bluuebunny/crossref_metadata_2025_split huggingface dataset:
OS10.5.A PERIVASCULAR CONTAINED T CELLS IN IDH-MUTANT ASTROCYTOMA CO-LOCALIZE WITH GEMISTOCYTIC TUMOR CELLS OF AN ASTROCYTE-STATE AND MACROPHAGES WITH AN IMMUNE-STIMULATORY PHENOTYPE
Authors: van Hijfte L , Geurts M , de Heer I , Vallentgoed W R , Balcioglu H E , Debets R , French P 
Published: September 2023

Abstract

BACKGROUND
Isocitrate dehydrogenase mutant (IDHmt) astrocytoma is considered a T cell excluded tumor, yet little is known regarding the underlying cues that may drive T cell exclusion. To aid better understanding of this mechanism in IDHmt astrocytoma, we systematically characterized the immune microenvironment of this tumor type.

METHODS
We performed and analyzed multiplex immunofluorescence stainings for lymphocytes, myeloid cells and IDH1-R132H of 1293 regions from 75 IDHmt astrocytomas. We consequently performed bulk, single nucleus and spatial RNA sequencing of samples from the same cohort. Integrative analysis of data from these platforms allowed for the identification of unique tumor and immune cell subsets that are crucial in the spatial organization of the immune microenvironment and T cell containment in IDHmt astrocytoma.

RESULTS
IDHmt astrocytoma showed three spatially distinct distribution patterns of T cells: regions with perivascular accumulation of T cells, regions with T cells in the tumor stroma and T cell absent regions. Perivascular accumulation of T cells, known as T-cell cuffs, was the most abundant pattern in our data. Spatially, there was a significant increase of tumor cells around T cell cuffs that showed a gemistocytic phenotype, a tumor cell type that is histologically characterized by a large eosinophilic cytoplasm and ectopically localized nucleus. Gemistocyte-high tumors (&gt; 20% of all tumor cells) demonstrated enhanced expression of genes related to immune cell migration and activation that were reliably expressed by tumor associated macrophages and T cells. Notably, gemistocytic tumor cells were identified as a distinct sub-cluster of the ‘astrocyte-like’ transcriptomic cell state with a phenotype that is linked to astrogliosis and expressed CD44, TNC and TGFB2. Moreover, gemistocytic tumor cells were spatially associated with a CD83+ subpopulation of tumor-associated macrophages that resemble reactive microglia in astrogliosis and show increased expression of the chemo-attractants CCL3, CCL4 and CCL4L2.

CONCLUSION
Using different modalities for spatial and single cell profiling of RNA and protein, we uncovered a previously unknown relationship between the immune system and tumor cells in IDHmt astrocytoma. We found that gemistocytic tumor cells showing an ‘astrocyte-like’ state co-occur with perivascular contained T cells and immune-stimulatory macrophages in IDHmt astrocytomas. The transcriptional profiles of these tumor cell subsets and macrophages point towards a CNS specific defense mechanism of glial cells associated with T cell exclusion.
OS13.6.A SURVIVAL IS ASSOCIATED WITH CIRCULATING B-CELLS AND AGE AMONG OLIGODENDROGLIOMA PATIENTS
Authors: Taylor J W , Warrier G , Hansen H , McCoy L , Rice T , Guerra G , Francis S , Clarke J , Bracci P , Kelsey K , Wrensch M , Molinaro A , Wiencke J 
Published: September 2023

Abstract

BACKGROUND
Though patients with oligodendroglioma have improved outcomes versus astrocytomas, there remains marked variability in their survival experience that is unexplained by known prognostic factors. Immunomethylomics uses DNA methylation as a substitute for flow cytometry to characterize immune cells in whole blood. We applied immunomethylomics to archived samples from patients with oligodendroglioma enrolled in the UCSF Adult Glioma Study (AGS) to assess the impact of immune-related characteristics on prognosis.

MATERIAL AND METHODS
We used immunomethylomics to evaluate immune cell fractions in archived whole blood from the AGS. We identified 141 patients with oligodendroglioma (IDH and TERT mutant, and 1p19q co-deleted) and compared them to other adult diffuse glioma subtypes (N = 347) and controls (N = 454).

RESULTS
The median age at diagnosis of the 141 oligodendrogliomas was 44 years (range 19 - 83), 57% were male, and 75% white. A total of 60% of patients had received chemotherapy, with or without radiation, prior to blood draw and 25% were taking dexamethasone at the time of sample collection. Oligodendroglioma patients had lower proportions of B cell fractions (total, memory, naïve) than people without glioma. Still, there were higher proportions of B cell (total, memory) and lymphocyte fractions (total, CD4-total and memory), basophil, eosinophil, and NK cells in oligodendrogliomas compared to other glioma subtypes. Median overall survival for the oligodendrogliomas was 17.5 years (95% CI 16.9 - NA) with median follow-up time of 12 years (95% CI 11 - 14). Recursive partitioning analysis delineated two statistically significantly different survival groups for oligodendroglioma patients based on an interaction between age at diagnosis and B naïve cell proportions. The group with the best outcome had a median survival of 24.2 years (95% CI 20.4 - NR) comprised of patients &lt;/= 42 years of age with a higher proportion of B naïve cells (&gt;1.91%) (N = 37). The group with the worse outcome had a median survival of 16.9 years (14.8 - NR) and included patients &lt;/= 42 years of age with a lower proportion of B naïve cells (&lt;/= 1.91%) (N = 28) OR patients &gt; 42 years of age (N = 76) (p = 0.00032). Additionally, proportions of lymphocyte fractions (CD4 and CD8 total, memory, and naïve) were significantly lower in the worse outcome group. Grade or prior treatment did not differ between the groups.

CONCLUSION
We identified an interaction between age and B naïve cell proportions that distinguished survival outcomes for patients with oligodendrogliomas. Immunomethylomics is a powerful tool for assessing immune status and these findings suggest that blood-immune characteristics may play a vital role in the prognosis of patients with gliomas.
P02.03.A INTEGRATIVE (EPI)GENETIC ANALYSIS OF INVASION-PROMOTING GENES IN HIGH-GRADE GLIOMA CELLS
Authors: Müller T , Mittelbronn M , Hau A 
Published: September 2023

Abstract

BACKGROUND
DNA methylation-based analyses of brain tumors including glioma is currently the gold standard for diagnostic classification. Aberrant epigenetic alterations can lead to inappropriate gene expression and thereby promote tumorigenesis. Glioblastoma (GBM), CNS WHO grade 4, is characterized by a very diffuse infiltrative growth into the adjacent brain parenchyma. Due to these highly invasive but also genetically heterogeneous and plastic cellular phenotypes, standard treatment, including maximum resection, radio- and chemotherapy, has limited efficacy. It can be assumed that intertumoral or molecular heterogeneity of gliomas on transcriptional level is caused by altered epigenetic changes within the individual tumor cells. Considering this, we hypothesize that tumor cell invasion being one of the critical hallmarks of highly malignant gliomas goes along with altered DNA methylation status and following from this changes in invasion-promoting transcript expression.

MATERIAL AND METHODS
We performed several in vitro screening approaches to describe the genetic, epigenetic and transcriptional status of invasion glioma cells. Within our first approach, we isolated fractions of glioma cells with increased invasive properties and performed Illumina® array-based methylation profiling as well as RNA sequencing analysis to identify methylation-driven, invasion-associated gene sets. During a second approach, the inducible CRISPR/Cas9 Synergistic Activation Mediator (SAM) system and a genome-wide sgRNA library were introduced in a serum-free glioma cell line. During CRISPR/Cas9 SAM mediated single-gene activation cells were subjected to a transwell invasion assay and sgRNA representation within the invasive glioma cell fraction was determined by next-generation-sequencing (NGS).

RESULTS
In this study, we integrated Illumina HumanMethylation850K methylation data, RNA-sequencing data and invasion-promoting sgRNA representation data. By systemic screening we identified an invasion-promoting gene signature and the correlating promotor DNA-methylation status. Future studies will be directed to further investigate the impact of identified genes in invasion of high-grade glioma cells.

CONCLUSION
This integrative experimental approach complements existing genome-wide inhibitory screens as well as incorporates for the first time DNA-methylation profiles and transcriptome analysis with gene-sets identified by CRISPR/Cas9 activation screen and thereby holds the potential to identify novel invasion-essential candidate genes.
P02.08.B ROLE OF THE PROTEIN NETWORK OF THE PARACASPASE MALT1 IN SELF-RENEWAL PROPERTIES OF GLIOBLASTOMA CELLS
Authors: Kerhervé M , Gavard J 
Published: September 2023

Abstract

BACKGROUND
Glioblastoma is the most common and most aggressive brain tumor in adults. Despite standard treatment combining surgery, radiotherapy and chemotherapy, relapse is inevitable, making this cancer a major public health issue. Within this tumor reside cells with a stem-like character, called GSCs for Glioblastoma Stem-like Cells, which maintain, initiate, and propagate the tumor, making them a potential target to hinder glioblastoma. These cells constitute a reservoir of heterogeneous cells capable of differentiating into neural lineage cell types. In this context, lysosomal homeostasis plays a key role in the maintenance of the GSC reservoir. Furthermore, previous studies demonstrated that the paracaspase MALT1 is a major regulator of lysosomal homeostasis: MALT1 is required for GSC viability as well as for the maintenance of stemness by modulating the abundance of lysosomal proteins. This current study addresses the issue of the balance between self-renewal and viability of GSCs. In addition, this project aims to identify and characterize an actor of self-renewal mechanisms that does not impact the survival-death balance of primary cells from patients.

MATERIAL AND METHODS
An initial analysis combining DepMap and String Network databases allowed us to select potential relays of the MALT1 activity in GSCs. A functional screening was then set up and performed in GSCs derived from patients to identify among 20 candidates those regulating cell expansion without impacting viability.

RESULTS
The strategy of functional screening allowed the selection of 14 genes with a global viability effect, while only 2 genes were affecting self-renewal while sparing viability in vitro. We next pursued with one of the identified targets, from the IKK family. The involvement of this serine/threonine protein kinase was then confirmed in multiple patient cells and in CRISPR-mediated knock-out. Future experiments will decipher the involvement of this kinase in the self-renewal properties of GSCs.

CONCLUSION
This study highlights a putative candidate specifically operating on the self-renewal of glioblastoma cells, that does not affect the life-death balance. Future investigations will permit to characterize and understand the underlying mechanisms.
P02.19.A ASSESSMENT OF HEAT SHOCK PROTEIN HSP70 EXPRESSION IN THE GLIOBLASTOMA STEM-LIKE CELLS
Authors: Shevtsov M , Yudintceva N , Nechaeva A 
Published: September 2023

Abstract

BACKGROUND
Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by poor prognosis. The search of biomarkers for early diagnosis and as targets and for tumor theranostics due to the aggressiveness of the disease is urgently needed. One of the promising targets for therapy of brain tumors is 70 kDa heat shock protein (Hsp70), which has been found on the plasma membrane of various tumor cells (including GBM) but not on normal cells. GBM, like other types of tumors, has cellular heterogeneity. In particular, it contains tumor stem-like cells (TSLCs), which are considered analogues of healthy stem cells and are largely responsible for disease progression, recurrence and mediate resistance to ongoing therapy. In the present work, the histological sections of human GBM were analyzed to identify and quantify the colocalization of the heat shock protein Hsp70 with TSLC markers using the Opal Multiplex analysis system.

MATERIAL AND METHODS
Histological sections were prepared in accordance with the standard protocol using postoperative material from patients with newly diagnosed GBM (n=15). Zones of necrosis and living tissue were identified on tumor sections. Based on a phenotyping map using the Pathology Views™ fluorescence image analysis software, Hsp70 colocalization was quantified with Nestin and Sox2 in the areas of necrosis and viable tissue employing primary antibodies (anti-Hsp70, anti-Nestin and anti-Sox2). The preparations were stained using an Opal 3-Plex Manual Detection Kit according to the manufacturer's protocol.

RESULTS
The expression of Hsp70 and TSLC markers (Nestin and Sox2) was detected. The obtained quantitative data did not reveal significant differences in the distribution of cells expressing Hsp70 in the areas of necrosis and viable tissue (36 and 41%, respectively), which may indicate their uniform distribution in the tumor. Sufficiently high colocalization of Hsp70 with Nestin and Sox2 factors was detected mainly in the zone of viable tissue (57 and 30%, respectively).

CONCLUSION
Colocalization of Hsp70 with TSLC markers indicates the prospects for further studies of Hsp70, as well as its use for targeted therapy of malignant brain tumors.
P03.10.A IDENTIFYING DNA DAMAGE RESPONSE (DDR) RELATED FACTORS ESSENTIAL FOR RADIOTHERAPY RESPONSE IN GLIOBLASTOMA USING A NOVEL CRISPR/CAS9 LIBRARY, DDRKOL
Authors: Kok I , Bayraktar C , Durgun A , Aksu A C , Kayabolen A , Yedier Bayram O , Sur Erdem I , Bagci Onder T 
Published: September 2023

Abstract

The standard treatment of glioblastoma, which is the most common and aggressive primary brain tumor, includes combined radiotherapy (IR) and chemotherapy (temozolomide (TMZ)) after surgery. The basic working principle behind radiotherapy is to induce DNA double-strand breaks (DSBs), which is the most lethal form of DNA damage and the primary cause of cell death, and to finally kill cancer cells. Additionally, exposure to IR induces the activation of a multicomponent signal transduction network of DNA damage response (DDR). In this study, we developed a new CRISPR/Cas9-based tool to investigate the role of DDR mechanisms and identify novel genes affecting radiotherapy response in glioblastoma. We generated a focused sgRNA library termed DDRKOL (DNA Damage Response Knock Out Library) targeting the DDR pathway to identify novel modulators of TMZ and radiotherapy response with CRISPR/Cas9 screening in vitro. DDRKOL includes a total of 9000 sgRNAs targeting 888 genes (10 sgRNA/gene) containing all DNA damage-related pathways, curated from Gene Ontology Consortium, 35 sgRNA positive controls targeting cell essential genes, 90 non-targeting control sgRNAs, and 35 internal control genes. The composition and coverage of the library were validated, and DDRKOL was utilized to investigate modulators of TMZ response in U87MG cells. Our future work will involve characterizing different effects of DDRKOL members in radiotherapy response, in different subtypes of gliomas with different genetic

BACKGROUND
s. Cell response to radiation is important, therefore optimization experiments were performed with different radiation doses in A172, U373, and U373-60Gy cell lines to choose the optimal dose suitable for screening. Accordingly, 2 Gy radiation was chosen for large-scale screens. DDRKOL will serve as an important tool to study not only glioblastoma but also many other diseases that threaten global health. Discovering novel genes that affect the therapy response will be stimulating because it will help understand how to overcome therapy resistance and develop new strategies to design effective therapeutic strategies, which can ultimately translate into clinics.This study is supported by TUBITAK 1001 program (#221S439).
P03.13.B RADIOTHERAPY-INDUCED COGNITIVE IMPAIRMENT IN CANCER PATIENTS: IS SENESCENCE INVOLVED?
Authors: Kuil L E , de Gooijer M C , van Heijningen C , Compter A , van Tellingen O , Schagen S B 
Published: September 2023

Abstract

BACKGROUND
Patients with primary and metastatic brain tumors that survive may experience cognitive problems including progressive impairments in memory, attention, and executive function attributed to radiation-induced brain injury. Irradiation is known to bring tumor cells into senescence, a state of permanent proliferation arrest that is accompanied by metabolic changes and a secretory phenotype, which may cause inflammation. We hypothesize that senescence in the healthy brain, caused by whole brain radiotherapy (WBRT), plays an important role in cognitive problems.

MATERIAL AND METHODS
In this pilot experiment the effect of three different dosages (12, 30 and 60 Gy) of fractionated WBRT was assessed at different intervals after irradiation using male C57BL/6JRj mice. We performed senescence associated β-glucosidase (SA-β-gal) staining, cytokine ELISA assays, RT-qPCRs and immunohistochemistry to identify cellular senescence in the healthy brain.

RESULTS
Unexpectedly, no clear indications of induced cellular senescence in the brain could be observed by assessing SA-β-gal, cytokines and senescence markers on RNA and protein level. However, we found persistent DNA damage foci, microglial activation, a reduction in oligodendrocyte precursor cells and neurogenesis in the subvertricular zone.

CONCLUSION
No clear evidence for cellular senescence after WBRT was detected in the mouse brain, whereas profound effects were found on glia, neurogenesis and DNA damage. Difficulties in detecting cellular senescence might be explained by differences in presence of so-called markers for cellular senescence in the brain, or the rare nature of senescence in vivo. In future studies neurobiological changes will be assessed at later time points after WBRT, using a senescence reporter mouse and will be combined with behavioral studies.
P06.01.A HAS THE USE OF DUAL AGENT IMMUNOTHERAPY BEEN ALTERING THE INCIDENCE OF MELANOMA BRAIN METASTASES? A POPULATION-LEVEL ANALYSIS AND SINGLE-INSTITUTION VALIDATION
Authors: Salmanian S , Bhanja D , Wilding H , Moeckel C , Hong J , Dao J , Baroz A , Stahl C , Sivik J , Drabick J , Pameijer C , Mansouri A 
Published: September 2023

Abstract

BACKGROUND
Dual agent immunotherapy (dIT) has revolutionized the treatment of advanced melanoma, with recent clinical trials demonstrating its effectiveness in managing small, asymptomatic melanoma brain metastases (MBM) in the upfront setting. However, its potential role in preventing MBM has not been thoroughly investigated.

MATERIAL AND METHODS
A retrospective analysis was performed on the TriNetX database, which compiles clinical data from 92 healthcare organizations. Melanoma patients without MBM at diagnosis were divided into two groups: those who received ipilimumab alone (single immunotherapy, sIT) and those who received ipilimumab and nivolumab (dIT). The study compared the frequency of new asynchronous MBM diagnosis among the cohorts and analyzed the median overall survival (OS) of MBM patients using Kaplan Meier analysis and log-rank tests. Additionally, a retrospective cohort validation study was conducted on a sample of randomly selected patients treated at a single institution between 2012-2019. Electronic medical records were reviewed for demographics, melanoma treatment history, and clinical outcomes.

RESULTS
TriNetX identified a total of 732,555 melanoma patients, including 717,408 who did not receive immunotherapy (no-IT), 4,585 who received dual immunotherapy (dIT), and 4,101 who received single immunotherapy (sIT). The mean ages at melanoma brain metastases (MBM) diagnosis were 66.9, 61.5, and 63.2 years for each respective group. The prevalence of MBM was 1.5%, 9.6%, and 15.6% for no-IT, dIT, and sIT groups, respectively (p &lt; 0.0001). DIT was associated with a lower rate of MBM compared to sIT(OR [95%CI], 0.64 [0.61-0.77]). After propensity score matching, the median overall survival for MBM patients was not statistically different between the no-IT, dIT, and sIT groups (347, 400, 414 days, respectively). In the validation group of 90 patients (30 no-IT, 27 sIT, 34 dIT), predominantly male (61.5%), stage III (62.5%), the prevalence of MBM was 13.8%, 59%, and 38.2% for each respective group for developing MBM (OR [95%CI], 2.35 [0.84-6.60], p=0.08). The mean time between immunotherapy initiation and MBM was 305 days for dIT and 500 days for sIT for patients who developed MBM.

CONCLUSION
The results suggest that using a combination of ipilimumab and nivolumab immunotherapy could be effective in reducing the incidence of brain metastases in advanced melanoma patients. Further investigation is needed to explore the potential preventive role of dIT in MBM development in this patient population.
P06.16.A FRACTIONATED IRRADIATION ACTIVATES GLIOMA IMMUNE MICROENVIRONMENT AND BOOSTS ANTIGEN-SPECIFIC T CELL RESPONSE IN MALIGNANT GLIOMAS
Authors: Elmadany N , Michel J , Sonner J , Ovchinnikova S , Sanghvi K , Bernhardt I , Zhang X , Jähne K , Ott M , Sati A , Anders S , Breckwoldt M , Platten M , Sahm K 
Published: September 2023

Abstract
Several ongoing clinical trials employ vaccine, immune checkpoint inhibitors and/or adoptive T cell therapy to treat glioblastoma. However, the impact of irradiation as part of the standard of care on the immune microenvironment and response to immunotherapy is ill-defined. In the present study, we analyzed the post-irradiation changes in the GBM immune microenvironment, and further elucidated the impact of fractionated tumor irradiation (FIR) on the efficacy of T cell-mediated immunotherapies in experimental gliomas. FIR boosted the response of orthotopically-implanted GL261 tumors expressing the tumor-associated antigen glycoprotein (gp)100 to a gp100 vaccine. Furthermore, FIR enhanced the infiltration of GL261-gp100 gliomas by adoptively-transferred gp100 T cell receptor-transgenic T cells, which also displayed a more activated and less exhausted phenotype post-irradiation. Single cell transcriptomic studies of tumor-infiltrating CD45+ leukocytes revealed that FIR promotes the expression of leukocyte-endothelial adhesion molecules in T cells, and transcripts encoding proinflammatory M1-like genes in tumor-associated microglia and macrophages. Collectively, our preclinical findings support the use of FIR with to increase response to T cell-based immunotherapies.