bluuebunny/crossref_metadata_2025_split huggingface dataset:
OS01.4.A MODELING EGFR REGULATION IN GLIOBLASTOMA REVEALS ASSOCIATIONS WITH ITS LIGANDS
Authors: Ghisai S A , Barin N , van Hijfte L , de Heer I , van den Bent M J , Hoogstrate Y , French P J 
Published: October 2024

Abstract

BACKGROUND
Approximately half of the IDH-wildtype glioblastomas (GBM) exhibit EGFR amplification, with highly varying copy number levels. Additionally, extracellular mutations in EGFR occur, leading to either ligand hypersensitivity (R108K/A289V/G598V) or ligand-independence (EGFRvIII). Contrary to EGFR-mutated lung adenocarcinoma (LUAD), clinical trials with EGFR inhibitors have not yet improved survival outcome for GBM. Our research aimed to elucidate the molecular mechanisms of EGFR activation and regulation in GBM and make comparisons with LUAD.

MATERIAL AND METHODS
We devised Random Forest models to identify EGFR co-regulated genes and pathways in GBM by leveraging RNA-sequencing data from TCGA-GBM (n=142) and the Intellance-2/EORTC_1410 (n=211) trial. Additionally, we compared EGFR signaling patterns between GBM and LUAD, where LUAD RNA sequencing data (n=508) were collected from TCGA. Sources of expression signals were determined by screening both in-house and public single cell and single nucleus RNA sequencing (sc/snRNA-seq) data from GBM (n=28) and LUAD (n=45). Multiple primary and established GBM and LUAD cell lines were used for in vitro functional studies.

RESULTS
Among the significant EGFR predictor genes (TCGA-GBM: n=169, Intellance-II: n=166), we found two ligands (BTC and EREG). Expression of these ligands was inversely correlated with the EGFR amplification level and presence of ligand-sensitizing mutations. Samples with ligand-sensitive mutations showed a downregulation of genes involved in the RB and TP53 pathways (CDK4, MDM2, CDKN2A). Our analyses showed a tumor-type specific expression profile in GBM and LUAD when comparing tumors carrying activating mutations. Sc/snRNA-seq analysis showed that EGFR ligands were primarily expressed by non-malignant cells, but differed in their source of expression between tumor types: GBM (BTC: neurons, EREG: myeloid) and LUAD (BTC: cilia, EREG: myeloid). The level of expression of nearly all EGFR ligands was significantly higher in LUAD than in GBM, which may be related to the presence of ligand-sensitizing mutations in the latter. Functional analysis on primary cultured glioma- and established cell lines showed no overt differences between GBM and LUAD in EGFR internalization and ligand-induced transcriptional regulation.

CONCLUSION
EGFR ligands BTC and EREG showed a significant and negative association with EGFR expression in GBM. These findings emphasize the potential role of ligands in regulating EGFR, where EGFR activation seems to be triaged by the highly varying level of EGFR amplification, sensitivity of the receptor towards the different ligands and ligand expression levels. These findings provide a better understanding of EGFR regulation in GBM and might eventually be used for patient stratification.
OS05.5.A DOSE ESCALATED SPINE RADIOSURGERY FOR RADIORESISTANT SPINAL METASTASES UTILIZING SIMULTANEOUS INTEGRATED BOOST
Authors: Ghia A J , Bernard V , Iskander G , Rooney M , De B , Briere T M , Tatsui C E , Bishop A , Perni S , Tom M , Wang C , Beckham T , Yeboa D N , McAleer M , Li J , Amini B , Rhines L D 
Published: October 2024

Abstract

BACKGROUND
Spine stereotactic body radiosurgery (SSRS) delivers ablative doses of radiation with sub-millimeter precision leading to high rates of local control with minimal toxicity. Treatment dose, fractionation and technique vary from institution to institution. We present our experience delivering single fraction SSRS using simultaneous integrated boost (SIB) for radioresistant spinal metastases.

MATERIAL AND METHODS
This is a single institution retrospective review analyzing patients treated from 2006-2021 with single fraction SSRS delivering 24 Gy to the gross disease (GTV) and 16 Gy to the clinical target volume (CTV) at risk of harboring microscopic disease per International consensus guidelines. We assessed clinical endpoints in relation to patterns of failure, including local control (LC) and overall survival (OS), using univariate and multivariate analyses.

RESULTS
From 2006-2021, 395 sites from 285 patients were treated with SSRS. With a median follow-up of 18 months, the actuarial 1- and 2-year local control (LC) was 93% and 89%, respectively. The most common pattern of failure was epidural (67%). Indeed, multivariate analysis revealed the presence of epidural disease as a significant predictor of local failure (HR 3.42, 95% CI 1.71-6.85 p=0.001). Of the 39 sites with local failure, only 2 sites were within the CTV volume outside of the GTV volume. The median overall survival was 26 months and no radiation myelopathy was noted. Grade 3 vertebral fracture was noted in only 5% of treated sites (20 fractures out of 395 sites).

CONCLUSION
This is the largest experience on SSRS using SIB for radioresistant spinal metastases. SSRS with SIB delivered to 24 Gy in 1 fraction is associated with a high local control rate with low toxicity. Failures outside the GTV and within the CTV were very rare and clinically significant fracture risk was 5% suggesting that SIB is a reasonable approach for radioresistant spinal metastases treated with SSRS.
OS05.7.A RADIATION-INDUCED CONTRAST ENHANCEMENT AFTER PROTON THERAPY IN PATIENTS WITH GLIOMAS IN THE PRO-CNS STUDY
Authors: Werlenius K , Agrup M , Flejmer A , Gojon H , Jakobsson F , Bergström P , Dahl L , Parcaoglu F , Strandéus M , Kinhult S , Morhed E , Gustavsson M , Modin K , Heggebø L , Witt Nyström P , Rylander H , Store Jakola A , Brandal P , Blomstrand M 
Published: October 2024

Abstract

BACKGROUND
Although proton radiotherapy has been used in a large scale for quite some time, published data from trials confirming the safety of proton therapy are still scarce. Yet, there are indications of radiation induced contrast enhancement (RICE) being more common after proton radiotherapy than photon radiotherapy.

MATERIAL AND METHODS
Between August 2015 and August 2020, adult patients receiving proton therapy for primary CNS tumours at the Skandion clinic in Sweden were invited to PRO-CNS, a prospective multicentre observational cohort study. We identified 120 patients with glioma WHO grade 1-3 in the PRO-CNS study. The patients received a total dose of 50.4 Gy (RBE), 54 Gy (RBE) or 59.4-60 Gy (RBE) in 1.8-2.0 Gy (RBE) fractions. Discrimination between RICE and true tumour progression was assessed by experienced neuroradiologists and neurooncologists. So far, only the 45 patients from Gothenburg have been analysed.

RESULTS
Preliminary data from the Gothenburg cohort show RICE in 70% of patients receiving a dose of 59.4 Gy (RBE). This is a considerably higher incidence of RICE than previously reported in patients treated with photons to the same dose. For comparison, 18% of patients receiving 50.4 Gy (RBE) and 21% receiving 54 Gy (RBE) developed RICE. Furthermore, RICE seemed to be slightly more common among patients treated for oligodendroglioma than astrocytoma, but the numbers are too small to draw conclusions. Analysis of the whole cohort of 120 patients is ongoing with the intention to be presented in 2024.

CONCLUSION
Our preliminary results raise concern on toxicity after proton treatment to 59.4 Gy (RBE) in glioma patients. These are preliminary results and further evaluation is needed.
OS06.5.A ACCURATE AND COMPREHENSIVE EVALUATION OF MGMT PROMOTER METHYLATION BY NANOPORE SEQUENCING
Authors: Halldorsson S , Nagymihaly R M , Leske H , Brandal P , Panagopoulos I , Lund-Iversen M , Patel A , Sahm F , Vik-Mo E O 
Published: October 2024

Abstract

BACKGROUND
The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region is essential in evaluating the prognosis and predicting the drug response in patients with glioblastoma (GBM). In this study, we evaluated the utility of using nanopore long-read sequencing as a method for assessing methylation levels throughout the MGMT CpG-island, compared its performance to established techniques, and demonstrated its clinical applicability.

MATERIALS AND METHODS
We analysed 165 samples from CNS tumours, focusing on the MGMT CpG-island using nanopore sequencing. Oxford Nanopore Technologies (ONT) MinION and PromethION flow cells were employed for single sample or barcoded assays, guided by a CRISPR/Cas9 protocol, adaptive sampling or as part of a whole genome sequencing assay. Methylation data obtained through nanopore sequencing were compared to results obtained via pyrosequencing and methylation bead arrays. Hierarchical clustering was applied to nanopore sequencing data for patient stratification.

RESULTS
Nanopore sequencing displayed a strong correlation (R² = 0.91) with pyrosequencing results for the four CpGs of MGMT analysed by both methods. The MGMT-STP27 algorithm’s classification was effectively reproduced using nanopore data. Unsupervised hierarchical clustering revealed distinct patterns in methylated and unmethylated samples, providing comparable survival prediction capabilities. Nanopore sequencing yielded high-confidence results in a rapid timeframe, typically within hours of sequencing, and extended the analysis to all 98 CpGs of the MGMT CpG-island.

CONCLUSIONS
This study presents nanopore sequencing as a valid and efficient method for determining MGMT promotor methylation status. It offers a comprehensive view of the MGMT promoter methylation landscape, which enables the identification of potentially clinically relevant subgroups of patients. Further exploration of the clinical implications of patient stratification using nanopore sequencing of MGMT is warranted.
OS06.6.A LOCAL DELIVERY OF HRBMP4 AS AN ANTICANCER THERAPY IN PATIENTS WITH RECURRENT GLIOBLASTOMA: A FIRST-IN-HUMAN PHASE 1 DOSE ESCALATION TRIAL
Authors: Bos E M , Binda E , Verploegh I S , Wembacher E , Hoefnagel D , Balvers R K , Korporaal A L , Conidi A , Warnert E A H , Trivieri N , Visioli A , Zaccarini P , Caiola L , van Wijck R , van der Spek P , Huylebroeck D , Leenstra S , Lamfers M L M , Ram Z , Westphal M , Noske D , Legnani F , DiMeco F , Vescovi A L , Dirven C M F 
Published: October 2024

Abstract

BACKGROUND
This Phase 1 study evaluates the intra- and peritumoral administration by convection enhanced delivery (CED) of human recombinant Bone Morphogenetic Protein 4 (hrBMP4) - an inhibitory regulator of cancer stem cells (CSCs) - in recurrent glioblastoma.

MATERIAL AND METHODS
In a 3 + 3 dose escalation design, over four to six days, fifteen recurrent glioblastoma patients received, by CED, one of five doses of hrBMP4 ranging from 0·5 to 18 mg. Patients were followed by periodic physical, neurological, blood testing, magnetic resonance imaging (MRI) and quality of life evaluations. The primary objective of this first-in-human study was to determine the safety, dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of hrBMP4. Secondary objectives were to assess potential efficacy and systemic exposure to hrBMP4 upon intracerebral infusion. Dynamics of BMP signaling were assessed by RNAseq, and differentiation induction was investigated by immunofluorescence.

RESULTS
Intra- and peritumoral infusion of hrBMP4 was safe and well-tolerated. We observed no serious adverse events related to this drug. Neither MTD nor DLT were reached. Three patients had increased hrBMP4 serum levels at the end of infusion, which normalized within 4 weeks, without sign of toxicity. One patient showed partial response and two patients a complete (local) tumor response, which was maintained until the most recent follow-up, 57 and 30 months post-hrBMP4. Tumor growth was inhibited in areas permeated by hrBMP4.

CONCLUSION
Local delivery of hrBMP4 in and around recurring glioblastoma is safe and well-tolerated. Three patients responded to the treatment. A complete response and long-term survival occurred in two of them. This warrants further clinical studies on this novel treatment targeting glioblastoma CSCs.
OS07.3.A DYNAMIC METABOLIC SHIFTS RESHAPE GLIOBLASTOMA: IMPACTS ON SPATIAL STRUCTURE AND IMMUNE RESPONSE
Authors: Conrad N , Benotmane J , Villa G , Schnell O , Heiland D , Kueckelhaus J 
Published: October 2024

Abstract

BACKGROUND
The structured layers of the human neocortex stand in sharp contrast to the disordered arrangement found in malignant CNS tumors. Glioblastoma (GBM), with its diverse characteristics, presents formidable hurdles in both research and treatment, as successful approaches demand a uniform biological understanding. In this study, we capitalize on recent advances in spatial biology, employing integrated spatially resolved transcriptomics and metabolomics to identify the factors driving the unique architectural changes observed in GBM.

MATERIAL AND METHODS
We performed spatial metabolomics (MALDI) and transcriptomics (Visium n=43 and MERFISH n=25) of glioblastoma patients. Computational analysis was performed with the SPATA2 R package.

RESULTS
We found that most spatially recurrent pattern in glioblastoma emerge in a gradient-like organization,particularly influenced by proximity to areas of necrosis and hypoxia. Samples without hypoxic regions demonstrated less spatial organization. Metabolomic and transcriptomic profiling revealed enhanced responses to hypoxia coupled with reduced cell proliferation and elevated expression indicative of the S-phase in cell division in regions near necrosis. The vicinity to hypoxic areas appeared to be associated with increased chromosomal alterations as well as extracellular matrix remodeling and epithelial-to-mesenchymal transition, implying that the invasive capabilities of GBM cells originate from these hypoxic niches. By further integrating spatially resolved TCR-sequencing, we identified that hypoxic gradients drive defined T cell response with higher likelihood of T cell dysfunction within a distance of 235 μm (sd: 132 μm) to hypoxia. This suggests that the presence of necrosis, hypoxia, and hypoxia associated metabolites could serve as potential predictive markers for T-cell behavior and exhaustion in glioblastoma.

CONCLUSION
Our investigation provides a comprehensive examination of the impact of two common environments in glioblastoma: necrosis and hypoxia. We demonstrate that the intricate structure of glioblastoma is not merely shaped by genetic chance and random events. Rather, it is substantially molded by spatial proximity to critical elements such as necrosis and hypoxia. This understanding contributes to an advancing framework aimed at unraveling the complex biology of glioblastoma.
OS07.4.A INTERLEUKIN-6 INCREASES STRUCTURAL AND ELECTROPHYSIOLOGICAL CONNECTIVITY IN GLIOMA
Authors: Picart T , Krishna S , Daniel A , Dada A , Oten S , Hyer J , Hervey-Jumper S 
Published: October 2024

Abstract

BACKGROUND
In the setting of glioblastoma (GBM), the pleiotropic cytokine interleukin-6 (IL-6) upregulates PD-L1 expression through STAT3-dependant mechanisms. This leads to local immunosuppression, sustains tumor progression and may explain the limited efficacy of immune checkpoint inhibition. Consistently, IL6 knock-out in orthotopic murine glioma models diminishes tumor growth and increases survival. IL-6 increases synaptogenesis during development and contributes to neuronal network remodeling following focal spinal cord or brain injury. Yet, the contribution of IL-6 to glioma-induced circuit remodeling from a structural and electrophysiological perspective is poorly understood and is investigated herein.

MATERIAL AND METHODS
The expression of IL-6 was compared in magnetoencephalography (MEG)-based intratumoral regions of High- (HFC) and Low-Functional Connectivity (LFC) from GBM patients using bulk and single-cell RNA sequencing and immunofluorescence staining in a panel of gliomas with biological replicates (n=44 samples). In mouse postnatal cortical neurons and primary patient-derived glioblastoma cell co-cultures, the effects of exogenously applied recombinant human IL-6 and the humanized anti-IL-6 receptor (IL6R) antibody TOCILIZUMAB (TCZ) on Ki67, GAP43, and synaptic marker expression, tumor connectivity and neuronal activity were monitored. Co-culture analysis were validated with human and mouse cortical organotypic glioblastoma slices.

RESULTS
IL-6 was overexpressed in HFC compared to LFC regions, independently of tumor grade. In neuron and GBM co-cultures, the number of tumor microtubes per cell was significantly increased by the addition of recombinant IL-6 and decreased by TCZ treatment. The addition of recombinant IL-6 increased the expression of the tumor microtube marker, GAP43 and modified the expression profile of synaptic markers. Electrophysiological recordings, from multielectrode array (MEA), showed a significant increase in the neuronal activity metrics, such as weighted mean firing rate, network burst frequency and duration, and also the neuronal synchrony index following the addition of recombinant IL-6. Additionally, the proliferation index of GBM cells was significantly increased by exogenous recombinant IL-6 and decreased by TCZ treatment.

CONCLUSION
These findings suggest that IL-6-signaling increases structural and electrophysiological connectivity in gliomas and may contribute to neuronal network hyper-excitability and synchrony. Additional experiments are required to more precisely understand the IL-6-driven molecular mechanisms promoting glioma-induce circuit remodeling. These data will help to design new therapeutic options likely to improve the oncological and functional outcomes of patients treated for diffuse gliomas.
OS07.5.A MULTI-MODAL EXAMINATION OF SPATIAL HETEROGENEITY IN THE ASTROCYTOMA MICROENVIRONMENT
Authors: Lu T , Freytag L , Moffet J , Kriel J , Whittle J , Freytag S , Best S A 
Published: October 2024

Abstract

BACKGROUND
Astrocytoma is a diffuse and heterogeneous glioma that infiltrates the brain. Despite post-operative therapy including radiation and chemotherapy, 52% of patients’ tumours recur within five-years of surgery. These tumours are characterised by mutations in the IDH1 or IDH2 gene that confer metabolic alterations, most notably the production of the onco-metabolite 2-hydroxyglutarate. To reveal the metabolic alterations and their upstream regulation across the heterogeneous tumour microenvironment, we applied spatial metabolomics and spatial transcriptomics to sequential sections from resected tumour samples of patients with low grade astrocytoma.

METHODS
Spatial assessment of heterogeneity was performed on serial sections using spatial metabolomics (atmospheric pressure MALDI-TOF) and spatial transcriptomics (10X Genomics Xenium). This necessitated extensive optimisation of image registration across modalities and sequential sections to create three dimensional models to deeply interrogate heterogeneity within tumours.

RESULTS
Combining spatial transcriptomics and spatial metabolomics across 8 matched sequential slices of the same patient tumour, enabled the identification of metabolic niches associated with distinct cell type composition and transcriptional regulation. Strikingly, normal domains that were present at the leading edge of tumours could be identified both metabolically and transcriptomically.

CONCLUSIONS
Understanding the heterogeneity of astrocytomas on a spatial level reveals unique domains and organisation of tumour and immune cell types. Matching these transcriptomic and cell state information with metabolic insights leads to pathway insights that will be essential to the identification of druggable targets. Our pipeline combining spatial transcriptomics and metabolomics with tissue histology reveals these biologically important domains.
OS07.9.A NETWORK ANALYSIS IDENTIFIES CSF2RA’S GENETIC COMMUNITY AS A REGULATOR OF PROGRESSION IN THE INVASIVE REGION OF HIGH GRADE GLIOMA
Authors: Stevens V , Flick M , Sereduk C P , Plaisier C L , Barajas R F , Hu L S , Kannan K , Tran N L 
Published: October 2024

Abstract

BACKGROUND
High-grade glioma (HGG) is the most common and aggressive form of primary brain cancer with poor prognosis. Standard of care surgery employs Magnetic Resonance Imaging to identify the contrast enhancing (CE) core for resection, typically leaving behind a non-enhancing (NE) invasive rim of tumor which invariably contributes to recurrence. Recent multi-omics analyses identified predominant neuronal and immune signatures within NE tumor, which could provide key insights for combatting recurrence. We present the novel application of graph network analyses to integrate multi-omic data from a unique dataset of multi-regional spatially-localized CE and NE biopsy samples from a large cohort of patients with HGG.

MATERIAL AND METHODS
159 IDH-wildtype tumor biopsy samples from NE and CE regions of 74 glioma patients were profiled by RNA sequencing. Differentially expressed genes (DEGs) across NE and CE samples (n = 1159) were determined using the EdgeR package, and represented as nodes within a Neo4j graph network. Samples were hierarchically clustered for each gene (method = median), and edges were generated between gene nodes sharing ≥ 1 sample. Modularity optimization was used to identify communities of densely connected nodes. Genes occurring exclusively within NE communities were localized by cell type using external single-cell RNA sequencing datasets and identified as source node candidates for NE network random walks. These were performed with CSF2RA nodes as source (walk length = 5; 10,000 walks/source node). Random walks traversing samples with significantly elevated CSF2RA expression (one-tailed test) were kept for analysis. Genes occurring at high frequency (one-tailed test) within random walks were ranked by correlation with CSF2RA expression in NE samples and analyzed by gene set enrichment analysis (GSEA) for biological processes using clusterProfiler.

RESULTS
CSF2RA, an immune signaling receptor gene, was upregulated (logFC = 0.789) in NE samples and uniquely occurred within NE communities. We show its differential expression within the immune-enriched Glycolytic/Plurimetabolic (GPM) glioma subtype and confirm its enrichment in myeloid populations across single cell HGG datasets. NE network random walks identified 46 DEGs associated with CSF2RA. GSEA revealed upregulation of proliferative and metabolic signatures and downregulation of signaling pathways within CSF2RA’s network.

CONCLUSION
CSF2RA’s genetic network in NE tumor represents a potential mechanism for immunologic facilitation of glioma progression within this region. Given the expression of CSF2 ligand by glioma cells, which plays a crucial role in recruiting myeloid cells during peripheral cancer progression, modulating the expression of key genes in the CSF2RA network may be a promising approach for perturbing the crosstalk signals between immune and tumor cells in the NE region.