bluuebunny/crossref_metadata_2025_split huggingface dataset:
P02.07.A IMMUNOLOGICAL AND EPIGENETIC FEATURES OF RADIATION NECROSIS IN GLIOBLASTOMA PATIENTS
Authors: Mildenberger I C , Perez-Montessoro V , Bunse L , Sahm F , Ratliff M , Giordano F A , Platten M 
Published: September 2023

Abstract

BACKGROUND
Radiation necrosis is a rare but highly relevant complication after radiotherapy in glioblastoma patients. Immunological and epigenetic features of these delayed tissue changes are not yet understood and no reliable biomarker to identify patients at risk of developing radiation necrosis is available.

AIM
To assess methylation patterns, T cell receptor repertoires and immune cell infiltration in patients with radiation necrosis and identify possible predictive epigenetic and immunological features.

METHODS
405 glioblastoma patients who underwent tumor resection between 2008 and 2018 were retrospectively screened for histology-proven or MR-spectroscopy/FET-PET-diagnosed radiation necrosis (RN). After identifying patients with RN, a control cohort matched for age, sex and MGMT-status was established. For both cohorts, epigenetic profiles of primary tumor tissues were assessed via DNA methylation arrays. Immunohistochemistry was performed for CD3, CD8, CD68 and CD163. For patients with histology-proven RN, additional TCRß-bulk sequencing of DNA extracted from FFPE tissue of primary tumor and RN was performed.

RESULTS
14/405 (3.5%) patients were identified with RN. The MGMT-promotor was hypermethylated in 50% of patients. The most prominent methylation subclasses of primary tumor tissue of patients in the RN cohort were GBM RTK I and GBM RTK II (5/8). None of these tumors was classified as GBM MES, which in contrast was the most prominent subclass in the control cohort (4/8). T cell infiltration and microglia/macrophage density did not differ between RN cohort and control cohort (CD3: 15.04 vs 20.28 counts/ROI, p= 0.55; CD8: 7.32 vs 8.3, p= 0.82; CD68: 7.42 vs 11.86, p=0.13; CD163: 14.29 vs 16.2, p= 0.73). In total, 1762 unique productive TCRß sequences were identified, overall productive clonality was low (0.2, range 0.06-0.4). A clonal expansion of specific TCRs from primary tumor to RN was identified in 2 patients. A more clonal repertoire in RN was observed in 50% of patients. 2 TCRß sequences were shared between 2 different patients, no sequences were shared between more than 2 patients.

CONCLUSION
Our data indicate that the methylation subclasses RTK I and RTK II may constitute a risk factor for RN whereas a MES subclass may be protective. Immune cell profiles in primary tumors of GBM patients that develop radiation necrosis during their treatment do not differ from primary tumors of control patients. TCR repertoire shows an overall low abundance of TCRs with polyclonal repertoires, but clonal expansion could be detected in a subset of patients, suggesting antigen-specific T cell responses to RN. Further analysis and validation of these findings in a larger cohort is ongoing.
P02.09.A THE PLASTICITY AND SURVIVAL OF GLIOBLASTOMA STEM CELLS DEPEND ON GD3, WHOSE INHIBITION IS SYNERGISTIC WITH RADIO-CHEMOTHERAPY
Authors: Hein V , Baeza-Kallee N , Berges R , Appay R , Snacel-Fazy E , Tchoghandjian A , Figarella-Branger D , TABOURET E 
Published: September 2023

Abstract

BACKGROUND
Glioblastoma is the most frequent and aggressive primary brain tumor in adults. No curative treatment is available, and the identification of relevant therapeutic targets is an unmet medical need. Our team previously demonstrated that A2B5 positive cells have a stemness behavior, with self-renewal, multipotency ability, and tumor initiation ability in vivo. A2B5 is recognizing different gangliosides, including GT3 and its precursor GD3. However, A2B5 is an IgM, that cannot be used directly as a therapeutic strategy.Then, our aim was to demonstrate that GD3 was a relevant and actionable target for glioblastoma stem cells (GSC).

MATERIAL AND METHODS
We used the GCS lines we already derived from fresh patient samples. Then, we sorted GD3 positive cells from patient samples to derive new GCS lines and explore GD3 properties. We downregulated the GD3 synthase (ST8Sia1) by using Sh. The tumor cells were orthotopically engrafted in nude mice.

RESULTS
First, we showed that GD3 was expressed by 15 to 20% of cells from patient tumor bulks but not by normal brain. Then, we showed that 60% to 90% of GSC expressed GD3. Importantly, this fraction dropped between 20 to 40% after cell differentiation. We sorted GD3 positive cells from patient samples and showed their ability to exhibit stemness properties including the generation of neurospheres (expressing stem cell markers like SOX2 and nestin), clonogenicity, and tumorigenicity after orthotopic engraftment. We also demonstrated that GD3+ cells had plasticity properties with differentiation and dedifferentiation abilities depending on their culture medium. Then, by using shST8SIA1 in GSC lines, we showed that the reduction of ST8Sia1/GD3 was associated with a decreasing size of spheres, and reduced clonogenicity and migratory capacities. Moreover, the reduction of ST8Sia1/GD3 expression increased the temozolomide and radiotherapy sensitivity in vitro with a synergistic effect. Finally, the reduction of ST8Sia1/GD3 expression in U87 cell line was associated with an increasing mouse survival after orthotopic engraftment in nude mice.

CONCLUSION
Taken together, our results strongly suggest that GD3 and ST8SIA1 are essential for GSC properties. GD3 appears to be an interesting and actionable stem cell marker, opening promising therapeutic development for GBM patients.
P02.10.B REGULATION OF GLIOBLASTOMA CANCER STEM CELLS AND PROLIFERATION THROUGH VOLTAGE GATED SODIUM CHANNEL
Authors: Brandalise F , Cianci F , Biella C , Pastorelli E , Giammello F , Amat di San Filippo M , Mazzanti M 
Published: September 2023

Abstract

BACKGROUND
Glioblastoma Multiforme (GBM) is one of the most devastating cancer known. Despite decades of research, we still lack an efficient treatment. The heterogeneity in the cell-type composition along with the presence of a subpopulation of cells with high tumorigenic capacity named glioblastoma stem cells (GSCs), make GBM extremely hard to treat. An extensive body of works supports the hypothesis that an aberrant functional expression of membrane ion channels mediates the progression of solid cancer tumors. Potassium, calcium, and chloride channels have been largely correlated with carcinogenesis. However, little is known about the voltage-gated-sodium channel (Nav) in GBM. In fact, the role of this membrane ionic permeability in the GBM progression and relapse is yet to be unveiled.

MATERIAL AND METHODS
Experiments have been performed on human GSCs obtained from surgical specimen at the Neurosurgery Department of IRCCS-AOU San Marino IST (Genova, Italy), from patients who had not received therapies before intervention. The mRNA profile of the cell lines as well as the transcript expression of stemness markers were evaluated both in control condition and in the presence of the Nav channel blocker Tetrodotoxin (TTX, 30μM). The protein content for the stemness markers and their intermediates was quantified using Western-Blot analysis. Nav-mediated inward currents were recorded from single cells and measured in voltage clamp by applying consecutive voltage steps of +10 mV from a holding potential of -70 mV and up to +60 mV. Transient inward current was calculated on the peak subtracting the baseline leak currents. Current density (pA/pF) was calculated as the ratio between the peak current recorded at +20 mV and the capacitance of the cell. Resting membrane potential (RMP) was also assessed in each recorded cells.

RESULTS
We have identified a subpopulation of GBM cells (in GBM Proneuronal subtype) that functionally expressed TTX-sensitive inward currents. Transcriptomics investigation reveals the significant expression of SCN1A mRNA. We have shown that Nav density positively correlates with the RMP in GBM cells. Additionally, Nav blockade promotes glioma cell proliferation and G1/S accumulation. Pharmacological blockade of Nav-mediated currents has shown a significant impact on some stemness markers both at the mRNA and protein expression level. A regulatory downstream pathway, modulated by Nav has also been investigated.

CONCLUSION
Our evidences suggests that Nav-mediated currents is significantly expressed in a subpopulation of GBM cells. As a result, the present study intends to demonstrate how Nav plays a fundamental role during GBM progression and correlates with the tumor resistance to treatments.
P02.13.A THIRD-GENERATION SEQUENCING OF BRAIN TUMORS ON SINGLE-CELL LEVEL
Authors: Schoebe N , Ippen F M , Ritter M , Tang Y , Wick W , von Deimling A , Sahm F , Berghaus N 
Published: September 2023

Abstract

BACKGROUND
Intratumor heterogeneity (ITH) in brain tumors has been suggested to increase tumor adaptability and thus drive tumor progression and treatment resistance. Single cell sequencing has widened the range of possibilities in detecting clonal diversity and tumor evolution. As of now, most single cell experiments rely on next-generation sequencing (NGS) platforms. However, they detect only short RNA/DNA fragments that do not show the full picture of existing transcript isoforms or genetic variants. New, long-read sequencing methods such as Oxford Nanopore Technologies (ONT) or PacBio sequencing may now enable novel approaches to single cell experiments. We here set-out to complement existing single-cell analysis workflows and data pipelines with long-read sequencing approaches. We compare and integrate the data output in order to advance the understanding of ITH in brain tumors, paradigmatically demonstrated on meningioma.

MATERIAL AND METHODS
We set up a pipeline for single-cell/nuclei RNA long-read sequencing on ONT devices. First, libraries as derived from the 10x genomics workflow were employed, subsequently followed by independently generated libraries. To widen the insight into ITH on multiple levels, we next assessed single-cell/nuclei DNA for small somatic variants and methylation along a modified version of this platform.

RESULTS
Long-read RNA-sequencing yielded results comparable to short-read sequencing. Depending on sample quality, higher reads per cell and library were required for ONT sequencing to achieve similar discrimination of clusters by expression. On the other hand, cell-type specific isoforms could be identified. Specific small somatic variants known from bulk sequencing data were identified when the region was sufficiently covered in the RNA data, which was naturally more often achieved in long read than short-read data. Thus, cell-specific variant calling was enabled in a high-throughput pipeline. Maintaining the methylation information in DNA sequencing could so far only be set-up in a plate-based approach.

CONCLUSION
Using these approaches, we resolved features of ITH and clonal tumor evolution, aiding us in gaining a broader understanding of brain tumor biology. Furthermore, it showed novel approaches that could possibly be implemented in routine diagnostic procedures in the future. These novel techniques could be applied to other tumor entities and possibly further omics layers such as single cell proteomics and metabolomics could be added to gain a greater understanding of different tumor types and their clonal diversity.
P02.16.B COMPENSATORY CROSS-TALK BETWEEN AUTOPHAGY AND GLYCOLYSIS REGULATES SENESCENCE AND STEMNESS IN HETEROGENEOUS GLIOBLASTOMA TUMOR SUBPOPULATIONS
Authors: Senthil H , Martell E , Kuzmychova H , Kaul E , Venugopal C , Singh S K , Sharif T 
Published: September 2023

Abstract

BACKGROUND
Despite tremendous research efforts, the successful targeting of aberrant tumor metabolism in clinical practice has remained elusive. Tumor heterogeneity and metabolic plasticity may play a role in the clinical failure of metabolism-targeting interventions for treating cancer patients. Moreover, compensatory growth-related processes and adaptive responses exhibited by heterogeneous tumor subpopulations to metabolic inhibitors are poorly understood. Hence, a deeper understanding of the cellular adaptations in response to metabolic interventions is urgently required to develop more effective therapeutic options against deadly glioblastoma (GBM) brain tumors.

MATERIAL AND METHODS
Using clinically-relevant patient-derived GBM models, we discovered a cross-talk between glycolysis, autophagy, and senescence. Protein and mRNA expression of glycolytic enzymes and stemness markers were measured using western blot and bioinformatic analysis, respectively. We were able to measure senescence with the use of β-galactosidase staining and upregulation of cell cycle regulators, such as p21/CDKN1A and p16/CDKN2A. Autophagy flux and EGFP-MAP1LC3B+ puncta formation analysis was used to determine autophagy induction.

RESULTS
We found that stem cell-like GBM tumor subpopulations possessed higher basal levels of glycolytic activity and increased expression of several glycolysis-related enzymes including, GLUT1/SLC2A1, PFKP, ALDOA, GAPDH, ENO1, PKM2, and LDH, compared to their non-stem-like counterparts. Importantly, the mRNA expression of glycolytic enzymes positively correlates with stemness markers (CD133/PROM1 and SOX2) in patient GBM tumors. While treatment with glycolysis inhibitors induced senescence in stem cell-like GBM tumor subpopulations, these cells maintained their aggressive stemness features and failed to undergo apoptotic cell death. Moreover, we determined that inhibition of glycolysis led to the induction of autophagy in stem cell-like GBM tumor subpopulations, but not in their non-stem-like counterparts. Similarly, blocking autophagy in stem cell-like GBM tumor subpopulations induced senescence-associated growth arrest without hampering stemness capacity or triggering apoptosis while reciprocally upregulating glycolytic activity. Combinatorial treatment of stem cell-like GBM tumor subpopulations with autophagy and glycolysis inhibitors blocked the induction of senescence while drastically impairing their stemness capacity which drove cells towards apoptotic cell death.

CONCLUSION
These findings identify a novel and complex compensatory interplay between glycolysis, autophagy, and senescence that helps maintain stemness in heterogeneous GBM tumor subpopulations and provides a survival advantage during metabolic stress. This work was supported by the Cancer Research Society (CRS).
P02.21.A A BIOBANK OF IDH1-MUTANT CELL LINES FOR STUDYING TUMOR HETEROGENEITY, PROGRESSION AND TARGET IDENTIFICATION
Authors: Garcia L , Pineau D , Hideg S , Rigau V , Darlix A , Bauchet L , Duffau H , Hugnot J 
Published: September 2023

Abstract

BACKGROUND
Diffuse low grade gliomas are grade II brain tumors mainly affecting young patients. Most of these tumors have a mutation in the IDH1 gene (isocitrate dehydrogenase 1) gene causing differentiation defects and cell accumulation in the brain. These tumors are classified as astrocytomas or oligodendrogliomas and often progress into high grade gliomas (i.e IDH1-mutant grade IV astrocytomas and grade III oligodendrogliomas). We and others have shown that IDH1-mutant diffuse low grade gliomas contain 3 types of tumoral cells, resembling astrocytes, oligodendrocytes and neural progenitors, the formation of which is regulated by Notch signaling (Augustus et al, 2021, Cells, Suvà et al, 2020, Cancer Cell). There is a significant lack of cellular tools to study these tumors in vitro, hindering the development of new therapies. We aimed to address this by creating a fully annotated biobank of IDH1-mutant cell lines derived from different grades of tumors and carrying various rare mutations.

MATERIAL AND METHODS
We derived and collected 8 IDH1-mutant cell lines (4 astrocytomas grade II/III/IV and 4 grade III oligodendrogliomas). These cell lines were characterized using bulk RNA sequencing under different culture conditions, single-cell RNA sequencing to determine their cellular heterogeneity, and immunofluorescence and western blotting.

RESULTS
Our findings show that these IDH1-mutant cell lines exhibit different growth rates and morphologies and are phenotypically related to brain radial glial cells, oligodendrocyte progenitor cells, or more mature oligodendrocytes. Some of these cell lines are multipotent and contain astrocyte-like and oligodendrocyte-like cells. Our current work aims to purify different cell subpopulations to study their properties and plasticity. Additionally, we are analyzing pathways and differentially expressed genes involved in IDH1-mutant cell quiescence and malignant progression.

CONCLUSION
In summary, we present a novel and deeply-annotated biobank of 8 IDH1-mutant cell lines derived from different grades of gliomas, providing an invaluable resource to study the cellular and molecular heterogeneity of IDH1-mutant gliomas, their progression to high-grade gliomas, and the identification of new therapeutic targets. We anticipate that our biobank may facilitate the development of personalized medicine for patients with IDH1-mutant gliomas and accelerate the discovery of new therapeutic strategies.
P02.23.A MDM2/X ALTERATIONS IN A MULTICOHORT RETROSPECTIVE ANALYSIS: A POTENTIAL TARGET OF TREATMENT IN PATIENTS WITH PRIMARY BRAIN TUMORS
Authors: Gomez Puerto D , Vega Cano K , Molina Lores G , Salva de Torres C , Lopez Valbuena D , Mirallas O , Gonzalez M , Vieito M 
Published: September 2023

Abstract

BACKGROUND
Primary brain tumors (pBTs) are a heterogeneous group of neoplasms with limited therapeutic alternatives. Disruption of the TP53 tumor suppressor pathway occurs in most high-grade gliomas. While TP53 loss-of-function mutations are poorly targetable, in 14% of patients this disruption is due to a Mouse double minute 2 homolog (MDM2) amplification. We aim to describe the characteristics of patients with MDM2 amplification and analyze its clinical and molecular factors using a cohort of patients with NGS data.

MATERIAL AND METHODS
Patients currently enrolled in the institutionally approved, retrospective multicenter study ESCAT-pBTs, which includes patients with pBT profiled used NGS methods (Foundation Medicine®, local NGS, Caris®, Oncomine®, copy number alterations (CNA), and fusion panels) were analyzed to determine the presence of MDM2 alterations (MDM2alt) and clinical factors including WHO 2021 diagnosis, sex, age, ECOG and other molecular findings.

RESULTS
A total of 493 pts with NGS and CNA were collected between Feb 2018 and January 2023 at 5 hospitals in Spain. The median age was 50.3 (range, 3.3-83.8), 14.2% had ECOG ≥2, 39.1% were women, 26.9% were 40 or younger, 21.5% had IDH1mut and 24.3% were methylated. The diagnosis of GBM (WHO 2021) was present in 68.8% of the patients and 33.8% had mutations or CNV in TP53. The overall prevalence of MDM2alt was 8.5%(42 pts), 92.9% gene amplifications, and 7,1% mutations. No differences were found in gender proportion between MDM2alt and no MDM2alt (Female: 37.1% vs 39.4% p:0.64) and no major variance in the percentage of ECOG ≥2 (16.6% vs 17.6% p:0.77). Prevalence in patients with GBM was: 11.8% vs 1.3% in patients with other pBTs. Concurrent mutations in MDM2alt patients were: CDK-4 copy number gain in 76,2%; a similar proportion of mutations in PI3KCA/PTEN was observed compared with the non-MDM2alt population in the pts with GBM (38,3% vs 50%, p:0.15). No significant variation was found in EGFR amplifications (42,9% vs 32% p:0.10) and EGFR mutations GBM MDM2 and no MDM2alt respectively (19% vs 18,7% p: 0.84). The median TMB was 5.22 (range from 2.24 to 13 mutations/megabase) in pts with MDM2alt and 4.76 (range from 1.26 to 53.7 mutations/megabase). 97,6% of the MDM2alt population was TP53 wild type.

CONCLUSION
MDM2alt is a common genomic event in patients with molecularly defined GBM and has a low prevalence in other pBT. The presence of MDM2alt is associated in our series with other genomic events such as PTEN loss, low TMB, and a high degree of CDK4 amplification. No statistically significant differences were found between the GBM MDM2alt and no MDM2alt subgroup in terms of molecular alterations and clinical aspects studied. These findings should inform the further clinical development of treatment strategies for MDM2alt pts.
P02.25.A THE IMPACT OF PSEUDOPROGRESSION IN OVERALL SURVIVAL IN GLIOBLASTOMA, EXPERIENCE IN OUR CENTER
Authors: Iruarrizaga Ovejas E , Iza Rodríguez E , Mañe J , Novo Sukia E , Fernandez R 
Published: September 2023

Abstract

BACKGROUND
After surgery, standard treatment for glioblastoma (GBM) is temozolomide (TMZ), initially concurrent with radiotherapy (Ct-Rt), and subsequently as maintenance therapy. For adequate tumour response assessment,, magnetic resonance imaging (MRI) is performed one month after completing Ct-Rt. Instead, the radiologic images obtained in that MRI can be difficult to interpret because they may show radiation-induced pseudoprogression (psPD) rather than disease progression.

METHODS
We analyzed retrospectively clinical and tumor biology characteristics of patients (pts) with GBM treated in our center with Ct-Rt +/- adjuvant Qt and their relationship with psPD (reported by RANO criteria) and impact in overall survival (OS)

RESULTS
From November-2016 to October-2022, we collected data for 82 pts (median age: 60 years) who underwent Qt-Rt exclusively (25 patients) or Qt-Rt and adjuvant temozolomide (57 patients). Pts characteristics were: male/female 61.4/38.6%, performance status (PS) 0/1/2 10.5/71,9/17.5%, total surgery/subtotal/biopsy 21.1 / 52.6 / 26.3%, MGMT methylated/ not methylated/not realized 52.7/ 45.6 /1.8 %, TERT mutated/not mutated/not realized 29.8 / 7 / 36.8 / 63.2 %, ATRX positive/negative/not realized 75.4 / 14 / 10.5 %, P53: positive/negative 25 / 75 %. After Ct-Rt, MRI for evaluation of response showed: no evidence of disease 13.4%, partial response 4.9%, stable disease 34.1%, progressive disease 18.3% and psPD 26.8%. In multivariate analysis, significant difference of OS were observed in function of type of surgery (better OS in R0; p=0.015), performance status (better OS in PS 0-1; P=0.006), MGMT methylation (better OS in methylated; p=0.037), and ATRX status (better OS in negative; p=0.05). OS was algo significantly increased in pts treated with Qt-Rt and adjuvant Qt (17.7 +/- 1.7 months) vs Qt-Rt exclusively (11.35 +/-1.62 months) (p=0.013). In pts with psPD, OS was significantly superior compared with pts with progressive disease after Qt-Rt treatment (16.6 vs 11.3 months, p=0.048). In our cohort no differences between MGMT methylation status and psPD were observed (MGMT methylated 54.5%/ 45.5 not methylated).

CONCLUSION
It is essential to differenciate between PsPD and PD because it may have a clinical impact on chemotherapy-based treatment and OS, but it is also neccesary to know patient characteristics and tumor biology in order to identify patients that will beneficiate from TMZ. &lt;!--EndFragment--&gt;