Glioblastoma Research Articles

Impact of endogenous viral elements on glioma clinical phenotypes by inducing OCT4 in the host

IntroductionEndogenous viral elements (EVEs) are viral sequences integrated within the host genome that can influence gene regulation and tumor development. While EVEs have been implicated in cancer, their role in regulating key transcription factors in glioblastoma (GBM) remains underexplored. This study investigates the relationship between EVEs and the activation of OCT4, a critical transcription factor in GBM progression.MethodsWe utilized CancerHERVdb and HervD Atlas databases to identify potential interactions between EVEs and key genes involved in GBM. Data from 273 GBM patient samples in the TCGA database were analyzed to examine the correlation between OCT4 expression and mutations in glioma-related genes. Furthermore, glioblastoma stem cells (GSCs) were assessed for the expression levels of OCT4 and SOX2, and Pearson correlation analysis was performed.ResultsOur analysis revealed that OCT4 is a pivotal gene activated by EVEs in GBM. OCT4 expression was significantly correlated with mutations in key glioma-associated genes. Higher OCT4 levels were associated with poorer patient prognosis, higher tumor grades, and older age. Additionally, GSCs exhibited elevated expression of both OCT4 and SOX2, with a positive correlation observed between these two genes in GBM patients.DiscussionThis study highlights the potential role of EVEs in driving GBM progression through the activation of OCT4. The findings emphasize the importance of OCT4 in GBM malignancy and suggest that targeting EVE-mediated pathways may provide new therapeutic approaches for GBM treatment.

PATH-07. CLINICAL AND RADIOGRAPHIC FEATURES OF DIFFUSE GLIOMA MOLECULAR SUBTYPES IN ACCORDANCE WITH THE 2021 CNS5 WHO CLASSIFICATION

Abstract BACKGROUND The incorporation of molecular parameters into WHO CNS5 has led to reclassification of many gliomas. We undertook to describe presenting clinical and radiographic features of diffuse adult gliomas according to CNS5 category. METHODS We reviewed pathology reports, clinical and MRI data at presentation of 972 adult patients with glioma diagnosed between 1/2010-2/2022. Continuous variables were presented as median; categorical variables as numbers and percentages, and comparison were performed using chi-square test. RESULTS 736 patients had sufficient data for CNS5 re-classification and were included in the analysis: Grade 2 IDH-mutant (IDHmut) astrocytoma (A2) n=69, grade 3 IDHmut astrocytoma (A3) n=37, grade 4 IDHmut astrocytoma (A4), n=32, grade 2 oligodendroglioma (O2) n=60, grade 3 oligodendroglioma (O3) n=23, and grade 4 IDH-wildtype glioblastoma (GBM) n=515. Age at presentation significantly differed between grade 2&3 gliomas and grade 4 (mean age:39 versus 61.7; p<0.001); A4 were also younger than GBM (39.8 versus 63.1). A majority in all categories except grade 2 gliomas were male. Seizure was more common in IDHmut gliomas than GBM (58.4% versus 31.8%;p<0.001), while cognitive impairment was more common in GBM than in IDHmut gliomas (64.4% versus 34.4%;p<0.001) and in A4 (64% versus 40.6%;p=0.013). Focal deficits at presentation were more frequent in GBM than in IDHmut gliomas (74.2% versus 30.5%;p<0.001), but not significantly different compared to A4 (74.2% versus 58.1%;p=0.06). Contrast enhancement on MRI was more frequent in GBM compared to IDHmut gliomas (93% versus 48%;p<0.001), but similar frequency with A4 (93% versus 96%;p=1.00). The minimum ADC was significantly higher in IDHmut glioma including A4 than GBM(p<0.001). Calcification was more common in oligodendroglioma than astrocytoma and GBM (p<0.001), and more frequent in A4 than GBM(p=0.016). CONCLUSION Significant differences in the clinical presentation and radiologic features exist among CNS5 glioma subtypes, informing potential diagnosis, management and prognosis at initial presentation.

CNSC-08. PROGNOSTIC BIOMARKER DISCOVERY THROUGH PROTEOMIC ANALYSIS OF GLIOBLASTOMA MULTIFORME

Abstract BACKGROUND Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor known for its high recurrence rates and dismal survival outcomes. Despite advancements in surgical and therapeutic strategies, the prognosis for GBM patients remains poor. This study applies proteomic analysis to postoperative GBM tumor specimens to identify molecular markers that could enhance prognosis prediction. Through the analysis of protein expression profiles before and after tumor recurrence, we identified significant alterations in protein pathways via Gene Set Enrichment Analysis (GSEA), highlighting potential biomarkers for disease outcome prediction. METHODS We employed Sequential Window Acquisition of All Theoretical Fragment Ion Spectra (SWATH-MS) proteomics to analyze protein samples from both primary and recurrent GBM patients. Gene Set Enrichment Analysis (GSEA) was utilized to compare protein expression profiles and discern significantly altered pathways. RESULTS Analysis revealed 778 differentially expressed proteins (DEPs), with notable alterations in pathways such as glycolysis, interleukin signaling, and MAPK signaling. Proteins such as TOLLIP, ENO1, and ITGA2B emerged as potential prognostic markers. Validation through an online tumor prognostic analysis platform (ToPP), integrated with The Cancer Genome Atlas (TCGA) data, facilitated the development of a prognostic risk score model. This model effectively categorized patients into high and low-risk groups based on their protein expression profiles, enhancing the prediction of patient outcomes. The proteins and pathways identified offer new insights into the molecular dynamics of GBM postoperatively. The association between protein expression profiles and patient prognosis underscores the potential of these markers in developing customized therapeutic strategies. Additionally, the prognostic risk score model serves as a valuable tool for improving prognostic accuracy and could guide personalized treatment plans. CONCLUSION Proteomic profiling of GBM tumors significantly contributes to uncovering the molecular mechanisms driving this aggressive cancer and facilitates the development of dependable prognostic tools. These findings not only affirm the critical role of proteomics in cancer research but also advance personalized medicine strategies in GBM management.

STEM-19. TARGETINGGIHCG LNCRNA IN GLIOBLASTOMA STEM CELLS: A POTENTIAL THERAPEUTIC STRATEGY

Abstract Glioblastoma multiforme (GBM) remains the most lethal and prevalent primary brain tumor worldwide. Despite advances in understanding its biology and multimodal therapy options, the overall prognosis for GBM patients remains bleak, primarily due to the high recurrence rate, which is intimately linked to tumor resistance against standard therapeutic interventions. Glioblastoma stem cells (GSCs) contribute to therapeutic resistance and cellular heterogeneity through their self-renewal properties and ability to adapt. Thus, identifying new molecular targets driving GBM progression is crucial for developing effective therapies. Recent advancements in genome biology have revealed that long non-coding RNAs (lncRNAs) play roles in various biological functions, and the dysregulation of numerous lncRNAs has been shown to be associated with specific cancers. To identify GSC-associated lncRNAs, a comprehensive analysis was performed using single-cell RNA sequencing datasets from GBM tumors, GBM organoids, GSC-enriched GBM tumors, as well as normal human brain samples. A chromatin-enriched lncRNA, GIHCG, was identified as being highly expressed in GSCs compared to non-neoplastic neural stem cells. The GIHCG gene, located on human chromosome 12q14.1, is frequently amplified in glioblastoma and impacts overall survival. Bioinformatic and functional analyses revealed that GIHCG is amplified in 13% of GBM patient samples, and high expression of GIHCG is a negative prognostic marker for GBM patients, with significantly reduced overall survival compared to the low expression group. In addition, the depletion of GIHCG significantly reduced GSC proliferation and migration. Further, the knockdown of GIHCG decreased both mRNA and protein expression levels of critical GSC stemness factors, including SOX2, NESTIN, and OLIG2. Moreover, GIHCG depletion reduced the sphere formation capacity of GSCs, demonstrating the functional importance of GIHCG in the maintenance of GSC stemness. These results indicate that GIHCG is essential for GSC proliferation, migration, and stemness, underscoring its potential therapeutic value in RNA-based therapies for combating glioblastoma malignancy.

NCOG-41. COMPARATIVE ANALYSIS OF MORTALITY AND SHORT-TERM SIDE EFFECTS IN PATIENTS WITH GLIOBLASTOMA MULTIFORME TREATED WITH TEMOZOLOMIDE VERSUS TEMOZOLOMIDE PLUS BEVACIZUMAB CHEMOTHERAPY

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor in adults with limited treatment options and poor prognosis. Temozolomide (TMZ) chemotherapy is the standard treatment for GBM, but alternative therapies have shown limited success. Bevacizumab (BEV), a monoclonal antibody targeting vascular endothelial growth factor (VEGF), is used to restrict GBM growth and spread. This study evaluates the efficacy of combining TMZ with BEV compared to TMZ alone in managing GBM. METHODS A retrospective cohort study was conducted using TriNetX database data, focusing on patients diagnosed with GBM and receiving chemotherapy. Cohorts were defined by treatment: TMZ plus BEV versus TMZ alone. Propensity-score matching balanced baseline characteristics, including demographics and health conditions. Primary outcomes included mortality rates and short-term side effects within the first year of treatment, such as cognitive communication deficits, headache, dizziness, insomnia, and nausea/vomiting. RESULTS After matching, 922 patients were included in each cohort. There were no significant demographic differences post-matching. The mortality rate was higher in the TMZ plus BEV group compared to TMZ alone (51.30% vs. 36.98%, RR: 1.387, 95% CI: 1.249-1.541). However, TMZ plus BEV was associated with a lower incidence of cognitive communication deficits (1.95% vs. 4.34%, RR: 0.45, 95% CI: 0.26-0.779) and dizziness (7.27% vs. 10.52%, RR: 0.691, 95% CI: 0.513-0.93). No significant differences were found in headache, insomnia, or nausea/vomiting rates between the groups. CONCLUSION This study indicates that combining bevacizumab with temozolomide may increase mortality in GBM patients but reduce certain side effects such as cognitive deficits and dizziness. These findings emphasize the need to carefully consider the benefits and risks of different chemotherapy regimens for GBM and highlight the necessity for further research to optimize treatment strategies and improve patient outcomes.

CCRG-02. MICRORNA TARGETING LOGIC IN DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMA

Abstract microRNA (miR)-124 is one of the main drivers of mitotic exit during neurogenesis, which is recapitulated in miR-9/9* and -124 (miR-9/9*-124)-mediated direct neuronal reprogramming. In the reprogramming of various cell types, miR-9/9*-124 induce non-neuronal cell fate erasure before activating the neuronal program. Regardless of the different starting transcriptome landscapes, a core gene regulatory network (GRN) is always targeted by miR-9/9*-124, with cell cycle progression as the most significant pathway. We reason that miR-9/9*-124 can induce cell fate erasure in cancerous cells by repressing similar GRNs, using glioblastoma multiforme (GBM) as a paradigm. Mechanistic insights into GBM fate erasure by miR-9/9*-124 while inducing the postmitotic state would facilitate the understanding of GRNs that govern the tumorigenic identity of GBMs and potentially offer novel therapeutic implications. Indeed, miR-9/9*-124 readily induces mitotic exit in patient-derived GBM cell lines, represses GBM cell fates, and promotes neuronal identity over time as assessed by RNA-seq. Mice receiving GBM intracerebral xenografts with miR-9/9*-124 survived significantly longer than those receiving non-specific control miRNA. Global profiling of miRNA targeting activity by AGO2-eCLIP reveals that miR-124 binds to transcripts enriched in G1/S transition and mitogen response. Conversely, miR-506—a miR-124 homolog—despite of sharing identical seed sequences, has significantly less targeting activity and fails to stop cell cycle progression. We are currently dissecting how the difference in the miRNA 3’ sequence renders contrasting biological outcomes. Preliminary data suggests that the length of miRNA alone alters targeting specificity to cell cycle transcripts. However, different permutations of miRNA 3’ sequence, though sufficient to induce mitotic exit, are not all competent to achieve neuronal reprogramming, indicating that mitotic exit and neurogenic activities of miR-124 could be decoupled. We hope that understanding the grammar logic of miRNA targeting would provide deeper insights into how miRNAs can be leveraged to manipulate GBM identity.

PATH-02. MESENCHYMAL-TYPE GENETIC MUTATIONS ARE LIKELY PRE-REQUISITE FOR GLIOBLASTOMA TO METASTASIZE OUTSIDE THE CENTRAL NERVOUS SYSTEM: AN ORIGINAL CASE SERIES AND SYSTEMATIC REVIEW OF THE LITERATURE

Abstract Glioblastoma multiforme (GBM) is locally aggressive, yet it metastasizes outside the central nervous system (CNS) in only 0.4% of cases. Little is known about what enables this subset of GBMs to survive outside the CNS, so we conducted a PRISMA-compliant systematic review of metastatic GBM wherein we reviewed 3579 search results and 1080 abstracts, ultimately analyzing data from 139 studies and 211 unique patients. Additionally, we analyzed four modern-day cases treated at our institution. In our patients, primary GBM lesions revealed consistent mutations in NF1, TERT, TP53, CDK4, and RB1/PTEN genes. Unique to the metastatic lesions were amplifications in integral cell-cycle regulating genes such as p53 and PDGFRA/KIT, as well as increased vimentin and Ki-67 expression. These data support the hypothesis that glial cells likely underwent mesenchymal-like mutations prior to extra-CNS involvement. How these cells reach the extra-axial environment is still under debate. In our systematic review, we found that metastases were discovered near previous surgical sites in at least 36.9% of cases, suggesting tumor seeding into the scalp during surgery may facilitate mesenchymal-type mutation in some cases. However, this is unlikely to be sole mechanism of spread as there are several reports of spread without surgery, and other hypothesized mechanisms include spontaneously circulating cells and glymphatics. Other sites of GBM metastasis included bone (47.9%), lung (25.6%), lymph nodes (25.1%), and liver (14.2%). On average, metastases were diagnosed 12.1 months after the most recent resection, and the mean survival from discovery was 5.7 months. In sum, there is strong evidence that GBMs acquire novel mutations to survive outside the CNS, are spread spontaneously or iatrogenically, and portend a poor prognosis once disseminated. Routine genetic and molecular characterization of both primary and metastatic GBMs and widespread reporting will help further characterize mechanisms of spread for this rare but deadly pathology.

EXTH-65. EXPLORING ANTIHISTAMINES TO TREAT GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive and lethal primary malignant brain tumor with a 5-year survival of 6.8%. Interestingly, people with allergies, atopy, or asthma are less likely to develop GBM. A recent study recognized that high histamine receptor 1 (HRH1) expression is inversely related to overall and progression-free survival. METHODS We used a bioinformatics tool, iLINCS, which, after analyzing 99 GBM and 38 healthy samples, identified 36 drugs that can reverse GBM signatures. Based on the concordance score and blood-brain barrier permeability, we selected brompheniramine (Brom), a first-generation HRH1 antagonist, as a potential candidate. HRH1 expression analysis was done on human and mouse GBM cell lines by immunoblotting. The effect of Brom alone or in combination with temozolomide (TMZ) was investigated on proliferation, DNA damage and apoptosis in human (U118 and U251) and three syngeneic (EGFRvIII, p16-/- & GFAP Cre, PTEN+/-, p53R172H+/-, EGFRvIII & GFAP Cre, PTEN-/-, p53R172H-/- & GFAP Cre) cell lines. GBM orthograft mouse models were used to test the efficacy of combination therapy. RESULTS HRH1 is highly expressed in human and syngeneic GBM cells. This expression is further increased in TMZ-resistant cells. Interestingly, the addition of histamine, induced TMZ resistance in GBM cells. TMZ with Brom decreases proliferation in mouse and human GBM cells, and Brom is non-toxic to non-transformed cells. Combination treatment increases DNA damage and decreases the DNA repair mechanism, possibly by downregulating the expression of O6methylguanine-DNA methyltransferase (MGMT). In the GBM orthograft mouse model, the combination therapy reduced tumor volume and significantly increased survival compared to single-agent treatment. CONCLUSIONS Brom is non-toxic; Brom, in combination with TMZ, enhanced the anti-tumor efficacy of TMZ both in in vitro and in vivo GBM models. Further studies in spontaneous mouse models are planned.

PATH-30. CLINICAL OUTCOMES AND PREDICTIVE BIOMARKERS FOR IDH-WILDTYPE GLIOBLASTOMAS DEVELOPING HYPERMUTATION FOLLOWING TEMOZOLOMIDE TREATMENT

Abstract Current standard of care for IDH-wildtype glioblastoma (GBM) includes maximal safe resection, adjuvant radiation, and chemotherapy with the alkylating agent temozolomide. A subset of gliomas treated with temozolomide develop somatic hypermutation with a predominance of C>T transitions due to the alkylation effects on DNA. However, the frequency of occurrence, clinical ramifications, and predictive biomarkers for developing temozolomide-induced hypermutation (TMZ-HM) in GBM are unknown. Here we performed comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent GBM from 106 patients. Seven patients (7%) developed hypermutation at time of first recurrence, while an additional five patients (5%) developed hypermutation at a subsequent recurrence. Patients who developed TMZ-HM had a significantly longer interval between initial surgery and first recurrence (27.8 vs. 9.9 months, p<0.001). In contrast to IDH-mutant gliomas where TMZ-HM is associated with poor prognosis, those patients with IDH-wildtype GBM who developed TMZ-HM had significantly longer overall survival from both initial surgery (67.9 vs. 20.3 months, p<0.001) and from time of recurrence after development of hypermutation (30.9 vs. 9.5 months, p=0.001). There was no difference in oncogenic alteration frequency in initial GBM that subsequently developed TMZ-HM. However, 4 specific CpG sites out of 12 total interrogated sites in the promoter region of MGMT (a DNA repair enzyme) and 5 specific CpG sites in the promoter region of KCNQ1DN (a negative regulator of c-Myc) were significantly more hypermethylated in those GBM that developed TMZ-HM. We then generated a weighted risk score across these CpG sites that can be used to prospectively identify GBM likely to develop TMZ-HM associated with favorable survival. In summary, patients with GBM that develop TMZ-HM follow a more favorable clinical course, and specific MGMT and KCNQ1DN promoter hypermethylation patterns could serve as a biomarker to identify these patients who may potentially benefit from extended alkylating chemotherapy treatment.

EPID-16. GENOMIC ANCESTRY-RELATED RISK RATIOS FOR GLIOBLASTOMA INCIDENCE AMONG GLOBAL POPULATIONS AND RACIAL/ETHNIC GROUPS WITHIN THE UNITED STATES

Abstract Glioblastoma (GBM) incidence varies globally, elevated rates in Europe and lower rates in Asia, Latin America, and Africa. While partially attributable to national differences in cancer diagnosis and reporting practices, analogous patterns exist within diverse Western nations. Ancestry-associated differences in GBM risk allele frequencies (RAFs) could contribute, a hypothesis supported by our prior multiethnic genome-wide association studies (GWAS). Leveraging variant effect estimates from large GBM GWAS datasets and ancestry-stratified allele frequency data from diverse reference populations (ALFA, GNOMAD, 1000 Genomes Project), we estimate the extent of GBM incidence differences across populations that can be attributed to underlying variation in RAF at the seventeen known GBM risk loci. Aggregate differences in RAF between global populations (Europe, West-Africa, East Asia) were associated with a >20% reduction in the normalized GBM risk-ratio (RR) in Africans compared to Europeans (RR=0.78) and a >50% reduction in East Asians compared to Europeans (RR=0.47). The frequency of GBM risk alleles was generally highest in Europeans, but four alleles found most commonly in Africans are at loci regulating telomere maintenance (TERC, TERT, OBFC1/STN1, RTEL1). We repeated analyses using RAF data from U.S. populations (African-Americans, Latinos, and non-Hispanic Whites). Compared to non-Hispanic Whites, aggregate differences in RAF were associated with a 15% reduction in GBM risk among African-Americans (RR=0.85) and a 28% reduction among Latinos (RR=0.72). We conclude that a sizable proportion of the reduced GBM incidence in Latinos and Asians, relative to non-Hispanic Whites, can be accounted for by population-level differences in RAF at known loci. However, registry-based analyses consistently report a two-fold lower incidence of GBM in African Americans relative to non-Hispanic Whites which is poorly explained by RAF differences at known loci. Further work is needed to identify structural, environmental, and biological factors contributing to unresolved differences in GBM incidence.

EPID-34. THE EFFECT OF ANTIDEPRESSANT THERAPY ON GLIOBLASTOMA SURVIVAL: A SYSTEMATIC REVIEW AND META-ANALYSIS

Abstract BACKGROUND Symptoms of depression are highly prevalent among patients with glioblastoma (GBM) and have been associated with poor outcomes. In addition to managing depression symptoms, preclinical and clinical studies suggest that antidepressants may inhibit GBM progression and may be associated with improved outcomes. However, the effect of antidepressants on survival is unknown and results from existing studies are conflicting. OBJECTIVE We conducted a systematic review and meta-analysis to investigate the effect of antidepressant therapy on GBM survival. METHODS We searched PubMed, Embase, Scopus, PsycINFO, and Web of Science from inception to March 2024 for studies assessing the overall survival of patients with GBM treated with antidepressants. Prespecified Hazard ratios (HR), 95% confidence interval (CIs), median overall survival, and patient characteristics were extracted. Results were combined using a restricted maximum-likelihood estimation random-effects model. RESULTS We identified 6 observational studies meeting inclusion criteria with 8269 patients. Of these, 1093 (13%) were treated with antidepressant therapy at some point after their GBM diagnosis. Antidepressant therapy was not significantly associated with overall survival (HR 1.27, 95% CI 0.90-1.79, p=0.17). In a sub-analysis of studies that specified whether SSRIs were utilized, SSRI usage specifically was also not associated with overall survival (HR 1.08, 95% CI 0.58-2.03, p=0.81) We observed considerable heterogeneity (I2 = 92.7%, p<.001). CONCLUSIONS Antidepressants were not significantly associated with overall survival in patients with GBM. However, our meta-analysis was limited by significant heterogeneity observed across studies. The effect of antidepressants on outcomes in patients with GBM remains uncertain. We observed conflicting results in the literature, with some studies suggesting decreased survival. Therefore, the role of antidepressants in patients with GBM warrants further investigation.

EXTH-10. TARGETING EPHA3 AND EPHRIN A5: ADVANCED PRECISION THERAPY FOR GBM

Abstract Glioblastoma (GBM) is an aggressive brain cancer and is associated with very poor prognosis. Standard treatment involves surgical resection, post-operative radiation and TMZ chemotherapy. Further research into new therapeutic approaches which target chemo-resistant tumor propagating cells, are urgently required. The EphA3 receptor is frequently elevated in GBM, particularly in the mesenchymal subtype and expressed on tumor-initiating cells (Day et al. Cancer Cell 2013). Our data in GBM tissue shows that tumor cells expressing the high-affinity EphA3 ligand, ephrin A5; are distinct from EphA3 expressing cells. The ephrin A5-positive cells express the glial differentiation marker GFAP, are less proliferative and less stem cell-like. EphA3 is expressed on the vimentin-positive, highly proliferative cell population. Single cell RNA-sequencing revealed that EphA3 and ephrin A5 are highly expressed in recurrent GBM, and are markers of MES-like and AC-like cell states respectively. Ephrin A5 over-expression in-vivo led to extension of survival in orthotopic xenograft animal models. GBM aggressiveness and downregulation of stem cell markers. Spatial transcriptomics of xenograft tumors revealed a reduction in proliferation markers including Ki67, MCM7 and PCNA. We detected an increase in expression of AC-like cell-state with a concomitant reduction of the MES-like and NPC-like cell-states. Consistently, over-expression of ephrin-A5 in primary GBM lines led to a reduction in neurosphere formation, Ki67 staining and growth rates. Thus we propose that dual targeting of EphA3 and ephrin-A5 might better capture GBM tumour heterogeneity and lead to an extension GBM patient survival. Whilst better understanding the biology of ephrinA5 expression, we also aim to validate antibody-based approaches to perform dual targeting of EphA3 and ephrin A5, leading to an extension GBM patient survival.

EPCO-44. SPATIAL TRANSCRIPTOMICS ENABLES STATISTICAL MODELING OF SEX DIFFERENCE IN GENE CO-EXPRESSION NETWORK AND IMMUNE LANDSCAPE OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is characterized by inter- and intra-tumor molecular heterogeneity and demonstrated cell plasticity. Sex differences have been observed in various aspects: a male:female GBM incidence ratio is 1.6:1; standard of care treatments (debulking, radiation, and temozolomide) benefit females more than males. 14 human GBM samples from two study sites were subject to spatial profiling on the Vizgen Merscope platform to examine whether distinct biological and therapeutic niches in GBM were evident, to understand how the tumor microenvironments (TME) contributed to the heterogeneity of GBM, and to interrogate sex difference in the immune landscape of GBM. Multiple sets of spatial probes were designed to capture the cell identities (cancer stem cells, astrocytes, oligodendrocytes, microglia, macrophages, etc.) and the spatially-resolved expression patterns of genes with different functions. Two of the 14 GBM samples were also profiled on another FISH-based spatial platform (Veranome). The data from the two platforms exhibited a linear correlation. We explored the gene-gene co-expression network, and calculated the interaction probabilities between genes which were then compared for sex difference. We found that female samples showed significantly lower (two-fold) CD4+/CD8A+ T-cell count ratios than male samples. We also observed sample-specific spatial niches of different GBM cell subtypes and immune cell populations. Our large-scale spatial transcriptomics study revealed intrinsic sex-based differences in immune response to tumor and illuminated microenvironmental influences on GBM.

DDDR-38. TRANSLATIONAL STEPS OF THE ANTITUMOR PEPTIDE TAT-CX43266-283 AS A TREATMENT FOR GLIOBLASTOMA: EGFR AS A TREATMENT RESPONSE BIOMARKER AND RESULTS OF COMBINATION THERAPY IN A PRECLINICAL RESECTION MODEL

Abstract Glioblastomas (GBM) are the most malignant primary brain tumors, and they remain incurable. Despite the current standard of care (surgical resection of the tumor and administration of chemotherapy and radiotherapy) some GBM stem cells (GSCs) remain in the brain parenchyma causing tumor recurrence. The Src-inhibitory peptide TAT-Cx43266-283 has shown promising antitumor results in GBM preclinical models, reducing GSC viability and increasing survival in GBM-bearing mice. Here we have investigated some of the main gaps to overcome when translating preclinical results to the clinic, to promote the progress of TAT-Cx43266-283 as a clinical therapy to fight GBM. First, we investigated biomarkers of treatment response to design a more targeted therapy. We assessed cell viability after treatment with TAT-Cx43266-283 in 13 patient-derived GSC lines and we found a stronger treatment response in those GSCs that had alterations in the Epidermal Growth Factor Receptor (EGFR). This was further confirmed in several murine GBM lines. Importantly, we found that treatment with TAT-Cx43266-283 was more effective in vitro than temozolomide or the classical EGFR inhibitor, erlotinib. Moreover, we showed that EGFR participates in TAT-Cx43266-283 response together with Src, by decreasing EGFR and EGFRvIII activity in some GSCs and increasing survival in mice bearing tumors with EGFR alterations. Next, we studied the effect of TAT-Cx43266-283 in a clinical context by administering it in a murine model of tumor resection. We analyzed the histopathology of this GBM model, uncovering histological features typical of human GBM. We found that resection alone improved, although not significantly, GBM-bearing mice survival. However, tumors that regrew after resection exhibited more aggressive features. Importantly, the combination of tumor resection and TAT-Cx43266-283 achieved better survival outcomes and showed reduced invasive features. Altogether, these results serve as a base for future clinical investigations and support the therapeutic potential of TAT-Cx43266-283 as a treatment for GBM.

NCOG-12. TREATMENT AND MORTALITY OF PRIMARY BRAIN TUMORS: A SINGLE CENTER OBSERVATIONAL STUDY OF GLIOBLASTOMAS AND ASTROCYTOMAS

Abstract Glioblastoma multiforme (GBM) is a WHO grade IV malignant tumor with a relatively poor prognosis (median survival of 15 months). Astrocytomas are WHO grade I-III tumors with a better prognosis than GBM; their median survival ranging from 2-10 years. We assessed the response to various mainstay treatment modalities in patients with GBM and astrocytoma, expecting to find that surgery and stereotactic radiation (SRS) treatment confer greater survival benefits than non-surgical and whole brain radiation (WBRT) options. We analyzed 108 patients at St. Luke’s University Health Network (SLUHN) who were previously diagnosed with a primary brain tumor consistent with GBM (90) or astrocytoma (18) prior to January 1, 2023. Mainstays of treatment were surgery with or without different radiation modalities (WBRT and SRS, and WBRT+SRS). Glioblastoma typically affected older patients (65.5), had mostly single lobe involvement (86.7%), and was predominantly located in the frontal (41.1%) and temporal lobes (37.7%). Astrocytoma was slightly younger (51.2), less often single lobe (72.2%), and mostly involved the frontal (44.4%), temporal (44.4%), and parietal lobes (33.3%). GBM found the greatest benefit from WBRT (without co-occurring SRS) with an increase of 16.4 months to 20.2 months (p=.789). Surgery, regardless of radiation status, appeared to provide the greatest benefit for patients with astrocytoma increasing average lifespan from 36.7 months to 53.6 months (p=.181). Patients with glioblastoma that underwent surgery also had increased survival of 18.2 months compared with nonsurgical patients at 12 months (p=0.097). While we were interested to see the impact these treatment modalities had on patient survival, our sample size was small and our results lacked statistical significance. Future studies with greater sample sizes would help determine whether statistical significance is correlated with meaningful clinical outcomes.

DDDR-21. MPT0G521 AS A DUAL INHIBITOR OF LSD1 AND HDAC: A POTENTIAL THERAPEUTIC AGENT AGAINST PARENTAL AND TEMOZOLOMIDE-RESISTANT GLIOBLASTOMA MULTIFORME

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is an aggressive brain tumor with limited treatment options and poor prognosis. Resistance to temozolomide (TMZ), the standard chemotherapy for GBM, further complicates treatment. This study investigates the effects of MPT0G521, a dual inhibitor of LSD1 and HDAC, on both parental and TMZ-resistant GBM cells to determine its potential as a therapeutic agent. MATERIALS AND METHODS TMZ and MPT0G521 were introduced in our study. Human GBM cell lines A172 and patient-derived primary GBM cells Pt#3, along with their TMZ-resistant counterparts, were cultured under specified conditions. Assays conducted include cell proliferation (MTT), colony formation, cell cycle analysis via flow cytometry, and western blot analysis for protein expression. Statistical significance was evaluated using ANOVA. RESULTS MPT0G521 exhibited significant anti-proliferative activity and toxicity against both parental and TMZ-resistant GBM cells in a dose- and time-dependent manner. Treatment with MPT0G521 led to reduced cell viability and colony formation. Cell cycle analysis revealed G2M phase arrest and increased subG1 phase, indicating cell cycle disruption and apoptosis. Western blot analysis showed upregulation of histone H3 methylation and acetylation, confirming the dual inhibitory action of MPT0G521 on LSD1 and HDAC. Additionally, MPT0G521 modulated markers of cell cycle and apoptosis, including increased levels of cleaved caspase-3 and PARP. CONCLUSION MPT0G521 demonstrates potential as an effective therapeutic agent against GBM by inhibiting cell proliferation, inducing cell cycle arrest, and promoting apoptosis in both parental and TMZ-resistant cells. These findings support further investigation of MPT0G521 as a promising treatment for GBM, particularly in cases resistant to standard chemotherapy.

EXTH-85. ASSESSMENT OF ANTI-CD69 ANTIBODY THERAPY ALONE OR IN COMBINATION WITH ANTI-PD-1 IN MURINE GBM

Abstract BACKGROUND Glioblastoma multiforme (GBM) is an aggressive central nervous system malignancy with a dismal prognosis. There is continued interest in trying multiple immunotherapy modalities for this intractable cancer. Cluster of differentiation 69 (CD69), is an early marker of T and NK cell activation. CD69 play multiple roles in T cell-mediated immunoregulation with immune inhibitory role in antitumor immunity. Previous preclinical work in non-CNS malignancies has demonstrated that targeting CD69 can improve clinical outcome and anti-tumor immunity. We aimed to assess anti-CD69 Ab therapy alone or in combination with anti-PD-1 in murine GBM. METHODS We explored transcriptomic data from 163 GBM patients using the TCGA database. We then tested the therapeutic value of anti-CD69 immunotherapy in a preclinical model of GBM and assessed the ideal timing of treatment with anti-CD69 Ab by evaluating survival and immune tumor-microenvironment by flow cytometry. Two treatment-timings with anti-CD69 Ab were further evaluated either alone, or in combination with anti-PD1 Abs and compared to control and to anti-PD1 only treatment. Survival data was collected. RESULTS CD69 expression was significantly increased in GBM patients tissues as compared with normal brains and increased CD69 expression was associated with worse progression-free survival. Treatment-timing with single dose anti-CD69 Ab at day 1 or three doses at days 4, 8 and 12 post tumor-injection minimally improved the median survival time of animals. In addition, effective CD69 blockage and significantly elevated PD-1 expression on NK cells was found in these timings, making them good candidates to evaluate anti-CD69 Ab therapy alone or in combination with anti-PD-1 Ab. However, when assessing these treatment-timings with anti-CD69 Ab alone or in combination with anti-PD1 no survival benefit was found. CONCLUSION This is the first study assessing anti-CD69 Ab therapy alone or in combination with anti-PD-1 in murine GBM. While some effect was found on the cellular level, this didn’t translate to survival benefit and results are overall negative.

BIOM-07. SURVIVAL-ASSOCIATED GENETIC VARIATIONS IN TAIWANESE GLIOBLASTOMA MULTIFORME PATIENTS - A RETROSPECTIVE NGS STUDY

Abstract This retrospective study, conducted at Taichung Veterans General Hospital in Taiwan, employed Next-Generation Sequencing (NGS) technology to analyze genetic variations associated with survival in patients with glioblastoma multiforme (GBM). The study encompassed 30 newly diagnosed GBM patients, divided into two groups based on survival duration: long-term (over two years) and short-term (under two years). The findings are outlined below: Key Mutations: Significant mutations included CHEK2, IDH1, and TP53, along with variations in the TERT promoter and TP53 RNA splicing sites. Correlation Between Mutations and Survival: Contrary to Western data, TP53 mutations were more prevalent in the long-term survival group, suggesting that regional genetic differences may influence GBM prognosis. Therapeutic Effectiveness: The application of bevacizumab was significantly associated with improved survival rates, indicating its potential efficacy in treatment. The study highlights the critical role of personalized medicine in the treatment of GBM in Taiwan and points to the necessity of further research into the genetic characteristics and treatment responses of GBM patients in Taiwan and potentially other non-Western regions. These insights underline the importance of continuing to explore effective personalized treatment strategies and enhancing our understanding of genetic determinants in diverse populations. Moreover, the prevalence of certain mutations in Taiwanese patients, which differ from those commonly observed in Western populations, prompts a reevaluation of global treatment frameworks and suggests the potential for region-specific therapeutic protocols. The implication of these findings extends beyond immediate clinical applications, providing a foundation for future clinical trials and research into genetic markers that could dictate treatment choices. In conclusion, this study not only underscores the importance of individualized treatment plans based on genetic profiling but also highlights the variations in genetic mutations across different populations, which could have profound implications for the management of GBM worldwide. Further investigation into these disparities and their implications on treatment efficacy is essential for advancing GBM treatment and improving patient outcomes globally.

DDDR-18. TARGETTING GLIOBLASTOMA MULTIFORME USING A MULTIACTION ANTICANCER ANTI-INFLAMMATORY THERAPEUTIC STRATEGY

Abstract Glioblastoma multiforme (GBM) represents a significant challenge in adult brain tumor management, characterized by its aggressive nature and poor prognosis despite multimodal therapies. Current standard chemotherapy, Temozolomide (TMZ), offers limited survival benefits. In a recent breakthrough, the cyclic peptide cyclo-((2-Nal)-Leu-Ser-(2-Nal)-Arg) acetate (c2), initially developed for prostate cancer, demonstrates remarkable promise against GBM. By selectively inhibiting the inflammatory phospholipase enzyme sPLA2IIA, c2 significantly impedes GBM cell proliferation, surpassing TMZ efficacy in multiple resistant cell lines. Moreover, c2 exhibits potential to inhibit tumor growth and metastasis, confirmed in 3D tumoroid and wound healing assays. Mechanistic insights reveal modulation of crucial cellular pathways, including viral entry, cytoskeletal structure, and signaling cascades. Preclinical investigations demonstrate c2’s ability to penetrate the blood-brain barrier and attenuate tumor progression markers. At multiple levels, c2 is shown to target inhibition of ERBB2 and EGFR signalling and also have impacts on HIF1-NF-kB axis. This study proposes c2 as a potent anti-GBM candidate, warranting evaluations for its pharmacological profile, safety, and efficacy. With its potential to address the urgent need for effective GBM therapies, c2 holds promise as a significant advancement in GBM treatment, offering hope for improved patient outcomes.

CSIG-32. MODULATION OF HALLMARKS OF GLIOBLASTOMA MULTIFORME BY BIOACTIVE PHYTOCHEMICALS: A PRECLINICAL MECHANISTIC EVALUATION

Abstract Glioblastoma multiforme (GBM) is an aggressive malignancy with extensive neovascularization, an immunosuppressive tumor microenvironment, and high recurrence rates. Bioactive plant secondary metabolites (phytochemicals), many from fruits and vegetables, have been extensively studied for GBM prevention and treatment. The objective of this study is to determine the effects of phytochemicals on GBM hallmarks and associated molecular mechanisms of action through evaluation of in vitro and in vivo studies. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed to collect articles using PubMed, ScienceDirect and Scopus. While 1,344 articles were screened, 115 in vitro and 36 in vivo studies met our inclusion criteria. The Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) review of bias tool assessed in vivo study bias. In vitro, 49 alkaloids, 10 phenolics, 2 sulfur- compounds, and 5 terpenoids modulated GBM hallmarks of cell growth, invasion, cell cycle progression, neovascularization, and apoptosis. The PI3K/Akt/mTOR pathway was suppressed by 13 phytochemicals. Apoptosis was induced by 15 phytochemicals activating caspase-3, whereas 11 compounds inhibited Bcl-2. Cell cycle modulation occurred through decreased cyclin-dependent kinases and increased p53, p21, and p27. In vivo, 17 alkaloids, 5 phenolics, 2 sulfur-compounds, and 2 terpenoids had effects on GBM hallmarks. Invasion and neovascularization were inhibited through MMP, HIF-1α, and VEGF modulation. Six phytochemicals induced apoptosis in vivo through caspases and Bcl-2. Utilizing the SYRCLE review of bias tool, the average score quality of animal studies is 10% with many indicators of bias unclear. Various phytochemicals show promise to prevent and treat GBM due to their effects on numerous GBM hallmarks. Study limitations include the inclusion of literature reporting effects of single agents and lack of clinical studies. Future research is warranted to fill gaps in our analysis before recommending phytochemicals for prevention and treatment of GBM, a perilous malignancy.