Primary Glioblastoma Multiforme Research Articles

A rare case of atypical intradural extramedullary glioblastoma diagnosed utilizing next-generation sequencing and methylation profiling: illustrative case.

Primary spinal cord tumors, especially primary spinal cord glioblastoma multiforme (PSC-GBM), are exceptionally rare, accounting for less than 1.5% of all spinal tumors. Their infrequency and aggressive yet atypical presentation make diagnosis challenging. In uncertain cases, a surgical approach for tissue diagnosis is often optimal. A 76-year-old male presented with a rapidly progressing clinical history marked by worsening extremity weakness, urinary retention, and periodic fecal incontinence alongside diffuse changes on neuraxis imaging. The patient, in whom subacute polyneuropathy was initially diagnosed, received multiple rounds of steroids and intravenous immunoglobulin without clinical improvement. Histopathological review of the biopsy tissue yielded an initial diagnosis of spindle cell neoplasm. Next-generation sequencing (NGS) is done routinely on all neuropathology specimens at the authors' institution, and methylation profiling is pursued in difficult cases. Ultimately, NGS and methylation profiling results were essential to an integrated final diagnosis of GBM. PSC-GBM is a rare but highly aggressive occurrence of this tumor. Prolonged back pain, rapid neurological decline, and imaging changes warrant the consideration of lesional biopsy for precise disease characterization. In inconclusive cases, NGS has proved invaluable for clinical clarification and diagnosis, underscoring its importance for integrated diagnoses in guiding appropriate treatment strategies.

Predominance of MGMT promoter methylation among Pakistani glioblastoma patients.

Glioblastoma multiforme (GBM), the most prevalent subgroup of neuroepithelial tumors, is characterized by dismal overall survival (OS). Several studies have linked O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation to OS in GBM patients. However, MGMT methylation frequencies vary geographically and across ethnicities, with limited data for South Asian populations, including Pakistan. This study aimed to analyze MGMT promoter methylation in Pakistani GBM patients. Consecutive primary GBM patients diagnosed ≥ 18 years-of-age, with no prior chemotherapy or radiotherapy history, were retrospectively selected. DNA was isolated from formalin-fixed-paraffin-embedded tissues. MGMT promoter methylation was analyzed using methylation-specific PCR. Clinical, pathological, and treatment data were assessed using Fisher's exact/Chi-squared tests. OS was calculated using Kaplan-Meier analysis in SPSS 27.0.1. The study included 48 GBM patients, comprising 38 (79.2%) males and 10 (20.8%) females. The median diagnosis age was 49.5 years (range 18-70). MGMT methylation was observed in 87.5% (42/48) of all cases. Patients with MGMT methylation undergoing radiotherapy or radiotherapy plus chemotherapy exhibited significantly improved median OS of 7.2 months (95% CI, 3.7-10.7; P < 0.001) and 16.9 months (95% CI, 15.9-17.9; P < 0.001), respectively, compared to those undergoing surgical resection only (OS: 2.2 months, 95% CI, 0.8-3.6). This is the first comprehensive study highlighting a predominance of MGMT methylation in Pakistani GBM patients. Furthermore, our findings underscore the association of MGMT methylation with improved OS across diverse treatment modalities. Larger studies are imperative to validate our findings for better management of Pakistani GBM patients.

Abstract 387: Ionizing radiation induces lipid metabolism-associated vulnerabilities in glioblastoma multiforme

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to its current standard-of-care regimen, which includes surgical resection followed by adjuvant ionizing radiation and temozolomide. Therapy resistance can be attributed to the abundance of genomic alterations in GBM tumors that evolve over time, which contribute to intra-and inter-tumoral heterogeneities that render targeted therapies as inadequate treatment options. Thus, there is an urgent unmet need for effective therapies targeting primary and recurrent GBM. Here, we investigated the impact of ionizing radiation on the transcriptome of patient derived GBM tumor spheres to identify key alterations and biological pathways involved in therapy resistance. Materials and methods: We acquired four independent human GBM tumor sphere cell lines, which were derived from patients and cultured in serum-free media. Tumor spheres were treated with either a sham radiation or a single dose of 10Gy. Using poly-A enrichment whole transcriptome sequencing, we evaluated transcriptional alterations occurring in GBM tumors spheres at 96h post-radiation. To do so, we performed differential expression analysis and gene set enrichment analysis (GSEA) to identify top differentially expressed genes and significant changes in biological phenomena. Additionally, we evaluated the cell viability response of these GBM tumor spheres to radiation. Results and discussion: PCA analysis of the four human GBM tumor sphere cell lines demonstrates that cell type has a substantial impact on variation among samples compared to non-irradiated and irradiated treatment conditions. Upon analysis of genes in common that are either upregulated or downregulated followed by GSEA, we identified enrichment of genes associated with inflammatory responses and ferroptosis repression (e.g., NUPR1, PTGS1, AOX1), and depletion of genes involved in fatty acid metabolism (e.g., INSIG1, PLA2G3, ACAT2). Furthermore, the GBM tumor sphere cell viability responses allowed us to stratify our models into radiosensitive and radioresistant subgroups. Conclusion: Radiosensitive human GBM tumor spheres exhibit transcriptional alterations in genes linked to inflammation and fatty acid metabolism to a greater extent compared to radioresistant tumor spheres. Our study characterizes the radiation response of patient derived GBM models, which is to be leveraged for combating therapeutic resistance. Citation Format: Arianna Richelle Izawa-Ishiguro, Subhiksha Nandakumar, Nicholas Carbone, Annalisa V. Ferrotta, Raashed Raziuddin, Kristen C. Vogt, Daniel A. Heller, Ingo K. Mellinghoff. Ionizing radiation induces lipid metabolism-associated vulnerabilities in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 387.

Identification of TMZ resistance-associated histone post-translational modifications in glioblastoma using multi-omics data.

Glioblastoma multiforme (GBM) is among the most aggressive cancers, with current treatments limited in efficacy. A significant hurdle in the treatment of GBM is the resistance to the chemotherapeutic agent temozolomide (TMZ). The methylation status of the MGMT promoter has been implicated as a critical biomarker of response to TMZ. To explore the mechanisms underlying resistance, we developed two TMZ-resistant GBM cell lines through a gradual increase in TMZ exposure. Transcriptome sequencing of TMZ-resistant cell lines revealed that alterations in histone post-translational modifications might be instrumental in conferring TMZ resistance. Subsequently, multi-omics analysis suggests a strong association between histone H3 lysine 9 acetylation (H3K9ac) levels and TMZ resistance. We observed a significant correlation between the expression of H3K9ac and MGMT, particularly in the unmethylated MGMT promoter samples. More importantly, our findings suggest that H3K9ac may enhance MGMT transcription by facilitating the recruitment of the SP1 transcription factor to the MGMT transcription factor binding site. Additionally, by analyzing single-cell transcriptomics data from matched primary and recurrent GBM tumors treated with TMZ, we modeled the molecular shifts occurring upon tumor recurrence. We also noted a reduction in tumor stem cell characteristics, accompanied by an increase in H3K9ac, SP1, and MGMT levels, underscoring the potential role of H3K9ac in tumor relapse following TMZ therapy. The increase in H3K9ac appears to enhance the recruitment of the transcription factor SP1 to its binding sites within the MGMT locus, consequently upregulating MGMT expression and driving TMZ resistance in GBM.

Genetic mutation patterns among glioblastoma patients in the Taiwanese population - insights from a single institution retrospective study.

This study utilized Next-Generation Sequencing (NGS) to explore genetic determinants of survival duration in Glioblastoma Multiforme (GBM) patients. We categorized 30 primary GBM patients into two groups based on their survival periods: extended survival (over two years, N = 17) and abbreviated survival (under two years, N = 13). For identifying pathogenic or likely pathogenic variants, we leveraged the ClinVar database. The cohort, aged 23 to 66 (median: 53), included 17 patients in Group A (survival >2 years, 10 males, 7 females), and 13 patients in Group B (survival <2 years, 8 males, 5 females), with a 60% to 40% male-to-female ratio. Identified mutations included CHEK2 (c.1477 G > A, p.E493K), IDH1 (c.395 G > A, p.R132H), and TP53 mutations. Non-coding regions exhibited variants in the TERT promoter (c.-146C > T, c.-124C > T) and TP53 RNA splicing site (c.376-2 A > C, c.376-2 A > G). While Group A had more mutations, statistical significance wasn't reached, likely due to sample size. Notably, TP53, and ATR displayed a trend toward significance. Surprisingly, TP53 mutations were more prevalent in Group A, contradicting Western findings on poorer GBM prognosis. In Taiwanese GBM patients, bevacizumab usage is linked to improved survival rates, affirming its safety and effectiveness. EGFR mutations are infrequent, suggesting potential distinctions in carcinogenic pathways. Further research on EGFR mutations and amplifications is essential for refining therapeutic approaches. TP53 mutations are associated with enhanced survival, but their functional implications necessitate detailed exploration. This study pioneers genetic analysis in Taiwanese GBM patients using NGS, advancing our understanding of their genetic landscape.

Transduction Efficiency of Zika Virus E Protein Pseudotyped HIV-1gfp and Its Oncolytic Activity Tested in Primary Glioblastoma Cell Cultures.

The development of new tools against glioblastoma multiforme (GBM), the most aggressive and common cancer originating in the brain, remains of utmost importance. Lentiviral vectors (LVs) are among the tools of future concepts, and pseudotyping offers the possibility of tailoring LVs to efficiently transduce and inactivate GBM tumor cells. Zika virus (ZIKV) has a specificity for GBM cells, leaving healthy brain cells unharmed, which makes it a prime candidate for the development of LVs with a ZIKV coat. Here, primary GBM cell cultures were transduced with different LVs encased with ZIKV envelope variants. LVs were generated by using the pNLgfpAM plasmid, which produces the lentiviral, HIV-1-based, core particle with GFP (green fluorescent protein) as a reporter (HIVgfp). Using five different GBM primary cell cultures and three laboratory-adapted GBM cell lines, we showed that ZIKV/HIVgfp achieved a 4-6 times higher transduction efficiency compared to the commonly used VSV/HIVgfp. Transduced GBM cell cultures were monitored over a period of 9 days to identify GFP+ cells to study the oncolytic effect due to ZIKV/HIVgfp entry. Tests of GBM tumor specificity by transduction of GBM tumor and normal brain cells showed a high specificity for GBM cells.

Expression of molecular markers and synergistic anticancer effects of chemotherapy with antimicrobial peptides on glioblastoma cells.

Glioblastoma multiforme (GBM) is the most aggressive and fatal malignant primary brain tumor. The enhancement of the survival rate for glioma patients remains limited, even with the utilization of a combined treatment approach involving surgery, radiotherapy, and chemotherapy. This study was designed to assess the expression of IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX in glioblastoma tissue from 11 patients. We investigated the anticancer impact and combined effects of cathelicidin (LL-37), protegrin-1 (PG-1), with chemotherapy-temozolomide (TMZ), doxorubicin (DOX), carboplatin (CB), cisplatin (CPL), and etoposide (ETO) in primary GBM cells. In addition, we examined the effect of LL-37, PG-1 on normal human fibroblasts and in the C6/Wistar rat intracerebral glioma model. For this study, 11 cases of glioblastoma were evaluated immunohistochemically for IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cells viability and to determine cytotoxic effects of LL-37, PG-1 and their combination with chemotherapy in primary GBM cells. Synergism or antagonism was determined using combination index (CI) method. Finally, we established C6 glioblastoma model in Wistar rats to investigate the antitumor activity. Peptides showed a strong cytotoxic effect on primary GBM cells in the MTT test (IC50 2-16 and 1-32μM) compared to chemotherapy. The dual-drug combinations of LL-37 + DOX, LL-37 + CB (CI 0.46-0.75) and PG-1 + DOX, PG-1 + CB, PG-1 + TMZ (CI 0.11-0.77), demonstrated a synergism in primary GBM cells. In rat C6 intracerebral GBM model, survival of rats in experimental group (66.75 ± 12.6days) was prolonged compared with that in control cohort (26.2 ± 2.66days, p = 0.0008). After LL-37 treatment, experimental group rats showed significantly lower tumor volumes (31.00 ± 8.8 mm3) and weight (49.4 ± 13.3mg) compared with control group rats (153.8 ± 43.53mg, p = 0.038; 82.50 ± 7.60 mm3, respectively). The combination of antimicrobial peptides and chemical drugs enhances the cytotoxicity of chemotherapy and exerts synergistic antitumor effects in primary GBM cells. Moreover, in vivo study provided the first evidence that LL-37 could effectively inhibit brain tumor growth in rat C6 intracerebral GBM model. These results suggested a significant strategy for proposing a promising therapy for the treatment of GBM.

IFITM3 promotes glioblastoma stem cell-mediated angiogenesis via regulating JAK/STAT3/bFGF signaling pathway

Interferon-induced transmembrane protein 3 (IFITM3) has been previously verified to be an endosomal protein that prevents viral infection. Recent findings suggested IFITM3 as a key factor in tumor invasion and progression. To clarify the role and molecular mechanism of IFITM3 in Glioblastoma multiforme (GBM) progression, we investigated the expression of IFITM3 in glioma datasets culled from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA). Primary GBM stem cells (GSCs) were cultured and identified in vitro. Loss-of-function and gain-of-function experiments were established by using shRNAs and lentiviral vectors targeting IFITM3. Co-culture system of GSCs and vascular endothelial cells was constructed in a Transwell chamber. Tube formation and spheroid-based angiogenesis assays were performed to determine the angiogenic capacity of endothelial cells. Results revealed that IFITM3 is elevated in GBM samples and predictive of adverse outcome. Mechanistically, GSCs-derived IFITM3 causes activation of Jak2/STAT3 signaling and leads to robust secretion of bFGF into tumor environment, which eventually results in enhanced angiogenesis. Taken together, these evidence indicated IFITM3 as an essential factor in GBM angiogenesis. Our findings provide a new insight into mechanism by which IFITM3 modulates GBM angiogenesis.

Dual blockade of EGFR and PI3K signaling pathways offers a therapeutic strategy for glioblastoma

BackgroundGlioblastoma multiforme (GBM) is a devastating disease that lacks effective drugs for targeted therapy. Previously, we found that the third-generation epidermal growth factor receptor (EGFR) inhibitor AZD-9291 persistently blocked the activation of the ERK pathway but had no inhibitory effect on the phosphoinositide 3-kinase (PI3K)/Akt pathway. Given that the PI3K inhibitor GDC-0084 is being evaluated in phase I/II clinical trials of GBM treatment, we hypothesized that combined inhibition of the EGFR/ERK and PI3K/Akt pathways may have a synergistic effect in the treatment of GBM.MethodsThe synergistic effects of cotreatment with AZD-9291 and GDC-0084 were validated using cell viability assays in GBM and primary GBM cell lines. Moreover, the underlying inhibitory mechanisms were assessed through colony formation, EdU proliferation, and cell cycle assays, as well as RNA-seq analyses and western blot. The therapeutic effects of the drug combination on tumor growth and survival were investigated in mice bearing tumors using subcutaneously or intracranially injected LN229 xenografts.ResultsCombined treatment with AZD-9291 and GDC-0084 synergistically inhibited the proliferation and clonogenic survival, as well as induced cell cycle arrest of GBM cells and primary GBM cells, compared to monotherapy. Moreover, AZD-9291 plus GDC-0084 combination therapy significantly inhibited the growth of subcutaneous tumors and orthotopic brain tumor xenografts, thus prolonging the survival of tumor-bearing mice. More importantly, the combination of AZD-9291 and GDC-0084 simultaneously blocked the activation of the EGFR/MEK/ERK and PI3K/AKT/mTOR signaling pathways, thereby exerting significant antitumor activity.ConclusionOur findings demonstrate that the combined blockade of the EGFR/MEK/ERK and PI3K/AKT/mTOR pathways is more effective against GBM than inhibition of each pathway alone, both in vitro and in vivo. Our results suggest that AZD-9291 combined with GDC-0084 may be considered as a potential treatment strategy in future clinical trials.396yfhm1sdcH9_KhVkr-McVideo

Primary spinal glioblastoma multiforme in a pediatric patient: A case report

Primary spinal glioblastoma multiforme in a pediatric patient: A case report

Spatially Resolved Whole Transcriptome Analysis of Histologically-Characterized Tissue Microarray of Patient-Matched Primary and Recurrent Glioblastomas to Identify Underlying Mechanisms of Treatment Resistance

Glioblastoma multiforme (GBM) is a lethal CNS malignancy. Radiation therapy increases overall survival, but tumors often recur in high-radiation dose regions. Additionally, recent investigations have underscored the importance of intra-tumoral heterogeneity as a driver of GBM biology. The purpose of this investigation is to characterize transcriptome differences in primary and recurrent GBM patient clinical samples using a digital spatial profiling approach to better appreciate treatment resistance mechanisms. To address the lack of understanding of molecular mechanisms of resistance in GBM, patient-matched primary and recurrent GBM pathological specimens were identified within the brain tissue biorepository and tissue cores were selected for generation of a tissue microarray (TMA). Hematoxylin and eosin staining with histomorphological (cellular tumor, pseudopalisading necrosis, invasive edge, and perivascular inflammation) scoring were performed in a blinded fashion for every core. This array was then molecularly characterized using digital spatial profiling of the transcriptome. Quality assurance including filtering of lowly expressed genes followed by downstream analyses of the data were performed using the manufacturer's recommended methods within their Bioconductor library. Gene Set Enrichment Analysis (GSEA) was then performed on the ranked gene lists. After recommended filtering, 6171 genes and 248 regions of interest remained for downstream analysis representing 22 unique patients across four different tumor histomorphological types. Significance testing revealed 679 genes that were differentially expressed between primary and recurrent tumor samples (at FDR<1%). On GSEA analysis, the chromosomal positional locus that contains genes most strongly up-regulated is 12q14, a locus that was previously identified as genomically amplified in multiple patient-derived xenograft lines after radiation selection. Additionally, recurrent tumors display a transcriptional profile more similar to the mesenchymal subtype, whereas primary tumors have a more classical transcriptional phenotype. The epithelial-to-mesenchymal transition pathway is particularly strongly up-regulated in recurrent tumors. Recurrent selection at previously identified genomic loci and molecular pathways underscores a possible conserved set of pathways for treatment resistance. This analysis has yielded a set of gene and molecular pathways that will guide future work in our lab targeting treatment resistance using novel therapeutics and radiation techniques in GBM. Future directions include assessing the feasibility of mapping these clinical samples onto our previously generated panel of comprehensively characterized patient-derived xenograft lines.

A Rare Case of Primary Glioblastoma Multiforme in the Cerebellum, Case Report and Literature Review

Glioblastoma multiforme (GBM) is a highly aggressive and malignant tumor that can arise in the central nervous system, including the cerebellum. We presented a case of GBM in cerebellum who was the 27 years - old male presented with gate ataxia and dysphagia. MRI demonstrated brain mass in right cerebellum with enhancement. Patient underwent surgical resection, followed by radiation therapy and chemotherapy. Further researches are needed to identify optimal treatment strategies and improve outcomes for patients with GBM in the spinal cord. Close monitoring and follow-up care are essential to manage potential complications and optimize quality of life for affected individuals.

Characterization of tumor microenvironment in glioblastoma multiforme identifies ITGB2 as a key immune and stromal related regulator in glial cell types

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor characterized by inter and intra-tumor heterogeneity and complex tumor microenvironment. To uncover the molecular targets in this milieu, we systematically identified immune and stromal interactions at the glial cell type level that leverages on RNA-sequencing data of GBM patients from The Cancer Genome Atlas. The perturbed genes between the high vs low immune and stromal scored patients were subjected to weighted gene co-expression network analysis to identify the glial cell type specific networks in immune and stromal infiltrated patients. The intramodular connectivity analysis identified the highly connected genes in each module. Combining it with univariable and multivariable prognostic analysis revealed common vital gene ITGB2, between the immune and stromal infiltrated patients enriched in microglia and newly formed oligodendrocytes. We found following unique hub genes in immune infiltrated patients; COL6A3 (microglia), ITGAM (oligodendrocyte precursor cells), TNFSF9 (microglia), and in stromal infiltrated patients, SERPINE1 (microglia) and THBS1 (newly formed oligodendrocytes, oligodendrocyte precursor cells). To validate these hub genes, we used external GBM patient single cell RNA-sequencing dataset and this identified ITGB2 to be significantly enriched in microglia, newly formed oligodendrocytes, T-cells, macrophages and adipocyte cell types in both immune and stromal datasets. The tumor infiltration analysis of ITGB2 showed that it is correlated with myeloid dendritic cells, macrophages, monocytes, neutrophils, B-cells, fibroblasts and adipocytes. Overall, the systematic screening of tumor microenvironment components at glial cell types uncovered ITGB2 as a potential target in primary GBM.

Establishment of a 3D Model to Characterize the Radioresponse of Patient-Derived Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite modern, multimodal therapeutic options of surgery, chemotherapy, tumor-treating fields (TTF), and radiotherapy, the 5-year survival is below 10%. In order to develop new therapies, better preclinical models are needed that mimic the complexity of a tumor. In this work, we established a novel three-dimensional (3D) model for patient-derived GBM cell lines. To analyze the volume and growth pattern of primary GBM cells in 3D culture, a CoSeedisTM culture system was used, and radiation sensitivity in comparison to conventional 2D colony formation assay (CFA) was analyzed. Both culture systems revealed a dose-dependent reduction in survival, but the high variance in colony size and shape prevented reliable evaluation of the 2D cultures. In contrast, the size of 3D spheroids could be measured accurately. Immunostaining of spheroids grown in the 3D culture system showed an increase in the DNA double-strand-break marker γH2AX one hour after irradiation. After 24 h, a decrease in DNA damage was observed, indicating active repair mechanisms. In summary, this new translational 3D model may better reflect the tumor complexity and be useful for analyzing the growth, radiosensitivity, and DNA repair of patient-derived GBM cells.

Genetic Variants Impacting Angiogenesis Signaling Pathways in Glioblastoma Multiforme: A Systematic Review of Mutations and Polymorphisms.

Several signaling pathways are involved in the process of angiogenesis, which is one of the most important hallmarks of glioblastoma multiforme (GBM). Identifying related gene variants can help researchers work out what causes anti-angiogenesis drug resistance. The goal of this systematic analysis was to identify all mutations and polymorphisms involved in angiogenesis pathways in GBM and their impact on clinical outcomes. The keywords include glioblastoma, angiogenesis, signaling pathway, mutation, polymorphism, and related terms used to search ISI, PubMed, and Scopus for relevant articles published up to January 2022. The PRISMA protocol was used to conduct our systematic review. The related articles were taken into consideration. The risk of bias in the associated articles was surveyed, as well as the article scoring. Two authors collaborated on data extraction. The inclusion criteria were included in 32 articles out of a total of 787 articles. VEGF, HIF1a, EGFR, PI3K, and MAPK are the pathways that have been studied the most. IDH1, VEGF, VEGFR, EGFR, and HIF1a are the genes with the highest frequency of mutations or polymorphisms. In conclusion, this study found that angiogenesis in primary or recurrent GBM is linked to gene changes in eleven signaling pathways. However, some of these gene mutations have been researched numerous times in relation to angiogenesis, while others have only been studied once. Understanding these changes will help us employ combination therapies more effectively for GBM patients' survival and personal medicine.

Alpha-Lipoic Acid, Auraptene, and Particularly Their Combination Prevent the Metastasis of U87 Human Glioblastoma Cells.

The primary malignant brain tumor glioblastoma multiforme (GBM) is most commonly detected in individuals over 60 years old. The standard therapeutic approach for GBM is radiotherapy combined with temozolomide. Recently, herbal products, such as alpha-lipoic acid (ALA) and auraptene (AUR), have shown promising anticancer effects on various cancer cells and animal models. However, it is not well understood how ALA, AUR, and their combination in GBM work to combat cancer. Thus, the purpose of this study was to investigate the antimetastatic effects of the ALA-AUR combination on U87 human glioblastoma cells. The inhibitory effects of ALA, AUR, and the ALA/AUR combination on the migration and metastasis of U87 cells were evaluated using a wound healing test and gelatin zymography. The expression levels of matrix metalloproteinase MMP-2 and MMP-9 were assessed at the transcriptional and translational levels using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Our findings revealed that combination therapy reduced cell migration and metastasis, which was indicated by the reduction in MMP-2/-9 expression both at mRNA and protein levels, as well as their enzymatic activity in U87 cells. This study demonstrated that the combination of ALA and AUR effectively inhibited the migration and metastasis of U87 cells. Thus, given their safety and favorable specifications, the combination of these drugs can be a promising candidate for GBM treatment as primary or adjuvant therapy.

A Preliminary Study Indicating Improvement in the Median Survival Time of Glioblastoma Multiforme Patients by the Application of Deuterium Depletion in Combination with Conventional Therapy.

Glioblastoma multiforme (GBM) and malignant gliomas are the most common primary malignant brain tumors. Temozolomide (TMZ) chemotherapy plus radiation therapy (RT), admi-mistered after debulking surgery, increased the median survival time (MST) from 12.1 months with RT alone merely to 14.6 months, respectively. In this study, the actions of deuterium-depleted water (DDW) on the survival of GBM patients who also received conventional therapies was investigated. Without changing the conventional treatment, the daily fluid intake of the patients was wholly replaced with DDW in 1.5-2 L per day volume to reduce the D concentration in their bodies. The primary endpoint was the MST. The 55 patients involved in this study, who received conventional treatment and consumed DDW, showed a longer MST (30 months) compared to the historical control (12.1-14.6 months). There was a massive difference between the two genders in the calculated MST values; it was 25 months in the male subgroup (n = 33) and 42 months in the female subgroup (n = 22), respectively. The MST was 27 months without TMZ treatment (38 patients) and 42 months in the TMZ-treated group (17 patients), respectively. For the selected 31 patients, who consumed DDW in the correct way in addition to their conventional treatments, their MST was calculated as 30 months. Within this group, the 20 subjects who had relapsed before DDW treatment had 30 months of MST, but in those 10 subjects who were in remission when DDW treatment started, their MST was 47 months. In the subgroup of patients who began their DDW treatment parallel with radiotherapy, their MST was again 47 months, and it was 25 months when their DDW treatment was started at 8 weeks or later after the completion of radiotherapy. Altogether, these survival times were substantially prolonged compared to the prospective clinical data of patients with primary GBM. Consequently, if conventional therapies are supplemented with D depletion, better survival can be achieved in the advanced stage of GBM than with the known targeted or combination therapies. Application of DDW is recommended in all stages of the disease before surgery and in parallel with radiotherapy, and repeated DDW courses are advised when remission has been achieved.

The Prognostic Value of the Treatment and Outcome in Patients with Glioblastoma: A Retrospective Cohort Study

The classification of central nervous system (CNS) tumors by the 2021 World Health Organization (WHO) has led to significant changes in tumor taxonomy. One of the most significant changes is that isocitrate dehydrogenase (IDH) mutant forms of glioblastoma multiforme (GBM) have been differentiated into separate entities, no longer allowing entries to be classified as not otherwise specified (NOS). As a result, this entity only comprises the most aggressive adult-type tumors and established prognostic factors no longer apply. Glioblastoma (GBM) IDH-wildtype CNS WHO grade 4 typically presents necrosis and/or microvascular proliferation and molecular alterations. Herein, we aimed to classify glioblastoma cases to establish a patient survival pattern based on age, gender, the number of masses, tumor location, functional localization, presence of shift, the volume of edema and necrosis, extent of surgery, radiotherapychemotherapy protocol, and isocitrate dehydrogenase (IDH) presence, affecting overall survival were determined retrospectively. A total of 433 patients &gt;20 years old with primary GBM were treated in a single institution between 1996 and 2019. The median survival was 9±0.62 [95% CI 7.78-10.21] months and the survival rate after diagnosis was 39.4% in 1st year, 17% in 2nd year, and 5% in 3rd year. Statistically, age, tumor location, edema, and necrosis were indicated as independent preoperative predictors of prognosis, and younger age at diagnosis, the left temporal and the right occipital location, maximal tumor resection, and administration of temozolomide adjuvant chemotherapy were revealed favorable prognostic factors. Keywords: Glioblastoma, Overall survival, Prognosis, Surgery

Abstract 1604: Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2

Abstract Interleukin 13 receptor subunit alpha 2 (IL13RA2) is one of the two major receptors for the cytokine interleukin 13 (IL-13). While IL13RA1 has low affinity for IL-13 and it is expressed ubiquitously in humans, IL13RA2 has high binding affinity to IL-13 and its expression in normal tissues is mainly restricted to the testes. IL13RA2 was initially regarded as a decoy receptor because it has a short cytoplasmic tail with no known signaling motifs, and its binding to IL-13 does not lead to activation of the JAK/STAT6 pathway. However, recent studies demonstrated that IL-13-mediated IL13RA2 signaling occurs via STAT6-independent pathways. IL13RA2 is reported to be expressed at a high frequency in glioblastoma multiforme (GBM) as well as in other solid tumors including melanoma, renal cell carcinoma and adrenocortical carcinoma and is correlated with poor prognosis. ADCT-211 is an antibody-drug conjugate composed of HuCl47, a humanized IgG1 antibody directed against human IL13RA2, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the PBD dimer cytotoxin SG2000 (drug to antibody ratio ~ 1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-211 in human cancer cell lines and xenograft models, to determine its safety, tolerability and pharmacokinetic (PK) in the rat and to measure IL13RA2 expression by immunohistochemistry (IHC) in human tumor specimens. HuCl47 showed specific binding to recombinant IL13RA2, while it did not bind to IL13RA1. In vitro, ADCT-211 had highly potent and targeted cytotoxicity against various IL13RA2-expressing solid cancer cell lines. In vivo, a single dose of ADCT-211 at 10 mg/kg was able to completely eradicate tumors in the subcutaneous (sc) A375 human melanoma xenograft model and resulted in 8/8 tumor-free survivors (TFS) at the end of the study on day 57. In the sc U251 human glioblastoma xenograft model, a single dose of ADCT-211 at 3.75 mg/kg showed potent and durable antitumor activity and it resulted in 5/10 partial responders and 5/10 complete responders, 2 of which were TFS at the end of the study on day 51. In an MTD study in male rats, ADCT-211 was stable and tolerated up to 24 mg/kg single dose, with exposure data being indicative of a linear PK profile with a half-life of 12-19 days. Expression of membranous IL13RA2 was confirmed by IHC in a panel of primary and refractory GBM samples and malignant melanoma, highlighting them as potential indications for the clinical development of ADCT-211. In conclusion, ADCT-211 demonstrated potent and specific in vitro and in vivo anti-tumor activity and it was stable and well tolerated in the rat, warranting further development of ADCT-211 into the clinic in IL13RA2-expressing cancers. Citation Format: Francesca Zammarchi, Karin Havenith, Ben Leatherdale, Britanny Roberts, Lara Montolio, Afroze Patel, Narinder Janghra, Pedro Alves, Kristina Zaitseva, Cecile Oblette, Ian Kirby, Lolke de Haan, Patrick H van Berkel. Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1604.

Abstract 6442: Role of TWEAK-Fn14 axis in driving immunosuppression in glioblastoma multiforme

Abstract Cellular heterogeneity within glioblastoma multiforme (GBM) is a key driver of resistance to standard-of-care therapeutic approaches such as DNA damage (temozolomide, TMZ), angiogenesis inhibition (Bevacizumab, BM), radiation necrosis, and surgical resection. As such, understanding and targeting heterogeneous tumor-associated populations is a critical aim of ongoing GBM research. Central nervous system (CNS) resident microglia constitute significant percentages of primary GBM samples, often up to 50% of bulk tumor. Additionally, glioblastoma-associated microglia have been implicated in intra-tumoral immunosuppression, and are thus a key barrier to successful implementation of immunotherapies that seek to enhance T-cell mediated anti-tumoral cytotoxicity. As such, insight into targetable pathways that upregulate immunosuppressive cytokines within GBM-associated microglia is necessary for overcoming immuno-therapeutic failure. Here, we identify expression of the cytokine TWEAK (TNFSF12) and its receptor Fn14 (TNFRSF12A) within the myeloid compartment of primary GBM samples via scRNA-seq. Furthermore, we demonstrate TWEAK-mediated reprogramming of GBM-associated microglia towards an immunosuppressive phenotype capable of inhibiting CD8+ T cell activation and proliferation. These findings unveil a potent, targetable axis of immunosuppression within GBM and are thus an exciting step towards enhancing efficacy of existing T-cell based anti-cancer strategies for GBM patients. Citation Format: Angad Beniwal, Ryan Eghlimi, Kenneth D. Alexander, Mylan R. Blomquist, Nhan Tran. Role of TWEAK-Fn14 axis in driving immunosuppression in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6442.