and cuts at approximately base pair 82 (g82). The second sgRNA recognized the reverse strand of exon 3 and cuts at base pair 165 (g165). Two sgRNAs, g82 and g165, created an 83bp deletion in the genomic sequence that can lead to the production of a non-functional PD-L1 protein. A homology-directed repair template (HDR) containing an in-frame stop codon was designed to enhance PD-L1 knockout specificity and efficiency. Both g82 and g165 were cloned into the CRISPR/Cas9 plasmid, and was co-transfected with the added HDR template. T7E1, qRT-PCR and western blot analysis determined that the dual sgRNA CRISPR/Ca9 system knocked out both endogenous (80%) and exogenous (64%) PD-L1 in U87 cells and PD-L1 overexpression U87 cells, respectively. Deletion of PD-L1 reduced U87 migration and proliferation, while PD-L1 overexpression promoted tumor growth and tumor-associated macrophage polarization. Together, deletion of both membrane and cytoplasmic PD-L1 altered the PD-L1-associated immunosuppressive environment and prevented tumor progression and migration. Thus, a dual sgRNA CRISPR/Cas9 gene-editing system is a promising avenue for anti-GBM immunotherapy.
NGMA-3. Use of multi-omics data to initialize and validate a causal model of glioblastoma stem cell signaling
Authors: Holtzapple Emilee , Miskov-Zivanov Natasa , Cochran Brent 
Published: July 2021

Abstract
Glioblastomas and glioblastoma stem cells are heterogeneous with respect to mutations, gene expression, and response to drugs. To make predictive responses of individual GBM stem cell lines to drugs, we have constructed a causal model of glioblastoma stem cell signaling. The core model was built starting from pathways identified from TCGA mutation data with the addition of the Jak/STAT, Hedgehog, and Notch pathways. Elements and relations between them were validated and extended using the PCNet interaction database and the INDRA database which includes machine read extractions from the biomedical literature. The result is a high confidence executable model consisting of 209 element and 370 rules of interaction between the elements. Stochastic simulations of the model provide dynamic (quantile) changes in time and responses to perturbations. The output provides activity of individual nodes as well as a cellular output state of cell cycle progression, apoptosis, or differentiation. To simulate the responses of individual cell lines to kinase inhibitors, the model was initialized using DNA sequencing data, RNA-seq, and reverse phase protein array (RPPA) data from each cell line. Comparing the results of the simulations to the drug responses of 11 different kinase targets, the model was 88% accurate in predicting effects on growth and survival. The model was further tested by comparing the effects of Mek inhibition of each of the cell lines in model to the results observed in the RPPA data which overlap by 127 elements. In this case, there was 62% concordance between the model and data when binned into quintiles. Discrepancies between the model predictions and the data are being investigated to determine whether the model logic or extent needs to be revised to improve the model. This modeling approach is a step toward developing algorithms for personalized therapeutics for GBM based on multi-omics data.
NGMA-6. Quantitative MGMT Promoter Methylation Index Indicates Non-Linear, Prognostic Effect in Glioblastoma
Authors: Gibson David , Ravi Akshay , Rodriguez Eduardo , Chang Susan , Bush Nancy , Taylor Jennie , Clarke Jennifer , Solomon David , Scheffler Aaron , Witte John , Okada Hideho , Berger Mitchel , Chehab Farid , Butowski Nicholas 
Published: July 2021

Abstract

Background
Epigenetic inhibition of the O6-methylguanine-DNA-methyltransferase (MGMT) gene has emerged as a clinically relevant prognostic marker in glioblastoma (GBM). Methylation of the MGMT promoter has been shown to increase chemotherapy efficacy. While traditionally reported as a binary marker, recent methodological advancements have led to quantitative methods of measuring methylation, allowing for clearer insights into methylation’s functional relationship with survival.

Methods
A CLIA assay and bisulfite sequencing was utilized to develop a quantitative, 17-point MGMT promoter methylation index derived from the number of methylated CpG sites. Retrospective review of 242 newly diagnosed GBM patients was performed in order to discern how risk for mortality transforms as promoter methylation increases. Non-linearities were captured by fitting splines to Cox proportional hazard models, plotting smoothed residuals, and creating survival plots. Covariates included age, KPS, IDH1 mutation, and extent of resection.

Results
Median follow-up time and progression free survival were 15.9 and 9 months, respectively. 176 subjects experienced death. A one-unit increase in CpG methylation resulted in a 4% reduction in hazard (95% CI 0.93–0.99, P&lt;0.005). Moreover, GBM patients with low levels of methylation (1–6 CpG islands) fared markedly worse (HR=1.62, 95% CI 1.03–2.54, P&lt;0.036) than individuals who were unmethylated (reference group). Subjects with medium levels of methylation (7–12 sites) had the greatest reduction in hazard (HR=0.48, 95% CI 0.29–0.80, P&lt;0.004), followed by individuals in the highest methylation tertile (HR=0.62, 95% CI 0.40–0.97, P&lt;0.035).

Conclusion
The extent of MGMT methylation shares a non-linear relationship with survival, suggesting conformation of the marker’s protective effect. This finding challenges the current understanding of MGMT and underlines the clinical importance of determining its prognostic utility. Potential limitations include censoring, sample size, and extraneous mutations. Future research is warranted to examine whether the location of CpG site methylation contributes to a reduction in mortality hazard.
OPTC-1. Proteomic analysis of genetically stratified low-grade meningioma
Authors: Akther Yeasmin , Adams Claire , Sharma Vikram , Barros Claudia , Banton Matthew , Hanemann Oliver 
Published: July 2021

Abstract

Introduction
Meningioma are the most common primary intracranial tumour. According to WHO, ~80% tumours are benign grade I. Although, some grade I tumour clinically show aggressive behaviour. Radio-surgery are the main therapeutic approaches, chemotherapies are ineffective. Accurate biomarkers for clinical management are lacking. The mutational profile of low-grade meningioma is well-defined, with non-NF2 mutated tumours harbouring recurrent mutations in genes including TRAF7, KLF4, AKT1 and SMO. Here, we aim to identify novel biomarkers and therapeutic targets of genetically stratified low-grade meningioma by characterising the proteomic landscape.

Materials and methods
Meningioma specimens were stratified according to mutational background: AKT1E17K/TRAF7, KLF4K409Q/TRAF7 and NF2-/-. Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion and sample preparation for LC-MS/MS analysis. Raw mass spectrometry data files were processed by MaxQuant and Perseus software. Quantitative phospho-proteomics was performed using TMT-10plex labelling approach followed by motif analysis using motif-X algorithm. GO enrichment analyses were performed using DAVID against all human proteins.

Results and Conclusions
We have quantified 4162 proteins across all mutational meningioma subgroups and normal meninges (n=31). Hierarchical clustering analysis showed distinct proteomic profiles of mutational subgroups revealing clusters of differentially expressed proteins (DEPs). Comparative analysis showed 10 proteins were commonly significantly upregulated (log2 fold-change≥1; p&lt;0.05) among all mutational subtypes vs. normal meninges, indicating proteomic landscapes of mutational subtypes to be highly variable. In contrast, 257 proteins were commonly significantly downregulated (log2 fold-change≤-1; p&lt;0.05) and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1E17K/TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. Lastly, analyses of 6600 phospho-sites (n=8) predicted regulatory kinases including EGFR and PKCα. Several of these up-regulated proteins and kinases already verified via WB. Further validation and functional verification will allow us to identify potential drug targets/biomarkers for meningioma.
OMRT-1. Cannabidiol converts NFKB into a tumor-suppressor in glioblastoma with defined antioxidative properties
Authors: Volmar Marie , Cheng Jiying , Synowitz Michael , Schick Joel , Kälin Roland , Glass Rainer 
Published: July 2021

Abstract

Background
The transcription factor NFKB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma; GBM). Precise therapeutic modulation of NFKB-activity can suppress central oncogenic signalling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive.

Methods
In a pharmacogenomics study with a panel of transgenic glioma cells we observed that NFKB can be converted into a tumor-suppressor by the non-psychotropic cannabinoid Cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study we performed pharmacological assays, gene-expression profiling, biochemical and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics-analysis of human GBM-datasets.

Results
We found that CBD promotes DNA-binding of the NFKB-subunit RELA and simultaneously prevents RELA-phosphorylation on serine-311, a key residue which permits genetic transactivation. Strikingly, sustained DNA-binding by RELA lacking phospho-serine 311 was found to mediate hGSC-cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen-species (ROS), while high ROS-content in other tumors blocked CBD induced hGSC-death. Consequently, ROS-levels served as predictive biomarker for CBD-sensitive tumors.

Conclusions
This evidence demonstrates how a clinically approved drug can convert NFKB into a tumor-suppressor and suggests a promising repurposing option for GBM-therapy.
OTEH-4. Deeper insight into intratumoral heterogeneity by MRI and PET-guided stereotactic biopsies from glioblastoma patients
Authors: Anand Atul , Petersen Jeanette Krogh , Burton Mark , Larsen Martin Jakob , Andersen Lars van Brakel , Harwood Dylan Scott Lykke , Pedersen Christian Bonde , Poulsen Frantz Rom , Grupe Peter , Kruse Torben A , Thomassen Mads , Kristensen Bjarne Winther 
Published: July 2021

Abstract
Glioblastoma is one of the most aggressive cancers, but the molecular evolution is still not fully understood. We used PET imaging combined with deep sequencing of glioblastoma biopsies at both the RNA and DNA levels to get a deeper insight into molecular evolution. In the clinical setting, PET imaging provides information about metabolically active tumor areas, but the molecular interpretation is unclear. Our primary objective was to perform an intratumoral spatial comparison of biopsies from potentially aggressive and less aggressive areas in glioblastomas according to PET scans. Additionally, tissue from the tumor periphery was included. We used MRI, 11C-methionine(MET) PET, and 18F-FDG PET was used in combination to obtain a series of neurosurgical stereotactic biopsies from tumor areas with high MET and 18F-FDG uptake (hotspot), low MET and 18F-FDG uptake (coldspot), as well as tumor periphery of six glioblastoma patients that were processed for whole genome, exome, and transcriptome sequencing. Differential gene expression and gene ontology analysis showed that hotspots were enriched in gene sets associated with DNA replication, cell cycle, and ligand receptor interaction. Genome and exome analysis suggested hotspots and coldspots to have similar mutational profiles. However, a limited number of hotspot-specific mutations and fusion transcripts indicated that hotspot tumor cells developed from coldspot cells and point at the potential role of hotspot driver genes in glioblastoma. Our findings reveal that hotspots in glioblastomas represent a more advanced stage of molecular evolution than coldspots.
OTME-1. TAMEP are brain tumor parenchymal cells controlling neoplastic angiogenesis and progression
Authors: Kälin Roland , Cai Linzhi , Li Yuping , von Baumgarten Louisa , Schulz Christian , Hellmann Ines , Glass Rainer 
Published: July 2021

Abstract
Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor parenchymal cells may promote specific phases of disease progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Notably, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell population, by conditional Sox2-knockout, drastically reduced glioblastoma vascularization and size. Hence, TAMEP emerge as a tumor parenchymal component with a strong impact on glioblastoma progression.
OTME-8. Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis
Authors: Gritsch Simon , Mathewson Nathan , Ashenberg Orr , Perez Elizabeth , Marx Sascha , Reardon David , Regev Aviv , Suva Mario , Wucherpfennig Kai 
Published: July 2021

Abstract
T-cells are critical effector cells of cancer immunotherapies, but little is known about T-cell gene expression programs in diffuse gliomas. We leveraged single-cell RNA-seq to chart the gene expression and clonal landscape of tumor-infiltrating T-cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. Our analysis revealed subsets of T-cells that expressed several NK-cell receptors, in particular the inhibitory CD161 receptor (KLRB1 gene). KLRB1 was overexpressed by clonally expanded CD8 T-cells, and larger populations of T-cells expressed CD161 than PD-1. The CLEC2D ligand of CD161 was expressed by malignant cells and myeloid cells, and inactivation of KLRB1 enhanced anti-tumor T-cell function. KLRB1 was also expressed by substantial T-cell populations in multiple other human cancers. CD161 and other NK-cell receptors expressed by T-cells represent opportunities for immunotherapy of diffuse gliomas and other human cancers.
OTME-15. Physical Confinement Induces Diverse Transcriptomic Changes and Chemoresistance in Migrating Glioblastoma Cells
Authors: Battiste James , Babu Anish , Sharp Rachel , Scott Sydney , Dunn Ian , Glenn Chad , Kim Young-tae , Jones Kenneth 
Published: July 2021

Abstract
Glioblastoma multiforme (GBM) cells migrating in physically confined environments are affected by mechanical stress that potentially lead to transcriptomic changes. To simulate those stresses, microfluidic channels were made with micro-patterned polydimethylsiloxane (PDMS) replicating the physical microenvironment of white matter tracts by confining the cells in linear channels similar to the space between axons. We employed a combination of microarray transcriptomic profiling and single cell-sequencing analyses to investigate cells undergoing linear confined space migration (LCSM). GBM cells spontaneously migrate through confined spaces along 5x5 mm (height/width) microfluidic channels, 0.5 to 5 mm in length. Our previous studies demonstrated that cells migrating in LCSM are more resistant to treatment with temozolomide than the same cells growing in standard monolayer culture (SMC). Cells in confined migration evaluated by microarray-based transcriptomic profiling demonstrated that linear confined migration induces increased expression in pathways involving angiogenesis, cell adhesion, cell motility, DNA damage repair, extracellular matrix structure, HIF1α, and others. Single cell transcriptomic analysis could identify GBM cells in different migratory states (LCSM vs. SMC), and similar pathways were seen upregulated with additional changes in cholesterol biosynthesis pathways and cell cycle regulation pathways. Trajectory Inference aligned single cells according to changes in migration status and demonstrated transcript changes during LCSM were progressive but generally reversible on return to SMC. Pathway analyses showed alterations in the cholesterol biosynthesis pathway and cell cycle regulation in cell clusters of confined migrating cells. Molecular studies confirmed that cholesterol biosynthesis pathway regulatory genes (SQLE, MVD, and HMGCR) are upregulated during LCSM. Expression analysis demonstrated increased G1 phase delay in confined migrating cells (LCSM) confirmed by FUCCI expression analysis. We propose that migration in linear confined spaces like white matter structures produces significant transcriptome changes that produce chemoresistance as a new mechanism for treatment resistance of Glioblastoma.
OTME-18. Targeted CRISPR/Cas9 gene-editing regulates the brain tumor environment
Authors: Perez Joshua , Fierro Javier , Aguilar Rocio , Dou Huanyu 
Published: July 2021

Abstract
Glioblastoma multiform (GBM) is the most common malignant brain tumor. Recent immunotherapy has demonstrated potential to treat GBM. However, the immune suppressive tumor environment in the brain represents a significant barrier for the treatment of GBM. Overexpression of programmed death ligand-1 (PD-L1) in GBM tumor cells and macrophages plays a key role in GBM vitality, proliferation, and migration, while also suppressing the immune system. We developed a CRISPR/Cas9 gene-editing system to delete whole cell PD-L1. Human PD-L1 targeted sgRNA were cloned into CRISPR/Cas9 plasmids with or without an HDR templet. CRISPR/Cas9 were treated to human GBM U87 cells for 15, 30, 60, 120 and 240 minutes. The intracellular concentration of CRISPR/Cas9 exhibited a time-dependent increases. A GFP tagged CRISPR/Cas9 plasmid was developed to test the transfection efficacy. Higher levels of GFP+ U87 cells were observed at day 3. CRISPR/Cas9 showed a greater PD-L1 knockout at day 3. The PD-L1 reduction limited the proliferation of U87 cells. A scratch assay showed that PD-L1 deletion inhibited the migration of U87 cells. An in vitro GBM model was developed by co-cultivation of U87 cells and macrophages. CRISPR/Cas9 treated co-cultures changed the ratios of U87 cells and macrophages and polarized tumor associated macrophages (TAM) from M2 toward M1. CRISPR/Cas9 gene-editing effectively deleted PD-L1 in U87 cells. Successful deletion of PD-L1 prevented U87 cells growth and migration, and altered the TAMs plasticity and the tumor environment.