bluuebunny/crossref_metadata_2025 huggingface dataset:
OS12.5.A HIGH-GRADE GLIOMAS OCCURRING IN TEENAGERS AND YOUNG ADULT PATIENTS COMPRISE NOVEL MOLECULAR SUBGROUPS
Authors: Clarke M T , Pereira R , Mackay A , Grabovska Y , Clarke A , Nicoll J , Procter J , Moore A , Schagen J , Walker L , Roncaroli F , Ogunbigi O , Jacques T , Marshall L , Carceller F , Reisz Z , Al-Sarraj S , Vinci M , Kurian K , Ng H , Brandner S , Jones C
Published: September 2023

BACKGROUND

High-grade gliomas (HGG) arise in any central nervous system location, in adults and children, with a poor prognosis. HGGs in teenagers and young adults (TYA) are understudied; this project aimed to characterise these tumours and identify potential therapeutic targets.

MATERIAL AND METHODS

HGG samples (n=195, FFPE/FF, 13-30 years) were collected from national/international collaborators, excluding well-characterised entities (histone/IDH-mutant). DNA methylation profiling (Illumina EPIC BeadArrays, brain tumour classifier (MNP v12.5 R package), n=195) classified cases against a reference cohort. Calibrated scores guided workflows to characterise mutational landscapes including RNA-based ArcherDx fusion panel (n=112), whole exome sequencing (n=84), and histological reviews in parallel. Well-characterised entities (n=50, including IDH-mutant tumours (n=18), PXA (n=12)) were excluded.

RESULTS

Of the cases scoring >0.6, 64% classified as paediatric-type subgroups (pedHGG_RTK1A/B/C (30%), pedHGG_MYCN (10%), pedHGG_A/B (7%), pedHGG_RTK2A (6%)). 29% classified as subgroups more frequently seen in adults (GBM_MES (12%), GBM_RTK1/2 (3%)). 54% classified with a score <0.6. 38% cases were assigned to novel, recently identified, poorly-characterised subgroups with distinct methylation profiles and molecular features (HGG_B, HGG_E, ANTCON, GBM_CBM). Frequency comparisons to publicly available methylation data showed GBM_MES_ATYP tumours are TYA age-specific, in contrast to adult age-specific GBM_MES_TYP. Copy number profiling identified frequent changes across the cohort, including chromosomal gains (chr1q (54%), chr2 (22%), chr7 (41%)) and losses (chr10 (40%), chr13 (64%)). Focal amplifications included PDGFRA (12%), CDK4 (7%), MYCN/ID2 (3%), MYC (2%) and EGFR (0.7%) and the most common focal deletion was CDKN2A/p16 (12%). The most frequent glioma-associated somatic variants included TP53, PDGFRA, EGFR, and NF1. BCOR alterations were enriched compared to adult and paediatric reference cohorts. 10% cases showed a hypermutator phenotype, enriched in HGG_E. CDK4 amplifications were recurrent in GBM_RTK1 and GBM_CBM. Histology showed variable cytological and architectural features within novel subgroups.

CONCLUSION

TYA HGG comprise novel subgroups with distinct methylation profiles and molecular characteristics, representing opportunities to refine future treatment.
OS13.5.A MOLECULAR CHARACTERISTICS AND PROGNOSTIC BIOMARKERS OF CENTRAL NEUROCYTOMA
Authors: Krech M , Muench A , Teichmann D , Kuzman P , Münther M , Weber K , Wenger-Alakmeh K , Onken J , Vajkoczy P , Behling F , May S , Ntoulias G , Krauss J K , Atallah O , Esmaeilzadeh M , Mueller W C , Heppner F L , Koch A , Capper D , Kaul D , Paulus W , Plate K H , Beschorner R , Neumann J E , Hartmann C , Thomas C , Schweizer L
Published: September 2023

BACKGROUND

Central neurocytomas (CN) are intraventricular brain tumors predominantly arising in young adults. Long-term prognosis is favorable in the majority of cases, whereas a considerable proportion of patients experience disease progression. Progression risk is currently assessed morphologically based on the presence of histological signs of atypia and/or an elevated ki67 proliferation index (>3%). The molecular profile and etiology of CN remains elusive and molecular biomarkers for risk stratification have not been identified yet.

MATERIAL AND METHODS

We analyzed a retrospective multi-institutional cohort of 108 CNs by genome-wide DNA methylation profiling. DNA whole exome sequencing was performed in 12 cases. Follow-up information was available for 45 patients.

RESULTS

The histological diagnosis of neurocytoma was epigenetically confirmed in 96 cases. 12/108 cases (11%) were assigned to another methylation class based on the v12.5 Heidelberg Brain Tumor Classifier. Median age at diagnosis was 29 years, distribution among sexes was equal. Based on morphology and immunohistochemistry, tumors were diagnosed as classical CN (n = 71), CN with increased proliferation (n = 10) and atypical CN (n = 15). In 49/72 cases (68%, NA=24), an elevated Ki67 above 3% was reported. DNA whole exome sequencing revealed no recurrent genetic alterations. Most copy number profiles were balanced (n = 87), while some cases (n = 9) showed individual chromosomal alterations (e.g. chr. +5, n = 3). Tumor recurrence was observed in 16/45 (36%, NA = 51) of cases. Median progression-free survival (PFS) after the initial diagnosis was 72 months, none of the patients died (observation time: range 3 - 141 months). In 16 cases, surgery was followed by adjuvant radiotherapy, predominantly in patients with subtotal resection (STR: n = 11, 69%; GTR: n = 5). There was no significantly different outcome between classical and atypical CNs (n = 45; median PFS 73 months vs. 26 months, p-value = 0.067) and cases with low vs. elevated Ki67 (n = 45; median PFS NA vs. 72 months, p-value = 0.44).

CONCLUSION

Preliminary data of our study indicate that histological diagnosis and Ki67 proliferation rate were not associated with PFS in epigenetically defined neurocytomas. Further research using clinical and molecular covariates is necessary to identify novel biomarkers to predict recurrence risk and benefit from adjuvant radiotherapy.
OS13.7.A ONSET SYMPTOMS PREDICT SURVIVAL IN GLIOBLASTOMA PATIENTS -REAL WORLD DATA FROM 1719 PATIENTS IN THE SWEDISH BRAIN TUMOUR REGISTRY 2018-2021
Authors: Bruhn H , Rosenlund L , Tavelin B , Henriksson R
Published: September 2023

BACKGROUND

Glioblastoma, the most common malignant brain tumour in adults, still has a poor long term survival. Non-invasive clinical prognostic markers are important to more accurately predict a patient’s survival. The aim of our register-based real-life study was to explore if onset symptoms have a prognostic value for survival.

MATERIAL AND METHODS

Data on onset symptoms and survival, as well as known prognostic factors, were retrieved for all glioblastoma patients registered in the Swedish Brain Tumour Registry between 2018 and 2021. The prognostic impact of different onset symptoms were calculated using the Cox proportional hazard model.

RESULTS

Data from 1719 adults with pathologically verified glioblastoma were analysed. The median survival was 347 days (330-364). The two-year survival was 21.9 %. The most common onset symptoms were focal neurological deficits, cognitive dysfunction, headache, epilepsy, and signs of raised intracranial pressure, with some patients having multiple symptoms. Patients with initial cognitive dysfunction had a significantly shorter survival compared to patients without cognitive dysfunction (p<0.001). Patients with onset seizures had significantly longer survival compared to patients with no onset seizures (p<0.001). The differences in survival remained after Cox regressions adjusting for known prognostic factors. When comparing tumors of the same size, there was still a difference in survival between patients having or not having cognitive dysfunction.

CONCLUSION

This large real-life study shows that initial cognitive impairment is a negative, and epileptic seizure at onset is a positive prognostic factor for survival in glioblastoma patients.
P01.02.A WHOLE-BRAIN STRUCTURAL CONNECTIVITY PREDICTS COGNITIVE OUTCOMES IN PRETREATED PATIENTS WITH WHO GRADE 3 OR 4 GLIOMAS
Authors: Friedrich M , Stoffels G , Filss C P , Lohmann P , Mottaghy F M , Weiss Lucas C , Ruge M I , Shah N J , Caspers S , Langen K J , Fink G R , Galldiks N , Kocher M
Published: September 2023

BACKGROUND

Glioma patients frequently suffer from cognitive dysfunction potentially related to brain damage caused by tumor invasion or therapeutic intervention. We hypothesized that, as in other neurological disorders, the long-term outcome of cognitive functions in glioma patients critically depends on maintaining the structural connectivity of multiple interconnected networks.

PATIENTS AND METHODS

One hundred and twenty-one glioma patients (median age, 52 years; median ECOG score, 1) with histomolecularly characterized CNS WHO grade 3 or 4 gliomas were investigated after a median time of 14 months (range, 1-214 months) following first-line therapy, including resection and chemoradiation with alkylating agents. Hybrid amino acid PET/MR imaging using the tracer O-(2-[18F]fluoroethyl)-L-tyrosine, a brain-networks-based cortical parcellation into nodes, and advanced fiber tractography tools were used for constructing fiber-count-weighted connectivity matrices. Cognitive performance was measured by ten different tests concerning various cognitive domains. The connectivity matrices and the patients' test scores were applied to a machine learning-based model that identified critical fiber connections, cortical nodes, and involved networks for predicting cognitive performance in the assessed domains.

RESULTS

Compared to a matched cohort of healthy subjects, the cognitive scores of the patients were significantly lower in 9 of 10 cognitive tests. The most affected domains were executive function/concept-shifting (-72%), attention/processing speed (relative performance, -53%), followed by semantic word fluency (-25%), verbal working and semantic memory (-8 and -22%, respectively), and visual working memory (-11 to -20%). For each cognitive test, a subset of connections (median number, 254; range, 32-542) was identified whose fiber count sum showed a correlation with the actual scores in a linear model (median R2, 0.37; range, 0.16-0.44). Leave-one-out cross-validation confirmed the model's generalizability in 7 of 10 tests (median correlation coefficient for predicted compared to observed scores, 0.47; range 0.39-0.57). The distribution of critical cortical regions (node degree of ≥ 5 predictive edges), found in 90% of the cross-validation iterations, varied considerably between domains. These included predominantly left-sided cortical nodes of the visual, somatomotor, dorsal/ventral attention, and default mode networks. Highly critical nodes (degree of 15-20 predictive edges) included the default-mode network’s left temporal and bilateral posterior cingulate cortex.

CONCLUSION

These results suggest that the cognitive performance of pretreated glioma patients is strongly related to the structural connectivity within multiple brain networks and depends on the integrity of known network hubs.
P01.04.B LONG-TERM COGNITIVE TOXICITY IN SMALL CELL LUNG CANCER POPULATION: EARLY CLINICAL, NEUROIMAGING, AND SEROLOGICAL BIOMARKERS
Authors: Simó M , Vaquero L , Alemany M , Padrones S , Caravaca A , Padró A , Navarro A , Palmero R , Nadal E , Bruna J
Published: September 2023

BACKGROUND

Small cell lung cancer patients (SCLC) are treated with platinum-based chemotherapy, thoracic radiation, and some of them will also receive prophylactic cranial irradiation (PCI). Following therapy, nearly 45% of SCLC will exhibit moderate to severe cognitive impairment (CI). The aim of our study was to identify early biomarkers including clinical, serological and changes in brain structure associated with an increased risk of CI in SCLC population.

MATERIAL AND METHODS

Fifty-two SCLC were prospectively recruited from January 2019-December 2022 at our institution and evaluated at diagnosis using a neuropsychological battery (NPS), a structural MRI (T1-weighted), and serological markers including: a) cytokines (interleukin 1, 2, 6, 8, 10 and TNF alpha) and b) single nucleotide polymorphisms (SNP) of apolipoprotein E (APOE), multidrug resistance protein (MDR1) and brain-derived neurotrophic factor (BDNF). They were also clinically and NPS evaluated at 1-month following chemotherapy-before PCI, at 3-6 months, at one-year and at 2-3 years post-PCI. Voxel-based morphometry (T1-VBM) was used to analyze gray matter (GM) integrity.

RESULTS

Of the 52 patients included, 70% were male, median age was 65 years (46-82). Most (94%) had an ECOG PS ≤1. Half had hypertension (52%) and dyslipidemia (46%); one third (36%) moderate alcoholism and 25% were diabetic. Nearly half (54%) had limited stage and received cisplatin-based chemotherapy (52%). One third (29%, n=15) exhibited brain metastasis (BM): 4 at baseline (8%) and the other during follow-up (21%). Regarding smoking, median packs per year was 45 (12-100). Twenty-nine (56%) received cranial radiation, most of them (n=22, 42%) PCI (25Gy). Median overall survival was 18 months (2.5, 71); twenty-one (40%) were alive at the end of the study.Nearly half of our cohort (48%, n=25) exhibited CI: 31% (n=16) at baseline and another 17% (n= 9) during follow-up. CI group performed significantly worse in all the domains explored, especially in verbal memory (p < 0.001) and executive functioning (p < 0.001). Only smoking habit resulted significantly different between groups: CI patients were heavier smokers (p=0.04). VBM analysis showed that CI group exhibited significant GM damage in anterior and mid cingulate, superior, and inferior temporal gyrus, precuneus and fusiform gyrus. GM volume positively correlated with measures of cognitive functioning at baseline (all correlations, p <0.04). We found no differences regarding cytokines levels or SNPs of APOE, MDR1 and BDNF between groups.

CONCLUSION

This study confirms that nearly half of the SCLC population exhibit CI. Of greater interest, however, is the fact that most of them already exhibit CI before therapy that seem to correlate with GM alterations previously described in this population. In addition, smoking seems to be the most important clinical risk factor.
P01.10.B EMOTION RECOGNITION IN PATIENTS WITH LOW-GRADE GLIOMA: THE RELATIONSHIP WITH TUMOR LOCATION AND TUMOR VOLUME
Authors: Siebenga F F , van der Weide H L , Gelmers F , Rakers S E , Kramer M C A , Enting R H , Bosma I , van der Hoorn A , Jeltema H , Wagemakers M , Groen R J M , Spikman J M , Buunk A M
Published: September 2023

BACKGROUND

Patients with low-grade gliomas (LGG), generally function well at the time of diagnosis and have a favourable prognosis. However, LGG can have impact on cognitive functioning of patients. To date, little is known about social cognition (SC) in patients with LGG. Social cognitive functions allow the processing of complex social information, with as important element the ability to recognize other people’s emotional expressions. Inaccurate recognition or misinterpretation of emotional facial expressions can lead to impairments in social functioning, which can have adverse effects on quality of life. Therefore, the aim of the present study was to investigate emotion recognition in patients with LGG and the relationship with tumor location and tumor volume.

MATERIAL AND METHODS

125 patients with IDH mutated astrocytoma and oligodendroglioma grade 2 and 3, were matched with 169 healthy controls (HC). Tumor location and volume were evaluated pre-radiotherapy based on MRI scans. As a parameter for LGG volume the clinical radiotherapy target volume was used. SC was measured with a test for emotion recognition, i.e. the Facial Expressions of Emotion Stimuli and Tests (FEEST), after surgery. Descriptive statistics and between-group comparisons were performed.

RESULTS

On a group level, patients with LGG performed significantly lower on the FEEST total score compared to HC. Also, compared to norm data used in clinical practice, 32.8% of the patients showed an impaired performance. Larger LGG volume was significantly associated (p < 0.001) with poorer performance on emotion recognition. However, no direct association between LGG location (frontal, temporal, parietal) and emotion recognition was found.

CONCLUSION

In patients with LGG, impaired emotion recognition is often present before start of radiotherapy, and is related to larger LGG volume but not to location. Because of the relatively favourable prognosis of patients with LGG, maintaining quality of life is of high importance. Therefore, a thorough neuropsychological examination, including measurements for SC, is necessary to inform patients with LGG about possible changes in SC, and offer appropriate psychoeducation and care.
P02.01.A THE ONGOING WAY TO PROVIDE EVIDENCE OF THE ANCESTRAL FOUNDER CELL TO OVERCOME THERAPEUTIC RESISTANCE IN IDH-WILDTYPE GLIOBLASTOMA
Authors: Brognaro E
Published: September 2023

BACKGROUND

Multiple clinical aspects and accumulating indirect and direct experimental data are providing evidence that the supratentorial brain parenchyma becomes entirely and quiescently micro-infiltrated long before primary tumor bulk growth. Two diffuse micro-infiltrations occur in IDH-wildtype glioblastoma, the first long before primary bulk growth (the only one effective in giving rise to distant relapses) and the second, well known but ineffective, during bulk growth and evolution. Furthermore, they clearly indicate that there are two causes of therapeutic resistance, the second never yet therapeutically challenged.

MATERIAL AND METHODS

The collection of separate multiregional samples in the tumor bulk enabled us to reconstruct the phylogenetic tree of the primary tumor mass. However, the therapeutic solution to IDH-wildtype glioblastoma lies in the micro-infiltrated brain where the ancestral founder CSCs reside quiescently (they are invariably extinct in the tumor bulk at the time of diagnosis). We are using the four ethical methods, as part of normal glioblastoma surgery, to sample the micro-infiltrated brain parenchyma to identify and deeply characterize the dormant ancestral founder CSCs.

RESULTS

In the bulk, multiple separated samples allowed us to infer the phylogenetic tree of the tumor as well as driver and passenger events and the CSC variants. The ethical methods to sample the micro-infiltrated brain parenchyma are enabling us to identify and characterize the ancestral founder CSCs, which hibernate in the perivascular parenchymal niches of the supratentorial brain (their activation being the only cause of distant recurrences).

CONCLUSION

The first cause of therapeutic resistance is due to intratumor heterogeneity and the second to the silent presence in the whole supratentorial brain of quiescent founder CSCs. The IDH-wildtype glioblastoma is the only brain tumor that arises from the III layer of the SVZ and it is able to disseminate widely across the supratentorial brain parenchyma before starting its growth. Only the identification and characterization of the ancestral founder CSC will enable us to simultaneously overcome both causes of resistance and provide a therapeutic solution to this dreadful malignancy. Indeed, the ancestral founder CSC is identical to the premalignant progenitor of the SVZ and to the cell that activated in one (or more) brain location, gave rise to the primary tumor bulk(s) and transmitted the ancestral driver genomic alterations to all subsequent evolutionary CSC variants.
P02.02.B LOSS OF ELAVL2 PROMOTES MAINTENANCE AND TUMORIGENIC COMPETENCE IN GLIOBLASTOMA
Authors: Argento C M , Modelska A , Zasso J , Soldano A , Dassi E , Destefanis E , Ricci G , Mione M C , Idilli A I , Longhi S , Rosatti E F , Sighel D , Broso F , Quattrone A
Published: September 2023

BACKGROUND

IDH1 wild type glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults. It has been proven that it is driven by glioma stem cells (GSCs) that govern tumor growth and determine treatment resistance. GSCs transit between active and quiescent states, whose balance is likely dependent on both cell-autonomous and microenvironmental signals.

MATERIAL AND METHODS

transgenic cell lines were produced by retroviral infection following standard protocols, as well as zebrafish growths and orthotopic mouse brain transplants. Transgenic Drosophila lines were crossed with conventional procedures. Phenotypic evaluations, iClip protocol were conducted with standard methods as well.

RESULTS

in our study, we identified the gene ELAVL2, encoding the HuB protein, as commonly lost in GBM. We demonstrated that the ELAVL2 deletion occurs collaterally with the CDKN2A/B deletion, very close in the 9p21 region, but it is also present as an independent event. Rescue of HuB expression on a panel of ELAVL2-null GSCs affects their invasive ability and shifts their cell state toward differentiation, with loss of self-renewal and multipotency. A reduction in the proportion of quiescent cells determines an increased cytotoxicity of the drug temozolomide (TMZ).We validated these results in vivo, in two different animal models, showing that the tumorigenic potential of xenotransplanted ELAVL2-rescued GSCs is greatly reduced with respect to the ELAVL2-null GSCs in zebrafish and in the mice brain. We also analyzed the effects of ELAVL2 silencing, through RNAi, in a GBM genetic model in D. melanogaster, observing an increased brain volume when Rbp9, the ELAVL2 ortholog in Drosophila, is silenced. Profiling of HuB target mRNAs in GSCs highlighted genes associated with PTEN and stem cells related pathways. PTEN is upregulated in ELAVL2-rescued GSCs and mediates part of the effects of HuB, as PTEN knock-down in the same cells interferes with cell growth and TMZ-sensitivity. In our Rbp9-silenced Drosophila genetic model PTEN overexpression rescues the Rbp9 phenotype, reducing brain volume.

CONCLUSION

our results, both in vitro and in vivo, indicate that ELAVL2 acts as a tumor suppressor in GBM and its selective loss increases the fitness of GSCs, thus resulting in increased GBM aggressiveness. ELAVL2 promotes activation and neuronal commitment of quiescent GSCs and PTEN is one of the mediators of ELAVL2 in tumor development.
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

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

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

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

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

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

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

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

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. 
P03.01.A “TO BE SMART” IN FOLLOW-UP AFTER CRANIAL RADIOTHERAPY: REPORT OF TWO CASES
Authors: KAMER S , Kitis O , Acarer A , Eraslan C , Calli C , Anacak Y
Published: September 2023

BACKGROUND

Stroke-like Migraine Attacks after Radiation Therapy Syndrome (SMART) is a rare, delayed complication of radiation therapy and its real incidence is unknown. In this study, clinical and treatment features of two cases with clinical and radiological diagnosis of SMART are presented.

METHODS

Case-1: 49 years old male was diagnosed with grade-3 Oligodendroglioma; after gross total surgery treated with 60 Gy local cranial radiotherapy and temozolomide. Patient presented with acute left-sided weakness and numbness, episodic myoclonic jerking of the left arm and leg, 6 years after treatment. Contrast-enhanced MRI and MR angiography revealed no sign of a recurrent tumor but contrast uptake at right parietooccipital cortical giriform in the lobe of the right temporal lobe on post-contrast images (FLAIR and T1) and an increase in cortical blood volume are observed in perfusion MRI (Figure A-B). The clinical and radiologic diagnosis was SMART syndrome. Pulse dose steroids was planned as the primary treatment, and neurological symptoms was completely disappeared. After 3.5 years of the primary episode, patient re-applied with acute headache, homonymous hemianopsia of the left eye and left sided hemiparesia. Cranial MRI revealed the similar features and after treatment with steroids for 7 days complete radiological and clinical regression was experienced. Case-2: 60 years old female with a right occipital grade-3 oligodendroglioma treated with resection followed by local cranial radiotherapy (60 Gy) and temozolomide chemotherapy. Ten years after the treatment she presented with severe headache, expressive aphasia and homonymous hemianopsia of the left eye. Contrast-enhanced MRI and MR angiography revealed no sign of a recurrent tumor but contrast uptake at right occipital cortical giriform on post-contrast images (FLAIR and T1) and an increase in cortical blood volume are observed in perfusion MRI (Figurec. The diagnosis was SMART syndrome. After one week of steroid treatment she completely recovered. Similar complaints recurred 9 months after the first episode and completely recovered with steroids in one week. Radiological finding disappeared in 2 months.

CONCLUSION

SMART syndrome represents an acute-onset rare condition which may result in serious consequences if not diagnosed and treated properly. It usualy appears years after cranial radiation therapy. Clinical signs and radiological findings may resemble tumor recurrence and distinction from real tumor progression is essential. Treatment is symptomatic, most patients recover with steroids and radiological signs disappear completely, however SMART episodes may recur during the follow-up which can be treated similarly.