Intertumoral heterogeneity in glioblastoma—driven by both genomic and transcriptomic variation—complicates our understanding of how different tumor cell populations contribute to disease progression. Infiltrating tumor cells, which invade surrounding brain tissue and evade surgical resection, are thought to play a central role in recurrence. To address this, we aimed to characterize the gene expression profiles and cellular states of infiltrative tumor cells in glioblastoma. We performed high-plex spatial transcriptomics using the CosMx Spatial Molecular Imager (NanoString) on tumor tissue from eight glioblastoma patients. Formalin-fixed paraffin-embedded samples were selected to capture both the tumor core and invasive margin. A targeted panel of 1,000 genes enabled spatially resolved gene expression profiling at single-cell resolution, allowing precise identification and localization of malignant and non-malignant cell states. We show that malignant cells can be distinguished from non-malignant populations by using patient-specific clustering. Based on this annotation, we identified several known malignant states—including AC-, OPC-, NPC-, and MES-like cells—as well as a recently characterized glial-progenitor (GPC)-like state. This population co-expressed genes associated with both astrocytic and oligodendrocyte progenitor lineages and was found to be more proliferative than the traditional AC-like state. The GPC-like state was most enriched in the classical glioblastoma subtype and was strongly associated with EGFR amplification or mutation. Spatial analyses investigating malignant differences between tumor and infiltrated tissue showed heterogeneous infiltration patterns across patients. In the most extreme case, the dominant GPC-like population in the tumor core gave way to increased proportions of AC-like cells in infiltrated regions. Our study highlights diverging infiltration patterns across glioblastoma tumors, with indications of a GPC-like to AC-like transition occurring in classical-subtyped tumors. This shift is associated with a decrease in cell proliferation and may have implications for clinical treatment.

Abstract Cancer stem cells (CSCs) within glioblastoma (GBM) contribute to therapeutic resistance and tumor recurrence. Receptor tyrosine kinases (RTKs) such as VEGFR2, MET, and AXL are frequently dysregulated in GBM and implicated as GBM driver genes, making them attractive therapeutic targets. This study evaluates the efficacy of cabozantinib (XL184), a multi-kinase inhibitor targeting VEGFR2, MET, and AXL, in GBM CSCs and patient-derived xenograft (PDX), with a focus on uncovering resistance mechanisms and identifying opportunities for combination therapies. Nine genomically diverse GBM CSCs were treated with XL184 to assess drug sensitivity. Proteomics were used to evaluate changes in key signaling pathways. CSC-derived PDX from two proneural (HF2587, HF3016) and one classical (HF2927) GBM were treated with XL184 monotherapy. Tumor growth and survival were monitored. Immunohistochemical (IHC) analysis of CD31 expression to assess angiogenesis was performed with high-resolution microscopy. GBM CSCs exhibited a wide range of sensitivity to XL184 (IC50: 2μM - 34μM). Proteomics showed XL184-mediated inhibition of phospho-VEGFR2, AKT, and ERK, while sub-lethal XL184 doses upregulated phospho-MET and STAT3. In vivo, HF2587 (p=0.00022, n>18) and HF2927 (p=0.1847, n>12) xenografts responded to XL184 with reduced tumor growth and improved survival while HF3016 (p=0.061, n>16) was resistant to the monotherapy. HF2587 and HF2927 demonstrated a reduction in vessel density, measured by CD31 expression, compared to normal brain. The non-responsive HF3016 exhibited increased angiogenesis following therapy, a potential indicator of treatment resistance. These findings highlight the heterogeneity of XL184 response in GBM. Adaptive resistance through MET and downstream STAT3 activation supports combination therapies to overcome redundancy from pathway-specific negative feedback. Evidence of reduced angiogenesis imply that vasculature normalization is a key drug mechanism of action and support the theory of modulating the tumor microenvironment in glioma therapy.

Abstract BACKGROUND Glioblastoma (GBM) is well known to interact with surrounding tumor and stromal cells in a variety of ways to maintain its pathogenicity. Emerging evidence has identified tunneling nanotubes (TNTs), dynamic actin-rich structures that mediate direct intercellular communication, as critical facilitators of GBM progression and therapeutic resistance, yet mediators of TNT formation remain poorly understood. Myristorylated Alanine-Rich C Kinase Substrate (MARCKS), an actin-binding protein regulated by PKC, may influence TNT dynamics. We hypothesized that MARCKS effector domain (ED) phosphorylation via PKC regulates TNT formation and mitochondrial transfer between GBM cells and astrocytes, contributing to chemoresistance. METHODS We employed serum-free TNT-promoting co-culture models of PTEN-null GBM cells (patient-derived xenograft JX14 cells or U87) and normal human astrocytes (NHAs), using immunofluorescence confocal microscopy to detect MARCKS localization, and quantitative assays to measure TNT number, length, and mitochondria transfer. Pharmacological modulation of PKC with phorbol ester agonist (PMA) or inhibitor (Enzastaurin), and treatment with a MARCKS ED peptide (MED2), assessed effects on TNTs. Doxycycline-inducible U87 MARCKS ED mutants (WT, pseudo-phosphorylated [PP], non-phosphorylatable [NP]) were used to dissect MARCKS phosphorylation roles. CGGA database analysis examined correlations between MARCKS and TNT-related genes in primary classical GBM. RESULTS JX14 co-cultured with NHAs exhibited increased temozolomide resistance. MARCKS localized to heterotypic TNTs, and MED2 reduced TNT number, length, and mitochondrial transfer. PKC activation increased TNT formation and transfer, while inhibition produced opposite effects. U87-PP cells displayed abundant TNTs and transfer similar to PKC activation, whereas U87-NP cells showed reduced TNTs. MARCKS positively correlated with MyosinX, ACTB, ACTG1, and RHOT1 in GBM patient data. CONCLUSIONS MARCKS ED phosphorylation via PKC promotes functional TNT formation and mitochondrial trafficking between GBM cells and astrocytes, contributing to chemoresistance. Targeting MARCKS or PKC may represent a novel strategy to disrupt TNT-mediated resistance mechanisms in GBM.

Abstract A new mouse model for the classical subtype of human glioblastoma (GBM) has been bred using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion, the mouse homolog of human p14ARF/CDKN2A, in GFAP expressing cells. Transgenic mice develop intraparenchymal and/or leptomeningeal brain lesions with some spinal cord invasion as early as 1 month old and 95% of mice die by 6 months due to hydrocephalus and/or paralysis. Mice with high grade tumors have worse survival and similar features to human classical GBM such as necrosis, high levels of mitosis and infiltration of tumor cells into normal brain. Immunohistochemical analysis confirms EGFRvIII overexpression and p19-ARF loss in Cre-expressing cells, along with patchy positive GFAP and high proliferation by Ki67. Bulk RNA sequencing reveals GEC3 tumors cluster closely with human classical GBM and have significant upregulation of downstream markers in the JAK/STAT and P13K/AKT pathways. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and generate xenograft tumors by 3 weeks after intracranial injections into NODSCID mice. Xenograft tumors are reminiscent of the primary tumor, with similar histopathological features and immunohistochemical staining. Our spontaneous glioma model is a powerful tool for future studies focused on the role of the immune microenvironment on glioma recurrence and resistance.

Abstract Extra-axial glioblastoma is rare. We report the first case of multicentric extra-axial sellar glioblastoma mimicking a meningioma and systematic review on its pathogenesis following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. The case report follows the CAse REport guidelines. With this review a series of cases, including our case, is presented.Case presentation: A 73-year-old gentleman had an extra-axial lesion in the sellar region and a left clinoid mimicking a meningioma. Dense dural and vessel adhesions restricted resection to subtotal decompression. Histopathology was isocitrate dehydrogenase (IDH) wild-type glioblastoma.We searched databases as per the PRISMA guidelines for articles on extra-axial glioblastoma, leptomeningeal glioblastoma, and cases where glioblastoma mimic meningioma. We analyzed demographics, clinical presentation, surgical challenges, histopathology, and immunohistochemistry.We identified 793 articles, of which 9 articles, 12 patients (including our case), matched our inclusion criteria. Most of the patients were elderly with a mean age of 56.9 years (range; 33–74 years); male:female of 8:4. The majority had headaches without raised intracranial pressure. The most common mimicking pathology was meningioma. Adhesions to brain, major vessels, and pial invasion made complete excision impossible in most patients. IDH wild-type tumors had middle meningeal artery blush in angiogram.Extra-axial/primary-intracranial-leptomeningeal-glioblastoma is a tumor subtype with specific clinical/radiological features, extent of resection, and outcomes. As glioblastoma prognosis differs significantly from meningioma, extra-axial glioblastoma's identification is essential for decision-making and prognostication. There is an early trend suggesting that primary leptomeningeal glioma prognosis may not be worse than classic glioblastoma. This case adds to knowledge on pathogenic mechanisms and variable representation of glioblastoma.

Glioblastoma (GB) is a highly aggressive brain cancer with a median survival of under 2 years despite conventional therapy. Research efforts frequently use murine in vivo models to study cancer progression and tumor growth, as these models recapitulate critical aspects of the tumor microenvironment, immune responses, and the appearance of classical GB pathology. Here, we describe a detailed protocol for performing stereotaxic injections to establish GB tumor models in immunocompetent C57BL/6 mice. Stereotaxic surgery enables rapid and precise delivery of GB cells grown as 2D cultures or spheroids into the brain using anatomical landmarks without the need for image guidance. Specifically, GL261 murine cell lines are injected into the striatum, allowing subsequent assessment of behavior and physiology. On day 21 post surgery, animals are euthanized following perfusion and their brains extracted and fixed. Hematoxylin-eosin staining on brain slices enables measurement of tumor area and volume, while immunohistochemistry enables imaging of tumor proliferation markers. This paper provides detailed protocols for a reliable approach to generate in vivo GB models for studying tumor growth and cancer progression. Additionally, this method is adaptable for other GB cell lines, transgenic mice, or patient-derived xenografts broadening its applicability for GB research.

Purpose: Proteomics of glioma have notyet providedbiomarkers and pathways that would clearly discriminate glioma subgroups. Methods: 82 glioma biopsies were prospectively collectedand classified into six subgroups defined by methylomic classification:two low-grade glioma (LGG) and four high-grade glioma (HGG) subgroups.Proteins were extracted and processed for liquid chromatography–massspectrometry (LC–MS). Differentially expressed proteins (DEPs)between subgroups were annotated, and functional validation was performedusing inhibitor response assays in subtype-positive, patient-derivedglioblastoma single-cell suspensions. Results: 5057proteins were quantified for each sample. Tumor grading and IDH mutation status were the strongest discriminators fordifferential expression patterns. The glioblastoma IDH-wildtype subgroups showed diverse patterns of functions enrichedwith overexpressed DEPs: translation and cell cycle/telomere regulationin proneural glioblastoma (linked to cell proliferation), actin cytoskeleton,cell adhesion, and apoptosis regulation in classical glioblastoma(migration and invasion), and mitochondrial ATP synthesis in mesenchymalglioblastoma (metabolism). The most overexpressed proteins were correlatedwith survival and mRNA expression data. In vitro, inhibition of theseproteins led to reduced cell viability that differed among subgroups,albeit in a small patient-derived exploratory cohort. Conclusion: This mainly descriptive study on proteomics in glioma providesinsights into subgroup metabolism and potential biomarkers for furtherexperimental testing.

Abstract Glioblastoma is the most common and malignant type of primary brain cancer with a median patient survival of only 15 months. Tumors are typically categorized into three molecular subtypes—proneural, classical, and mesenchymal—each linked to specific genomic and microenvironmental factors. However, the functional and clinical implications of these subtypes remain poorly understood. Standard treatment involves surgical resection followed by chemotherapy and radiotherapy. Despite this, malignant cells have already at the time point of diagnosis infiltrated healthy brain tissue, driving tumor recurrence. Infiltrating cells have been found to maintain high proliferative rates while invading the brain (termed “go-and-grow”) but have also been shown be non-proliferative (termed “go-or-grow”). Here, we perform computational analyses on transcriptomic data from over 600 patients, supplemented with genomic data, to investigate infiltration patterns in glioblastoma tumors. We identify a proliferative astrocytic state strongly associated with the classical subtype and EGFR amplification and distinct from a non-proliferative astrocytic state. Further analysis showed subtype-specific infiltrative patterns between the tumor core and adjacent brain tissue. To validate these findings, we performed single-cell spatial transcriptomics on patient samples, revealing that classical tumors exhibit a “go-or-grow” phenotype, where core regions are proliferative and infiltrative regions are dormant. In contrast, proneural tumors maintain consistent malignant states and proliferation rates, reflecting a “go-and-grow” phenotype. These findings indicate a direct relationship between the genomic status of glioblastoma tumors and the transcriptomic profiles of their infiltrating malignant cells. Together, our study provides insights into genomically-driven infiltration patterns in proneural and classical glioblastomas, with potential implications for clinical treatment. Citation Format: Dylan Harwood, Sara Blaabjerg Artzi, Vilde Pedersen, Alessio Locallo, Maya Jeje Lü, David Scheie, Anne Dorte Nørøxe, Nadine Margaretha Hammouda, Ulrik Lassen, Joachim Lütken Weischenfeldt, Bjarne Winther Kristensen. Genomic heterogeneity drives distinct infiltration patterns in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 168.

Glioblastoma (GB), IDH wildtype, is an aggressive and malignant tumor that necessitate early surgical intervention upon radiological suspicion. Consequently, the natural growth dynamics of GB remain not fully understood. Previous studies have examined GB growth using multiple MRIs between initial radiological diagnosis of GB and preoperative MRI, but these studies have short-term intervals between MRIs (defined as shortGB-cohort). Therefore, the shortGB-cohort requires extrapolations to determine radiological initiation which harbors greater uncertainty. In this study, we focused on early-stage GB (eGB), characterized by small, nonspecific lesions on MRI that eventually progress to classic GB findings. We compared three cohorts: 49 cases of eGB. 68 cases of shortGB, and 23 cases of eGB from previous publications (other-eGB). Molecular analyses revealed that TERT promoter mutations, EGFR amplification/gain, CDKN2A hemi/homozygous deletion, and PTEN hemi/homozygous deletion were consistent between eGB and shortGB. Our analysis indicated that the radiological initiation of GB in eGB was -0.84 years (95% confidence interval (CI): -1.09 to -0.68), compared to -0.35 years (95%CI: -0.46 to -0.28) in shortGB. In eGB, factors such as age<65, MIB1>30%, TERTp mutation, and the presence of copy-number alterations (CNA) in EGFR/PTEN/CDKN2A, with or without TERTp mutation, correlated with faster tumor progression. In cases without these alterations, the estimated radiological initiation was -2.31 years (95%CI: -100 to -0.80), indicating slower progression. The ShortGB-cohort did not show any significant changes based on these factors, suggesting it does not actually reflect tumor origin. The other-eGB cohort presented radiological initiation between -0.92 and -0.15 year, consistent with our eGB-cohort. This study investigates the radiological origin of GB using extremely rare cases of eGB. This cohort minimizes the extrapolations, providing insights into the radiological pathophysiology and its association with molecular alterations in IDH-wildtype GB.

Abstract Glioblastoma (GB), IDH wildtype, is an aggressive and malignant tumor that necessitate early surgical intervention upon radiological suspicion. Consequently, the natural growth dynamics of GB remain not fully understood. Previous studies have examined GB growth using multiple MRIs between initial radiological diagnosis of GB and preoperative MRI, but these studies have short-term intervals between MRIs (defined as shortGB-cohort). Unless GB growth is a constant phenomenon, shortGB cannot estimate radiological initiation. In this study, we focused on early-stage GB (eGB), characterized by small, nonspecific lesions on MRI that eventually progress to classic GB findings. We compared three cohorts: 49 cases of eGB. 68 cases of shortGBt, and 23 cases of eGB from previous publications (other-eGB). Molecular analyses revealed that TERT promoter mutations, EGFR amplification/gain, CDKN2A hemi/homozygous deletion, and PTEN hemi/homozygous deletion were consistent between eGB and shortGB. Our analysis indicated that the radiological initiation of GB in eGB was -0.84 years (95% confidence interval (CI): -1.09 to -0.68), compared to -0.35 years (95%CI: -0.46 to -0.28) in shortGB. In eGB, factors such as age &amp;lt; 65, MIB1 &amp;gt; 30%, TERTp mutation, and the presence of copy-number alterations (CNA) in EGFR/PTEN/CDKN2A, with or without TERTp mutation, correlated with faster tumor progression. In cases without these alterations, the estimated radiological initiation was -2.31 years (95%CI: -100 to -0.80), indicating slower progression. The ShortGB-cohort did not show any significant changes based on these factors, suggesting it does not actually reflect tumor origin. The other-eGB cohort presented radiological initiation between -0.92 and -0.15 year, consistent with our eGB-cohort. This study investigates the radiological origin of GB using extremely rare cases of eGB, providing insights into the radiological pathophysiology and its association with molecular alterations in IDH-wildtype GB.

Abstract A new mouse model for the classical subtype of human glioblastoma (GBM) has been bred using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion, the mouse homolog of human p14ARF/CDKN2A, in GFAP expressing cells. Transgenic mice develop intraparenchymal and/or leptomeningeal brain lesions with some spinal cord invasion as early as 1 month old and 95% of mice die by 6 months due to hydrocephalus and/or paralysis. Mice with high grade tumors have worse survival and similar features to human classical GBM such as necrosis, high levels of mitosis and infiltration of tumor cells into normal brain. Immunohistochemical analysis confirms EGFRvIII overexpression and p19-ARF loss in Cre-expressing cells, along with patchy positive GFAP and high proliferation by Ki67. Bulk RNA sequencing reveals GEC3 tumors cluster closely with human classical GBM and have significant upregulation of downstream markers in the JAK/STAT and P13K/AKT pathways. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and generate xenograft tumors by 3 weeks after intracranial injections into NODSCID mice. Xenograft tumors are reminiscent of the primary tumor, with similar histopathological features and immunohistochemical staining. Our spontaneous glioma model is a powerful tool for future studies focused on the role of the immune microenvironment on glioma recurrence and resistance.

Abstract BACKGROUND Adult gliomas exhibit a high level of heterogeneity presenting a substantial challenge hampering development of targeted therapies. Bulk RNAseq studies have identified a mesenchymal gene expression signature across the glioma continuum, based on which a mesenchymal subtype of glioblastoma (MES-GBM) has been described. However, the mesenchymal phenotype of gliomas remains inconsistently defined and the origin of the mesenchymal signature has been debated). Single cell transcriptomic profiling has allowed resolution of gene expression profiles of tumor and non-tumor cells, leading to recent identification of cancer-associated fibroblasts in gliomas and improved understanding of the mesenchymal gene expression signature. METHODS We performed longitudinal scRNAseq analysis of organotypic human glioma slices from 8 patients cultured ex vivo for up to 3 weeks. RESULTS We found 2 of the 6 IDHwt glioblastomas exhibit the mesenchymal phenotype based on Consensus Methylation Profiling (NIH) and scRNAseq analysis, annotated as Mes-1 and Mes-2. The gene expression profiles of mesenchymal glioblastomas show limited concordance with that of either the proneural or classical glioblastoma signatures or of cell types in physiological human brain, with Mes-2 deviating more extensively from non-mesenchymal gliomas. Instead, single cell transcriptomics suggests a hyper-secretory phenotype and overlaps broadly with that of cancer-associated fibroblasts (CAFs) and both express FGF2, other growth factors, and cytokines. Notably, Mes-2 cells express minimal to no levels of genes associated with brain cell lineages, such as PTPRZ1 and SCG3, which are abundantly expressed in other glioma subtypes. CONCLUSIONS Taken together, our data reveals extended depth of glioma heterogeneity that distinguishes MES-GBM from other glioma subtypes and suggests a phenotypic and lineage linkage between MES-GBM and glioma-associated fibroblasts. Thus, new diagnostic and treatment strategies leveraging auto- and paracrine mechanisms underpinning tumor and non-tumor interactions including disruption of the FGF2-FGFR1 circuitry may apply specifically to MES-GBM.

Abstract Therapy resistance in glioblastoma is in part attributed to intratumoral heterogeneity and treatment-resistant cell populations. Understanding heterogeneity requires more complete tumor sampling at diagnosis and at recurrence. We used a whole-tumor sampling approach to obtain 43 spatially mapped biopsies from 3 glioblastomas at diagnosis and recurrence, Pyclone and ClonEvol imputed clonal evolution from exome data, and weighted gene co-expression network analysis inferred gene expression programs from RNA sequencing. Across the cohort, tumor-wide clonal alterations representing evolutionarily early expansions included canonical changes (i.e. Chr7 gain, EGFR amplification) and a diverse set of copy number variations (Chr19/20 gain), driver mutations (i.e. PTEN, KDR), and fusions (LIMCH1::UCHL1, KANK::DOCK8). In 2/3 patients, the founding clone was the only subclone common to primary and recurrence samples and 37% of cancer drivers across the cohort appeared after evolutionary divergence of the primary and recurrent tumors. We identified thirty-two transcriptional programs, six of which were differentially expressed between timepoints. Programs enriched for mitochondrial activity, endothelium/pericytes and classical glioblastoma subtype signature were more highly expressed in primary samples. Programs enriched for mesenchymal-like state, oligodendrocytes and inhibitory interneurons were more highly expressed in recurrence samples. At diagnosis, 11/16 samples showed higher expression of the classical subtype program compared to mesenchymal-like, but at recurrence the mesenchymal program dominated in 18/18 samples. Expression of the programs correlated more strongly with position relative to the contrast-enhancing rather than T2 tumor centroid (p&amp;lt;0.001). These findings reveal novel diversity and complexity in the genetic roots of individual glioblastoma. Recurrences arose from subclones diverging early in evolution of the primary tumor and contained clonal drivers not detected in the primary. Expression data revealed transition from an intratumorally heterogeneous mixture of classical and mesenchymal signatures to tumor-wide mesenchymal at recurrence and suggests that the contrast enhancing centroid may serve as the biological center of the tumor.

Abstract BACKGROUND Isocitrate dehydrogenase (IDH)1/2 wildtype astrocytomas, classified as CNS WHO grade 4 tumors in the latest WHO classification of 2021, have high recurrence rates. The role of surgery in these tumors remains uncertain.. In this study the impact of the extent of resection (EOR) on progression free (PFS) and overall survival (OS) in astrocytomas IDH1/2 wt (CNS WHO Grad 4) is compared to glioblastoma IDH1/2 wt (CNS WHO Grad 4). METHODS Data acquisition was conducted as a multi-center retrospective analysis at 6 University Hospitals (2016-2020). Patients with IDH 1/2 wildtype diffuse astrocytoma (CNS WHO grade 2-4) were included in our study. Patients presenting with IDH wildtype glioblastoma from one institution served as controls. Primary outcome parameters were EOR (GTR; &amp;lt;5% residual tumor (RT), subtotal resection (STR; 5-75% RT), and biopsy (&amp;gt; 75% RT) and OS. RESULTS 160 patients with IDH-wildtype glioblastoma (CNS WHO grade 4) (45.6 % females) and 138 patients with IDH-wildtype diffuse astrocytoma (CNS WHO grade 2-4), (37.7 % females) were enrolled. The median age was 68 years (37-86 years) in the classic glioblastoma cohort and 55 years (20-87 years) in those with astrocytomas. GTR was achieved more often in patients with classic glioblastoma: 58.8% compared to those with diffuse astrocytoma 39.1% (p = 0.0001); STR (32.6%, p = 0.0048) and biopsy (28.3%, p = 0.0032) were more often in astrocytomas. The overall survival was 12.7±10.4 months in classic glioblastoma compared to 27.3±2.9 months in astrocytoma. GTR significantly increased OS in both entities (glioblastoma: 15.3±10.4 months, 95% CI 13.13 - 17.40, p = &amp;lt;0.0001; diffuse astrocytoma: 26.7±17.9 months, 95% CI 20.13 - 33.25, p = &amp;lt;0.0001). Further, also STR significantly increased OS in glioblastoma (11.46±11.03 months, 95% CI 7.55 - 15.37, p = 0.0257) and astrocytoma (18.8±13.54 months, 95% CI 14.47- 23.13, p = 0.0035). ROC analysis identified a residual cut-off tumor volume of &amp;gt;20% in the glioblastoma cohort and &amp;gt;60% in the difuse astrocytoma cohort to be associated with impaired OS. Multivariate logistic regression analysis identified age and EOR, not radio-chemotherapy, as independent predictors of OS in diffuse astrocytoma. MGMT methylation had no impact on OS (p = 0.11). CONCLUSION The presented data suggests that resection prolongs OS in patients with IDH-wildtype astrocytomas and is of higher value than radio-chemotherapy. Of note, STR also positively influences OS in these patients. These results underline the importance of surgical resection even if GTR is not achievable and thus mark a potential paradigm shift in clinical decision making.

Abstract BACKGROUND Glioblastoma (GBM) 5-year survival (LTS) and a 10-year survival (VLTS) rate are extremely low, reaching 7% and 4.7% respectively (2020 CBTRUS Statistical Report). The biological mechanisms that drive the prolonged clinical outcomes remain significantly understudied. MATERIAL AND METHODS As part of a recent study on LTS (PMID: 38423245), here we detail two out of the 17 VLTS cases, addressed with extensive molecular analyses. RESULTS Patient AR 10-046 was a 31-year-old female with a left fronto-parietal giant cell GBM (gcGBM), treated with surgery, radiotherapy and chemotherapy. After 6 months from the diagnosis, she recurred and underwent tumor exeresis and adjuvant TMZ, maintaining disease stability for a total of 243 months. According to the v12.5 BTC the tumor was assigned to the methylation subclass pedHGG_RTK1a subtype (score &amp;gt;0.99), frequently associated with Lynch syndrome (LS). Indeed, we detected MSH6 mutation in tumor DNA as well as in peripheral lymphocyte. We detected high Tumor Mutational Burden. Interestingly, the Copy Number Variation (CNV) plots showed chromosome 1q gain and chromosome 13 loss with no other typical GBM alterations.Patient AR 10-037 was a 70-year-old male with left temporal lobe GBM treated with surgery, adjuvant radiotherapy and chemotherapy, achieving a survival of 73 months. Histology was classical GBM. The tumor was assigned to the pedHGG_MYCN subclass with a suboptimal score (0.5). At CNV examination, apart from the canonical chromosome 7 gain and chromosome 10q loss, we observed MDM2 gene amplification and possible rearrangements on chromosome 12 and 18 with the typical aspect of a chromothripsis (CT), confirmed by RNASeq analysis. Our case presented CT on chr12 harboring two putative gene fusions: CPSF6::CPM and PTPRR::RAB3IP never reported in the literature. CONCLUSION We described two cases of VLTS patients with peculiar molecular profile, widening the scenario of clinical and molecular variability that can be found in such patients. Further studies are still needed to identify the biological mechanisms causing such prognosis, with a fundamental contribution from molecular analyses, including DNAm profiling, NGS and RNAseq. FUNDING RRC Italian Ministry of Health

Abstract BACKGROUND Glioblastoma (GBM) research urgently requires a new murine model for preclinical research. The highly used cell line, GL261, reliably forms glioma-like tumours when transplanted intracranially, yet these tumours do not recapitulate human GBM, particularly when applied as a model for immune therapy. The tumour immune microenvironment (TIME) of a GL261 tumour consistently responds to immune checkpoint inhibitors, such as αPD-1 and αCTLA-4, which have shown no significance in multiple clinical trials. MATERIAL AND METHODS We have developed a novel murine model of GBM which has been designed to address the shortcomings of GL261. Into an immortalised eGFP astrocyte C57BL/6 cell line mutations were introduced to create genetic profile similar to human GBM cells. One version of these astrocytes models a classical GBM cell, with a Pten knockdown and RAS V12 stable transfection. Pten/RAS V12 and GL261 cells were separately transplanted intracranially into C57BL/6 mice and developed for either two weeks (spectral flowcytometry) or until ethical endpoint (survival and histological analysis). RESULTS Intracranial tumours reliably formed in both GL261 and Pten/RAS V12 models, with median survivals of 19.50 and 20.00 days, respectively. However, key histological differences were seen in H&amp;E sections. The GL261 tumours were highly vascularised and showed clear borders with the healthy tissue. The Pten/RAS V12 tumours had very densely packed cell mass with more invasion into the surrounding tissue. Spectral flow cytometry indicated that macrophages and monocytes contributed predominately to the tumour immune infiltration of both models. Despite similar abundance of these cells, key inflammatory markers showed significant differences. The GL261 tumours myeloid compartment was largely comprised of classical monocytes (cMo). While the populations were heterogeneous, these cMo cells were characterised by high expression of Ly6C and MHCII, both markers associated with a proinflammatory, anti-tumour responses. Conversely, Pten/RAS V12 tumours showed significantly less of Ly6C+ MHCII+ cells, indicative of an immune suppressive environment comprised of anti-inflammatory TAMs and non-classical monocytes (ncMo). Additionally, GL261 tumours contained higher numbers of dendritic cells suggesting increased antigen presentation and greater potential for anti-tumour immune responses. Higher PDL1 expression was seen on infiltrating leukocytes in GL261 tumours, consistent with literature on GL261 demonstrating response to PD1 inhibitors. However, Pten/RAS V12 tumours showed minimal expression of PDL1, consistent with poor responses in GBM patients to immune checkpoint inhibitors. CONCLUSION Together, these data indicate that this novel model will be a more appropriate tool for GBM research to help understand how to better implement immune therapy.

Comprehensive molecular characterization of long-term glioblastoma survivors

Glioblastoma (GBM), a highly lethal and aggressive central nervous system malignancy, presents a critical need for targeted therapeutic approaches to improve patient outcomes in conjunction with standard-of-care (SOC) treatment. Molecular subtyping based on genetic profiles and metabolic characteristics has advanced our understanding of GBM to better predict its evolution, mechanisms, and treatment regimens. Pharmacological ascorbate (P-AscH−) has emerged as a promising supplementary cancer therapy, leveraging its pro-oxidant properties to selectively kill malignant cells when combined with SOC. Given the clinical challenges posed by the heterogeneity and resistance of various GBM subtypes to conventional SOC, our study assessed the response of classical, mesenchymal, and proneural GBM to P-AscH−. P-AscH− (20 pmol/cell) combined with SOC (5 µM temozolomide and 4 Gy of radiation) enhanced clonogenic cell killing in classical and mesenchymal GBM subtypes, with limited effects in the proneural subtype. Similarly, following exposure to P-AscH− (20 pmol/cell), single-strand DNA damage significantly increased in classical and mesenchymal but not proneural GBM. Moreover, proneural GBM exhibited increased hydrogen peroxide removal rates, along with increased catalase and glutathione peroxidase activities compared to mesenchymal and classical GBM, demonstrating an altered H2O2 metabolism that potentially drives differential P-AscH− toxicity. Taken together, these data suggest that P-AscH− may hold promise as an approach to improve SOC responsiveness in mesenchymal GBMs that are known for their resistance to SOC.

Abstract INTRODUCTION Prior to 2021, (classic) glioblastoma (GBM) required absence of IDH mutation and histologic features of hypercellularity, nuclear atypia, microvascular proliferation (MVP), and necrosis. Absence of histologic features can be a result of undersampling, with molecular, radiologic, and clinical features consistent with GBM. Accordingly, WHO 2021 defined molecular glioblastoma as diffuse glioma IDHwt, H3wt, with one or more of MVP, necrosis, TERT promoter mutation, EGFR gene amplification (EGFRamp), and/or +7/−10. We hypothesized that there is clinical and radiographic heterogeneity among tumors defined as molecular GBM. METHODS We identified 121 patients that met the criteria for molecular GBM. 121 Classic GBM, defined as IDHwt with hypercellularity, MVP or necrosis, were matched to molecular GBM cases by age, sex, and diagnosis date. All cases were reviewed for relevant histologic features and molecular features. Examined clinical variables included: first symptom, time from first symptom to date of pathological diagnosis, date of first MRI, tumor location, radiographic features of enhancement, diffusion or mass effect, time to progression and overall survival. Clinical, radiologic, and molecular features were compared between classic and molecular GBM. RESULTS Currently,160 cases have been reviewed. Preliminary results show that there is a longer time from first symptom to diagnosis for molecular GBM. Molecular GBM is significantly more likely to be discovered coincidentally, and without enhancement or radiographic necrosis. Classic GBM is significantly more likely to present with focal neurologic deficit as first symptom, regional mass effect on index imaging and have monosomy 10. CONCLUSION While pathologic undersampling accounts for a significant proportion of molecular GBM, our data suggest that there are significant clinical, radiographic, and outcome differences between classical and molecular GBM. Final analysis of the entire cohort will help better define this clinical spectrum.

Abstract A new mouse model for the classical subtype of human glioblastoma has been generated using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion (the mouse homolog of human p14-ARF/CDKN2A) in GFAP expressing cells. Transgenic mice develop intraparchenymal and/or leptomeningeal brain lesions with some spinal cord invasion as early as 1 month old and 95% of mice die by 6 months due to hydrocephalus and/or paralysis. Mice with high grade tumors have worse survival and similar features to human classical glioblastoma such as necrosis, high levels of mitosis, and infiltration of tumor cells into normal brain. Immunohistochemical analysis confirms EGFRvIII overexpression and p19-ARF loss in tumor cells, along with patchy positive GFAP and positivity for Olig2, S100β and NeuN, suggesting that tumor cells arise from a progenitor glial cell type. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and are able to generate xenograft tumors by 3 weeks after intracranial injections into NODSCID mice. Xenograft tumors are reminiscent of the primary tumor, with similar histopathological features and immunohistochemical staining. This novel mouse model can be used to study diffuse glioma with a leptomeningeal component.