Classical Glioblastoma Research Articles

Comprehensive molecular characterization of long-term glioblastoma survivors

Fewer than 5 % glioblastoma (GBM) patients survive over five years and are termed long-term survivors (LTS), yet their molecular background is unclear. The present cohort included 72 isocitrate dehydrogenase (IDH)-wildtype GBM patients, consisting of 35 LTS and 37 short-term survivors (STS), and we employed whole exome sequencing, RNA-seq and DNA methylation array to delineate this largest LTS cohort to date. Although LTS and STS demonstrated analogous clinical characters and classical GBM biomarkers, CASC5 (P = 0.002) and SPEN (P = 0.013) mutations were enriched in LTS, whereas gene-to-gene fusions were concentrated in STS (P = 0.007). Importantly, LTS exhibited higher tumor mutation burden (P < 0.001) and copy number (CN) increase (P = 0.013), but lower mutant-allele tumor heterogeneity score (P < 0.001) and CN decrease (P = 0.026). Additionally, LTS demonstrated hypermethylated genome (P < 0.001) relative to STS. Differentially expressed and methylated genes both enriched in olfactory transduction. Further, analysis of the tumor microenvironment revealed higher infiltration of M1 macrophages (P = 0.043), B cells (P = 0.016), class-switched memory B cells (P = 0.002), central memory CD4+ T cells (P = 0.031) and CD4+ Th1 cells (P = 0.005) in LTS. We also separately analyzed a subset of patients who were methylation class-defined GBM, contributing 70.8 % of the entire cohort, and obtained similar results relative to prior analyses. Finally, we demonstrated that LTS and STS could be distinguished using a subset of molecular features. Taken together, the present study delineated unique molecular attributes of LTS GBM.

Differential H2O2 Metabolism among Glioblastoma Subtypes Confers Variable Responses to Pharmacological Ascorbate Therapy Combined with Chemoradiation.

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.

PATH-32. COMPARISON OF CLINICAL, RADIOGRAPHIC AND TUMOR GENOMIC ALTERATIONS BETWEEN CLASSIC AND MOLECULAR GLIOBLASTOMA (IDH WILDTYPE)

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.

MODL-32. EGFRVIII OVEREXPRESSION AND LOSS OF MOUSE SPECIFIC CDKN2A IN GLIAL CELLS LEADS TO GLIOMAGENESIS IN A NOVEL MOUSE MODEL

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.

Identification of Excellent Prognosis IDH Wildtype Glioblastomas Using Genomic and Metabolic Profiling

High grade gliomas (HGGs) are aggressive brain tumors with altered cellular metabolism. HGGs can carry mutations in the tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase 1 (IDH1), conferring distinct biology and improved patient prognosis compared to IDH wildtype (wt) tumors. Using metabolomic analyses of tumor tissue, we previously showed that IDH wt and IDH mutant (IDH mut) tumors have unique metabolomic signatures that correlate with different survival outcomes. Among this cohort of 69 HGG samples, we identified two unique patient tumors that metabolically clustered with IDH mut tumors, but lacked both the IDH mutation and its product 2-hydroxyglutarate. We aimed to discover unique mutations in these two tumors that may impart an IDH mutant-like phenotype in the absence of an IDH1 or IDH2 mutation. Whole exome sequencing (WES) was performed on frozen tumor samples from two patients diagnosed as glioblastoma (GBM), IDH wt via Agilent v5 + IncRNA platform. Alignment to the hg38 genome and variant calling were completed using an accelerated implementation of GATK's BWA and MuTect2 algorithms from Sentieon. Variants were filtered based on supporting reads and variant allele thresholds, with synonymous variants and common SNPs removed. High-confidence variants were further filtered by membership in the four KEGG pathways associated with IDH1 and IDH2. Identified variants were corroborated with metabolomics data from the two unique IDH wt tumors compared with classical GBM IDH wt, oligodendrogliomas IDH mut and astrocytomas IDH mut to identify putative drivers of an IDH mutant-like metabolomic phenotype in these unique IDH wt tumors. Despite the lack of an IDH mutation, one patient survived 45.6 months and the other patient remains alive at last follow up 64 months post diagnosis, much longer than the 16-18-month median survival typical of patients with GBM IDH wt. WES of outlier IDH wt tumor samples revealed 65 unique mutations in the queried KEGG pathways, of which 34 had a variant allele frequency > = 0.15. These variants were processed in Gprofiler, confirming expected enrichment of the carboxylic acid metabolic biologic process, a functional gene set consisting of TCA genes, among these variants (p = 0.002, 3.6-fold enrichment). Accordingly, metabolite levels of intermediates of the TCA cycle, including malate and isocitrate were decreased in the outlier tumor samples compared to classic GBMs IDH wt (p<0.001). Presence of genetic alterations in key variants of the carboxylic acid metabolic biologic process (including ME1, GYP4F3, PTGIS, PFKL, PSPH, AKR1A1, HK2, NOS1) correlated with improved overall survival among GBM patients in the TCGA (p = 0.04). Laboratory validation of these findings in preclinical GBM models is ongoing. Disruption of the TCA cycle independent of an IDH mutation is associated with favorable survival in GBM. Pharmacologic inhibition of these pathways may be a promising strategy to improve GBM outcomes.

SYST-27 INTERROGATION OF THE TUMOR IMMUNE MICROENVIRONMENT REVEALS DISTINCT IMMUNOSUPPRESSIVE LANDSCAPES IN GLIOBLASTOMA

Abstract High-resolution sequencing technologies have revealed significant inter-and intra-tumoral molecular heterogeneity in glioblastoma (GBM), leading to an urgent need for novel targeted therapies. Our research using representative murine GBM models and various experimental techniques has shown that classical GBMs (e.g., EGFRvIII+, TIMElow) and mesenchymal GBMs (e.g., NF1ko/PTENko/p53ko, TIMEhigh) are both immunosuppressive but in distinct ways. TIMElow GBMs have a more impaired and less functional tumor vasculature, resulting in low infiltration of T cells with anergic features. TIMEhigh GBMs have a more intact BBB revealing overall more adaptive and innate immune cell infiltrates, but with highly dysfunctional T cells and immunosuppressive TME. Congruently, GSEA analysis of both human and murine GBM scRNA-seq data demonstrated enriched immunosuppressive signatures in the myeloid cell populations of TIMEhigh GBM compared to TIMElow GBM. Our findings have also revealed potential adaptive and intrinsic resistant mechanisms to antiangiogenic immunotherapy in TIMElow and TIMEhigh GBMs, respectively, with TIMElow GBMs exhibiting a transitory response to the treatment, while TIMEhigh GBMs being intrinsically resistant due to their intact tumor vasculature and reliance on dysfunctional T cells. We then tested the efficacy of different immunotherapies, including dendritic cell vaccination, a CD40 agonist, and a myeloid cell PI3K activity inhibitor in TIMElow and TIMEhigh tumors. Surprisingly, our results revealed that the response to these treatments can be either beneficial or detrimental and may depend on factors such as tumor antigenicity and TIME subtype. Overall, our study provides new insights into the intricate immune landscape of GBMs, emphasizes the importance of considering tumor subtypes as they may lead to potential unintended consequences of immunotherapies.

Glioblastoma and cerebral organoids: development and analysis of an in vitro model for glioblastoma migration.

It is currently challenging to adequately model the growth and migration of glioblastoma using two-dimensional (2D) in vitro culture systems as they quickly lose the original, patient-specific identity and heterogeneity. However, with the advent of three-dimensional (3D) cell cultures and human-induced pluripotent stem cell (iPSC)-derived cerebral organoids (COs), studies demonstrate that the glioblastoma-CO (GLICO) coculture model helps to preserve the phenotype of the patient-specific tissue. Here, we aimed to set up such a model using mature COs and develop a pipeline for subsequent analysis of cocultured glioblastoma. Our data demonstrate that the growth and migration of the glioblastoma cell line within the mature COs are significantly increased in the presence of extracellular matrix proteins, shortening the time needed for glioblastoma to initiate migration. We also describe in detail the method for the visualization and quantification of these migrating cells within the GLICO model. Lastly, we show that this coculture model (and the human brain-like microenvironment) can significantly transform the gene expression profile of the established U87 glioblastoma cell line into proneural and classical glioblastoma cell types.

MODL-16. SOMATICALLY ENGINEERED MOUSE MODELS RECAPITULATING CELLULAR AND MOLECULAR FEATURES OF HUMAN GBM

Abstract INTRODUCTION Mouse models are instrumental in advancing our understanding of glioblastoma (GBM), however many commonly used models have features that limit their practicality or applicability. These limitations can include an incomplete immune context, a requirement for complex breeding strategies, high cost of specialized lines, and unpredictability in where tumors arise. We sought to develop and validate a mouse modeling system for some of the major human GBM subtypes that overcame these practical challenges. METHODS In vivo electroporation was used to introduce genetic alterations into periventricular cells of early postnatal C57Bl6/j mice. PiggyBac transposon/transposase and CRISPR-Cas9 systems were used to overexpress (OE) or knock out (KO) genes associated with mesenchymal (Nf1-KO/Pten-KO/p53-KO), classical (EGFRvIII-OE/Cdkn2a-KO/Pten-KO), and proneural (Pdgfra-OE/Cdk4-OE/p53-KO) IDH-WT GBM subtypes. KO or OE was confirmed by Indel Detection by Amplicon Analysis and immunofluorescent staining (IF). Tumours were analyzed for histologic, immunohistochemical, and transcriptional (RNAseq) features. RESULTS Tumors arose with near complete penetrance and median survivals of 36 to 116 days amongst models. All tumors were GFAP-expressing high-grade gliomas, with distinct phenotypes associated with different genetic combinations. Nf1-KO/Pten-KO/p53-KO tumors were composed of spindle cells. EGFRvIII-OE/Cdkn2a-KO/Pten-KO and Pdgfra-OE/Cdk4-OE/p53-KO tumors showed prevalence of small, round cells. Molecularly, Nf1-KO/Pten-KO/p53-KO tumors were enriched for human mesenchymal signature and displayed more differentiated phenotype. The Nf1-KO/Pten-KO/p53-KO model also showed increased stromal cells and macrophages. The other models had mixed proneural and classical profiles and were enriched for OPC- and NPC-like gene signatures found in human GBM. Once tumors formed, cells from each model were transplantable into C57Bl6/j mice to generate subsequent tumors. CONCLUSION We developed and validated a rapid, versatile, and reproducible system to model GBMs. These models allow for controlled study of GBM pathogenesis, progression, and treatment response, and allow for robust generation of syngeneic implantable mouse models that can serve as valuable tools for preclinical testing.

A Quantitative Digital Analysis of Tissue Immune Components Reveals an Immunosuppressive and Anergic Immune Response with Relevant Prognostic Significance in Glioblastoma.

Objectives: Immunostimulatory therapies using immune checkpoint blockers show clinical activity in a subset of glioblastoma (GBM) patients. Several inhibitory mechanisms play a relevant role in the immune response to GBM. With the objective of analyzing the tumor immune microenvironment and its clinical significance, we quantified several relevant immune biomarkers. Design: We studied 76 primary (non-recurrent) GBMs with sufficient clinical follow-up, including a subgroup of patients treated with a dendritic cell vaccine. The IDH-mutation, EGFR-amplification, and MGMT methylation statuses were determined. Several relevant immune biomarkers, including CD163, CD8, PD1, and PDL1, were quantified in representative selected areas by digital image analysis and semiquantitative evaluation. The percentage of each immune expression was calculated with respect to the total number of tumor cells. Results: All GBMs were wild-type IDH, with a subgroup of classical GBMs according to the EGFR amplification (44%). Morphologically, CD163 immunostained microglia and intratumor clusters of macrophages were observed. A significant direct correlation was found between the expression of CD8 and the mechanisms of lymphocyte immunosuppression, in such a way that higher values of CD8 were directly associated with higher values of CD163 (p < 0.001), PDL1 (0.026), and PD1 (0.007). In a multivariate analysis, high expressions of CD8+ (HR = 2.05, 95%CI (1.02–4.13), p = 0.034) and CD163+ cells (HR 2.50, 95%CI (1.29–4.85), p = 0.007), were associated with shorter survival durations. The expression of immune biomarkers was higher in the non-classical (non-EGFR amplified tumors) GBMs. Other relevant prognostic factors were age, receipt of the dendritic cell vaccine, and MGMT methylation status. Conclusions: In accordance with the inverse correlation between CD8 and survival and the direct correlation between effector cells and CD163 macrophages and immune-checkpoint expression, we postulate that CD8 infiltration could be placed in a state of anergy or lymphocytic inefficient activity. Furthermore, the significant inverse correlation between CD163 tissue concentration and survival explains the relevance of this type of immune cell when creating a strong immunosuppressive environment. This information may potentially be used to support the selection of patients for immunotherapy.

Extracellular vesicles derived from glioblastoma promote proliferation and migration of neural progenitor cells via PI3K-Akt pathway

BackgroundGlioblastomas are lethal brain tumors under the current combinatorial therapeutic strategy that includes surgery, chemo- and radio-therapies. Extensive changes in the tumor microenvironment is a key reason for resistance to chemo- or radio-therapy and frequent tumor recurrences. Understanding the tumor-nontumor cell interaction in TME is critical for developing new therapy. Glioblastomas are known to recruit normal cells in their environs to sustain growth and encroachment into other regions. Neural progenitor cells (NPCs) have been noted to migrate towards the site of glioblastomas, however, the detailed mechanisms underlying glioblastoma-mediated NPCs’ alteration remain unkown.MethodsWe collected EVs in the culture medium of three classic glioblastoma cell lines, U87 and A172 (male cell lines), and LN229 (female cell line). U87, A172, and LN229 were co-cultured with their corresponding EVs, respectively. Mouse NPCs (mNPCs) were co-cultured with glioblastoma-derived EVs. The proliferation and migration of tumor cells and mNPCs after EVs treatment were examined. Proteomic analysis and western blotting were utilized to identify the underlying mechanisms of glioblastoma-derived EVs-induced alterations in mNPCs.ResultsWe first show that glioblastoma cell lines U87-, A172-, and LN229-derived EVs were essential for glioblastoma cell prolifeartion and migration. We then demonstrated that glioblastoma-derived EVs dramatically promoted NPC proliferation and migration. Mechanistic studies identify that glioblastoma-derived EVs achieve their functions via activating PI3K-Akt-mTOR pathway in mNPCs. Inhibiting PI3K-Akt pathway reversed the elevated prolfieration and migration of glioblastoma-derived EVs-treated mNPCs.ConclusionOur findings demonstrate that EVs play a key role in intercellular communication in tumor microenvironment. Inhibition of the tumorgenic EVs-mediated PI3K-Akt-mTOR pathway activation might be a novel strategy to shed light on glioblastoma therapy.9isrUz1Ju_9yzYdEacJBzAVideo

SINGLE-CENTER DESCRIPTIVE STATISTICAL ANALYSIS OF GLIAL TUMORS WITH REFERENCE TO GLIOBLASTOMAS

Gliomas are the most common primary intracranial brain neoplasms composing almost 80% of all cancerous brain tumours. Glioblastoma is the most common malignant brain tumour in the adult population and accounts in about 15% of all intracranial neoplasms. This observational analytical study gives a better overview of the current state of glial tumours in North Macedonia from 2021 to 2022 with special reference to glioblastomas. Epidemiological and statistical analysis of glial and other brain tumours in a specific time period with special reference to glioblastomas. This research is an observational statistical analysis that takes place on the Institute for Pathology in the Medical Faculty of ‘’Ss. Cyril and Methodius’’ University in Skopje using the digital data base of patients that had undergone surgery in the University Clinic of Neurosurgery and were diagnosed at the Institute of pathology during the time period 2021-2022. In the period from 2021 and 2022, at the Institute of pathology were diagnosed 106 (62%) glioblastomas out of which classic glioblastoma histomorphology confined 85 (80.19%) cases; 9 (8.49%) glioblastomas showed oligodendroglial component, 5 (4.72%) cases were gliosarcomas and 4 (3.77%) cases were diagnosed as giant cell glioblastomas. Three (2.83%) cases showed glioblastoma histomorphology with radionecrosis. When it comes to global statistics, we can conclude that Republic of North Macedonia follows the global trends regarding the incidence of glioblastoma associated with gender. Our results show that glioblastoma is more prevalent in males than females with the ratio male to female being 1.2:1, and this tumour is more prevalent in patients older than 55 years with the average age of diagnosis being 61 years. The results of this descriptive statistical analysis show that glial tumours, especially glioblastomas as most prevalent malignant tumour type deserves greater scientific attention which beside the impact on their diagnostic complexity, will expand the knowledge of this pathology field and strongly reflect on future scientific and diagnostic work. Keywords: Glioblastoma, glial tumors, statistics, analyzis, epidemiology. &nbsp; &nbsp

SMARCAL1 loss and alternative lengthening of telomeres (ALT) are enriched in giant cell glioblastoma

Subsets of high-grade gliomas, including glioblastoma (GBM), are known to utilize the alternative lengthening of telomeres (ALT) pathway for telomere length maintenance. However, the telomere maintenance profile of one subtype of GBM—giant cell GBM—has not been extensively studied. Here, we investigated the prevalence of ALT, as well as ATRX and SMARCAL1 protein loss, in a cohort of classic giant cell GBM and GBM with giant cell features. To determine the presence of ALT, a telomere-specific fluorescence in situ hybridization assay was performed on 15 cases of classic giant cell GBM, 28 additional GBMs found to have giant cell features, and 1 anaplastic astrocytoma with giant cell features. ATRX, SMARCAL1, and IDH1 protein status were assessed in a proportion of cases by immunohistochemistry and were compared to clinical-pathologic and molecular characteristics. In the overall cohort of 44 cases, 19 (43%) showed evidence of ALT. Intriguingly, of the ALT-positive cases, only 9 (47.4%) displayed loss of the ALT suppressor ATRX by immunohistochemistry. Since inactivating mutations in SMARCAL1 have been identified in ATRX wild-type ALT-positive gliomas, we developed an immunohistochemistry assay for SMARCAL1 protein expression using genetically validated controls. Of the 19 ALT-positive cases, 6 (31.5%) showed loss or mis-localization of SMARCAL1 by immunohistochemistry. Of these cases, four retained ATRX protein expression, while two cases also displayed ATRX loss. Additionally, we assessed five cases from which multiple temporal samples were available and ALT status was concordant between both tumor biopsies. In summary, we have identified a subset of giant cell GBM that utilize the ALT telomere maintenance mechanism. Importantly, in addition to ATRX loss, ALT-positive tumors harboring SMARCAL1 alterations are prevalent in giant cell GBM.

Early stage glioblastoma: retrospective multicentric analysis of clinical and radiological features.

The aim of this study was to report our experience with early stage glioblastoma (e-GB) and to investigate the possible clinical and imaging features that may be helpful to the radiologist to correctly diagnose this entity. We performed a retrospective research of patients diagnosed with glioblastoma at two hospitals during a 10-year period. We reviewed all pre-operative MR and included only patients with early stage GB lesions, characterized by hyperintense on T2-weighted signal, with or without contrast-enhancement at post-contrast T1-weighted images, without "classic" imaging appearance of GB (necrosis, haemorrhage, oedema). All preoperative MR were evaluated by an experienced neuroradiologist and information on patients' demographics, clinical presentation, follow-up, and histopathology results study were collected. When available, preoperative CT examination was also evaluated. We found 14 e-GBs in 13 patients (9 males, 4 females, median age 63years) among 660 patients diagnosed with GB between 2010 and 2020. In 10 lesions, serial imaging revealed the transformation of e-GB in classic glioblastoma in a median time of 3months. Clinical presentation included stroke-like symptoms, vertigo, seizures and confusion. Preoperative plain CT was performed in 8/13 cases and in 7 e-GBs presented as a hyperdense lesion. Ten out of 14 lesions transformed in classic GB before surgical intervention or biopsy. All lesions revealed typical immunohistochemical pattern of primary glioblastoma. E-GB is a rare entity that can often lead to misdiagnosis. However, the radiologist should be aware of its imaging appearance to suggest the diagnosis and to request close imaging follow-up, hopefully improving the prognosis of this very aggressive disease.

Downregulation of both mismatch repair and non-homologous end-joining pathways in hypoxic brain tumour cell lines.

Glioblastoma, a grade IV astrocytoma, has a poor survival rate in part due to ineffective treatment options available. These tumours are heterogeneous with areas of low oxygen levels, termed hypoxic regions. Many intra-cellular signalling pathways, including DNA repair, can be altered by hypoxia. Since DNA damage induction and subsequent activation of DNA repair mechanisms is the cornerstone of glioblastoma treatment, alterations to DNA repair mechanisms could have a direct influence on treatment success. Our aim was to elucidate the impact of chronic hypoxia on DNA repair gene expression in a range of glioblastoma cell lines. We adopted a NanoString transcriptomic approach to examine the expression of 180 DNA repair-related genes in four classical glioblastoma cell lines (U87-MG, U251-MG, D566-MG, T98G) exposed to 5 days of normoxia (21% O2), moderate (1% O2) or severe (0.1% O2) hypoxia. We observed altered gene expression in several DNA repair pathways including homologous recombination repair, non-homologous end-joining and mismatch repair, with hypoxia primarily resulting in downregulation of gene expression. The extent of gene expression changes was dependent on hypoxic severity. Some, but not all, of these downregulations were directly under the control of HIF activity. For example, the downregulation of LIG4, a key component of non-homologous end-joining, was reversed upon inhibition of the hypoxia-inducible factor (HIF). In contrast, the downregulation of the mismatch repair gene, PMS2, was not affected by HIF inhibition. This suggests that numerous molecular mechanisms lead to hypoxia-induced reprogramming of the transcriptional landscape of DNA repair. Whilst the global impact of hypoxia on DNA repair gene expression is likely to lead to genomic instability, tumorigenesis and reduced sensitivity to anti-cancer treatment, treatment re-sensitising might require additional approaches to a simple HIF inhibition.

Gliosarcoma in patients under 20\u2009years of age. A clinicopathologic study of 11 cases and detailed review of the literature

BackgroundGliosarcoma is a rare variant of IDH- wild type glioblastoma with both glial and mesenchymal differentiation. It accounts for approximately 2% of glioblastomas and has a poor prognosis similar to that of classic glioblastoma. It is seen mostly between 40 and 60 years of age with a mean age over 50 years. Pediatric gliosarcoma is even rarer than gliosarcoma in adults. We describe the clinicopathological features of gliosarcoma in patients under 20 years of age and determine whether there are significant differences from gliosarcoma in adults. We also present detailed review of published literature on pediatric gliosarcoma.MethodsSlides of gliosarcomas in patients under 20 years of age were reviewed. Clinicopathological features were noted in detail and follow up was obtained.ResultsEleven cases of gliosarcoma were reported in patients under 20 years of age. Ages ranged from three to 19 years (mean age 13 years). Frontal, parietal and temporal lobes were the commonest locations. Mean and median tumor size was six and five cm respectively. All 11 cases demonstrated the classic biphasic pattern. In 10 cases, glial component was astrocytic and was highlighted on GFAP. Sarcomatous component in most cases resembled fibrosarcoma and was high grade in 72.7%. Glial areas were reticulin poor while sarcomatous areas were reticulin rich. In over 45% cases, bizarre tumor giant cells were seen in the sarcomatous areas. In 1 case, sarcomatous areas showed extensive bone and cartilage formation. Other histologic features included hyalinized blood vessels, hemorrhage, infarction, gemistocytic cells, rhabdoid cells etc. Follow up was available in nine patients, five received chemoradiation post resection while three received radiotherapy only. Prognosis was dismal and eight patients died within one to 14 months following resection.ConclusionsGliosarcomas in patients under 20 comprised 13% of all gliosarcomas reported during the study period. Frequency and mean age were higher compared to other published reports. Pathological features were similar to those described in literature. Clinicopathological features and prognosis of pediatric gliosarcomas were similar to adult gliosarcomas.

Local anesthetics impair the growth and self-renewal of glioblastoma stem cells by inhibiting ZDHHC15-mediated GP130 palmitoylation

BackgroundA large number of preclinical studies have shown that local anesthetics have a direct inhibitory effect on tumor biological activities, including cell survival, proliferation, migration, and invasion. There are few studies on the role of local anesthetics in cancer stem cells. This study aimed to determine the possible role of local anesthetics in glioblastoma stem cell (GSC) self-renewal and the underlying molecular mechanisms.MethodsThe effects of local anesthetics in GSCs were investigated through in vitro and in vivo assays (i.e., Cell Counting Kit 8, spheroidal formation assay, double immunofluorescence, western blot, and xenograft model). The acyl-biotin exchange method (ABE) assay was identified proteins that are S-acylated by zinc finger Asp-His-His-Cys-type palmitoyltransferase 15 (ZDHHC15). Western blot, co-immunoprecipitation, and liquid chromatograph mass spectrometer-mass spectrometry assays were used to explore the mechanisms of ZDHHC15 in effects of local anesthetics in GSCs.ResultsIn this study, we identified a novel mechanism through which local anesthetics can damage the malignant phenotype of glioma. We found that local anesthetics prilocaine, lidocaine, procaine, and ropivacaine can impair the survival and self-renewal of GSCs, especially the classic glioblastoma subtype. These findings suggest that local anesthetics may weaken ZDHHC15 transcripts and decrease GP130 palmitoylation levels and membrane localization, thus inhibiting the activation of IL-6/STAT3 signaling.ConclusionsIn conclusion, our work emphasizes that ZDHHC15 is a candidate therapeutic target, and local anesthetics are potential therapeutic options for glioblastoma.

Differentiating Giant Cell Glioblastoma from Classic Glioblastoma With Diffusion-Weighted Imaging

Differential diagnosis of giant cell glioblastoma (GC) and classic glioblastoma (GBM) using conventional radiological modalities is difficult. This study aimed to use diffusion-weighted imaging (DWI) to distinguish GC from GBM and thereby improve the accuracy of preoperative assessment of patients with GB. The clinical, magnetic resonance imaging, and pathologic data of 12 patients with GC and 21 patients with GBM were retrospectively analyzed. Independent sample t tests were used to compare the minimum apparent diffusion coefficient (ADCmin) and the normalized apparent diffusion coefficients (nADC) of the 2 tumor types. Receiver operating curve (ROC) analysis was used to assess the diagnostic efficacy of ADCmin and nADC values. Compared with that of the classic GBM group, the ADCmin (0.98 ± 0.14 vs. 0.80 ± 0.19×10-3 mm2/second, P= 0.007) and nADC (1.42 ± 0.25 vs. 1.17 ± 0.25, P= 0.011) of the GC group were significantly higher. ROC curve analysis showed that the maximum area under the curve of ADCmin and nADC were 0.800 ± 0.080 and 0.778 ± 0.082, respectively. The sensitivity, specificity, and accuracy distinguishing GC and classic GBM was best (83.33%, 76.19%, and 78.79%, respectively) when ADCmin= 0.84×10-3 mm2/second (maximum area under the ROC, 0.800). Its positive and negative predictive values under this condition were 88.89% and 66.67%, respectively. By distinguishing GC from classic GBM, the ADCmin parameter of DWI can improve the accuracy of the preoperative differential diagnosis of the 2 tumor types.

Genetic driver mutations introduced in identical cell-of-origin in murine glioblastoma reveal distinct immune landscapes but similar response to checkpoint blockade.

Glioblastoma (GBM) is the most aggressive primary brain tumor. In addition to being genetically heterogeneous, GBMs are also immunologically heterogeneous. However, whether the differences in immune microenvironment are driven by genetic driver mutation is unexplored. By leveraging the versatile RCAS/tv-a somatic gene transfer system, we establish a mouse model for Classical GBM by introducing EGFRvIII expression in Nestin-positive neural stem/progenitor cells in adult mice. Along with our previously published Nf1-silenced and PDGFB-overexpressing models, we investigate the immune microenvironments of the three models of human GBM subtypes by unbiased multiplex profiling. We demonstrate that both the quantity and composition of the microenvironmental myeloid cells are dictated by the genetic driver mutations, closely mimicking what was observed in human GBM subtypes. These myeloid cells express high levels of the immune checkpoint protein PD-L1; however, PD-L1 targeted therapies alone or in combination with irradiation are unable to increase the survival time of tumor-bearing mice regardless of the driver mutations, reflecting the outcomes of recent human trials. Together, these results highlight the critical utility of immunocompetent mouse models for preclinical studies of GBM, making these models indispensable tools for understanding the resistance mechanisms of immune checkpoint blockade in GBM and immune cell-targeting drug discovery.

A contemporary perspective on the diagnosis and treatment of diffuse gliomas in adults

Gliomas are intrinsic brain tumours, which are classified by the World Health Organization (WHO) into different grades of malignancy, with glioblastoma being the most frequent and most malignant subtype (WHO grade IV). Mutations in the isocitrate dehydrogenase (IDH) 1 or 2 genes are frequent in lower (WHO II/III) grade tumours but typically absent in classical glioblastoma. IDH mutations are associated with a better prognosis compared with IDH wild-type tumours of the same WHO grade. Following detection of a tumour mass by imaging, maximum safe surgery as feasible is commonly performed to reduce mass effect and to obtain tissue allowing histopathological diagnosis and molecular assessment. Radiotherapy has been the mainstay in the treatment of diffuse gliomas for several decades. It provides improved local control, but is not curative. Furthermore, several randomised trials have shown that the addition of alkylating chemotherapy, either temozolomide or nitrosourea-based regimens, to radiotherapy results in prolonged survival. Tumour-treating fields (TTFields) have emerged as an additional treatment option in combination with maintenance temozolomide treatment for patients with newly diagnosed glioblastoma. Treatment at recurrence is less standardised and depends on the patient&rsquo;s performance status, symptom burden and prior treatments. Bevacizumab prolongs progression-free survival in newly diagnosed and recurrent glioblastoma, but does not impact overall survival. However, in Switzerland and some other countries, it is still considered a valuable treatment option to reduce clinical symptom burden. Given the generally poor outcome for these patients, various novel treatment approaches are currently being explored within clinical trials including immunotherapeutic strategies such as immune checkpoint inhibition and the brain-penetrant proteasome inhibitor marizomib.

PHIP drives glioblastoma motility and invasion by regulating the focal adhesion complex

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.