Marker In Glioblastoma Research Articles

Abstract B069: Spatial and single-cell proteomic landscaping of the hypoxic microenvironment in glioblastoma

Abstract Glioblastoma (GBM) is a fatal adult solid tumour with median overall survival of 18-20 months post-diagnosis; contributing factors to therapeutic inefficacy include acquisition of genomic alterations post-therapy, immune evasion, deregulated hypervascularization, and tumor microenvironmental factors such as hypoxia. Combined with extensive inter- and intra-tumoral heterogeneity at bulk and single-cell level, hypoxia contributes to a gradient of molecular alterations that are specific to different cell populations that make up tumour bulk and reside in specific niches. Hitherto, high-dimensional histopathologic analyses of hypoxic regions within GBM tissue have not been performed. We took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study wherein the exogenous hypoxia marker, pimonidazole (PIMO), was administered to GBM-patients preoperatively. Tissue specimens were subjected to imaging mass cytometry, high-resolution imaging, and serial immunohistochemistry using a panel of markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. Our findings showed that PIMO staining is associated with histopathologic features of hypoxia and correlates with specific metabolic, immune, and stemness markers in GBM; specific lymphocyte populations were depleted from hypoxic regions alongside alterations in macrophagic and microglial landscape in a niche-specific manner; hypoxia reduced the proportion of proliferating glioma initiating cells and altered the proliferative, transcriptional, and translational capacity of different cell populations; microvessel density was reduced in hypoxic microenvironment. Cytometry by time-of-flight further validated the altered proportions of specific immune cell types enriched in hypoxic populations of GBM microenvironment. Our study is the first to report use of PIMO to interrogate spatial, single-cell, and phenotypic architecture associated with tissue hypoxia and altered expression of biomarkers associated with hypoxia/glycolysis, immune infiltration, proliferation, and stemness. Identification of targetable biomarkers and mediators of hypoxia-driven habitats in GBM may provide direction for future immunotherapeutic research. Citation Format: Shreya Gandhi, Sheila Mansouri, Olivia Singh, Mark Zaidi, Ronald Wu, Andrew Gao, Bradley Wouters, Gelareh Zadeh. Spatial and single-cell proteomic landscaping of the hypoxic microenvironment in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B069.

IMMU-46. CROSS-TALK OF INNATE IMMUNE CELLS IN GLIOBLASTOMA PATHOPHYSIOLOGY: UNDERSTANDING NLRP3 AND NLRP12

Abstract Glioblastoma (GBM) are malignant gliomas with an average survival of less than 14 months after diagnosis, despite surgery, chemotherapy, and radiotherapy. Tumor survival is aided by the heterogeneous cell populations in the tumor microenvironment. Two crucial innate immune cells of the brain, namely microglia and macrophages, favor a tumor-promoting microenvironment IN GBM. The nucleotide-binding domain leucine-rich repeat-containing receptor (NLR) family in astrocytes and microglia cells recognizes pathogen- and damage-associated molecular patterns that induce inflammation. NLRP3, upon activation, leads to cleavage and activation of Caspase-1, causing the proteolytic cleavage-mediated activation and conversion of pro-IL-1ß and pro-IL-18 to IL-1ß and IL-18, leading to pyroptosis. NLRP12 downregulates inflammatory responses in microglia and macrophages by regulating the NF-kB pathway. NLRP3 and NLRP12 have tumor-promoting and anti-tumour functions in various cancers. NLRP12 is a prognostic marker for glioblastoma, and its increased expression correlates with poor survival in GBM patients. However, the cellular and molecular mechanisms of NLRP3 and NLRP12 in GBM pathophysiology are largely unknown. This study aims to understand the effect of NLRP3 and NLRP12 expression in GBM, to interpret the pathophysiology, and to help develop future targeted therapeutic interventions. To understand the baseline expression of NLRP3 and NLRP12 in cell lines and patient-derived GBM cells, PCR, western blotting, and Immunocytochemistry were performed comparing WT and nlrp3-/- and nlrp12-/- cells. Differential expression of NLRP3 and NLRP12 in patient-derived cells can be compared to GBM, astrocyte, and microglia cell lines. This may be the underlying cause for intra and inter-tumor heterogeneity and resultant differential therapy outcomes. To understand cell-to-cell communication in GBM-like tumor microenvironments, heterocellular 3D organoids, and patient-derived tumor organoids were developed. Cell viability in 3D patient-derived tumor organoids decreased by up to 10% with Doxorubicin and 40% with Carboplatin treatment decreases compared to untreated controls. This suggests the scope of developing drugs targeting inflammation regulatory genes.

Molecular Profile as an Outcome Predictor in Glioblastoma along with MRI Features and Surgical Resection: A Scoping Review.

Glioblastoma (GBM) is one of the most aggressive malignant tumors of the brain. We queried PubMed for articles about molecular predictor markers in GBM. This scoping review aims to analyze the most important outcome predictors in patients with GBM and to compare these factors in terms of absolute months of survival benefit and percentages. Performing a gross total resection for patients with GBM undergoing optimal chemo- and radiotherapy provides a significant benefit in overall survival compared to those patients who received a subtotal or partial resection. However, compared to IDH-Wildtype GBMs, patients with IDH-Mutant 1/2 GBMs have an increased survival. MGMT promoter methylation status is another strong outcome predictor for patients with GBM. In the reviewed literature, patients with methylated MGMT promoter lived approximately 50% to 90% longer than those with an unmethylated MGMT gene promoter. Moreover, KPS is an important predictor of survival and quality of life, demonstrating that we should refrain from aggressive surgery in important brain areas. As new therapies (such as TTFs) emerge, we are optimistic that the overall median survival will increase, even for IDH-Wildtype GBMs. In conclusion, molecular profiles are stronger outcome predictors than the extent of neurosurgical resection for GBM.

Clinical significance and potential mechanism of AEBP1 in glioblastoma.

Glioblastomas (GBM), the most common primary brain tumor, lack accurate prognostic markers and have a poor prognosis. Our study was designed to identify effective biomarkers for GBM prognosis analysis and development of precise treatments. Differentially expressed genes (DEGs) between GBM patients and controls were analyzed from the Xena database and GEPIA. Based on the screened DEGs, univariate COX and LASSO regression analysis were performed to identify the most relevant genes associated with GBM prognosis. Genes highly expressed in GBM patients were selected to construct receiver operating characteristic analysis and enrichment analysis was constructed on groups of high and low expression of adipocyte enhancer-binding protein 1 (AEBP1). CIBERSORT, ssGSEA and ESTIMATE were used to perform immune infiltration analysis. About 3297 DEGs were identified using data from Xena database; 8 prognostic genes were identified. AEBP1, which plays a role in neuronal differentiation and development, was positively correlated in GBMs with immune infiltration; its high expression in cancer patients is associated with short overall survival and advanced tumor staging. This study suggests that AEBP1 could serve as a prognostic marker for GBMs and that patients with high expression may have a better response to immunotherapy.

Preoperative prediction of MGMT promoter methylation in glioblastoma based on multiregional and multi-sequence MRI radiomics analysis

O6-methylguanine-DNA methyltransferase (MGMT) has been demonstrated to be an important prognostic and predictive marker in glioblastoma (GBM). To establish a reliable radiomics model based on MRI data to predict the MGMT promoter methylation status of GBM. A total of 183 patients with glioblastoma were included in this retrospective study. The visually accessible Rembrandt images (VASARI) features were extracted for each patient, and a total of 14676 multi-region features were extracted from enhanced, necrotic, “non-enhanced, and edematous” areas on their multiparametric MRI. Twelve individual radiomics models were constructed based on the radiomics features from different subregions and different sequences. Four single-sequence models, three single-region models and the combined radiomics model combining all individual models were constructed. Finally, the predictive performance of adding clinical factors and VASARI characteristics was evaluated. The ComRad model combining all individual radiomics models exhibited the best performance in test set 1 and test set 2, with the area under the receiver operating characteristic curve (AUC) of 0.839 (0.709–0.963) and 0.739 (0.581–0.897), respectively. The results indicated that the radiomics model combining multi-region and multi-parametric MRI features has exhibited promising performance in predicting MGMT methylation status in GBM. The Modeling scheme that combining all individual radiomics models showed best performance among all constructed moels.

Prognostic relevance of high expression of kynurenine pathway markers in glioblastoma

Glioblastoma (GBM) continues to exhibit a discouraging survival rate despite extensive research into new treatments. One factor contributing to its poor prognosis is the tumor's immunosuppressive microenvironment, in which the kynurenine pathway (KP) plays a significant role. This study aimed to explore how KP impacts the survival of newly diagnosed GBM patients. We examined tissue samples from 108 GBM patients to assess the expression levels of key KP markers—tryptophan 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenase (IDO1/2), and the aryl hydrocarbon receptor (AhR). Using immunohistochemistry and QuPath software, three tumor cores were analyzed per patient to evaluate KP marker expression. Kaplan–Meier survival analysis and stepwise multivariate Cox regression were used to determine the effect of these markers on patient survival. Results showed that patients with high expression of TDO2, IDO1/2, and AhR had significantly shorter survival times. This finding held true even when controlling for other known prognostic variables, with a hazard ratio of 3.393 for IDO1, 2.775 for IDO2, 1.891 for TDO2, and 1.902 for AhR. We suggest that KP markers could serve as useful tools for patient stratification, potentially guiding future immunomodulating trials and personalized treatment approaches for GBM patients.

Si-rhodamines with fine-tuned reactivities for tracking formaldehyde in glioblastoma

Formaldehyde (FA), an essential signaling molecule and an emerging neurotoxin in the brain, regulates various physiological and pathological processes. The abnormal metabolism of FA is associated with extensive brain diseases, including glioblastoma. However, FA probes with systematically fine-tuned reactivities are still very challenging. A group of silicon-substituted rhodamine-based FA probes was designed with fine-tuned structures and FA activities for tracking FA in vivo. With systematic structural manipulation, we have quantitatively revealed the chemical origins of the distinct FA reactivities in probes. Moreover, we successfully tracked the dynamic of FA in live cells, organs, and live animals by the designed probe. The vivid upregulation of FA in heterotopic xenografted and orthotopic glioblastoma by fluorescence imaging with the designed FA probe has proved that FA can be a potential molecular marker for glioblastoma.

Genomic landscape of glioblastoma without IDH somatic mutation in 42 cases: a comprehensive analysis using RNA sequencing data.

Glioblastoma is a malignant brain tumor with a poor prognosis. Genetic mutations associated with this disease are complex are not fully understood and require further elucidation for the development of new treatments. The purpose of this study was to comprehensively analyze genetic mutations in glioblastomas and evaluate the usefulness of RNA sequencing. We analyzed 42 glioblastoma specimens that were resected in routine clinical practice and found wild-type variants of the IDH1 and IDH2 genes. RNA was extracted from frozen specimens and sequenced, and genetic analyses were performed using the CLC Genomics Workbench. The most common genetic alterations in the 42 glioblastoma specimens were TP53 mutation (28.6%), EGFR splicing variant (16.7%), EGFR mutation (9.5%), and FGFR3 fusion (9.5%). Novel genetic mutations were detected in 8 patients (19%). In 12 cases (28.6%), driver gene mutations were not detected, suggesting an association with PPP1R14A overexpression. Our findings suggest the transcription factors SOX10 and NKX6-2 are potential markers in glioblastoma. RNA sequencing is a promising approach for genotyping glioblastomas because it provides comprehensive information on gene expression and is relatively cost-effective.

Potential diagnostic and drug target markers in glioblastoma

Glioblastoma multiforme (GBM) IDH-wildtype is the most prevalent brain malignancy in adults. However, molecular mechanisms, which leads to GBM have not been completely elucidated. Granulocyte colony-stimulating factor (GCSF), Granulocyte colony-stimulating factor receptor GCSFR, and Signal transducers and activators of transcription 3 (STAT3) have been involved in the occurrence and development of various cancers, but their role in GBM is little known. Herein, we have investigated the gene and protein expression of GCSF, GCSFR, and STAT3 in 21 tissue biopsy samples and also in tumor associated normal tissue (TANT) samples derived from glioblastoma patients, which revealed significantly differential expression of these genes. To validate our findings, we performed a comprehensive integrated analysis of transcriptomic and proteomic profiling of respective genes by retrieving GBM RNA-sequence data from Genome Atlas Databases. GO and KEGG analysis revealed enrichment in disease-related pathways, such as JAK/STAT pathway activation, which were associated with GBM progression. We further performed computational docking analysis of potential drug candidate Nisin against GCSF, and the results were validated in vitro through cytotoxic activity assay using a human glioblastoma cell line SF-767 in a dose-dependent manner. Our comprehensive analysis reveals that GCSF augments glioma progression, and its blockade with anticancer bacteriocin peptide Nisin can potentially inhibit the growth and metastasis of GBM.

A systematic review of high impact CpG sites and regions for MGMT methylation in glioblastoma [A systematic review of MGMT methylation in GBM

BackgroundMGMT (O 6 -methylguanine-DNA methyltransferase) promoter methylation is a commonly assessed prognostic marker in glioblastoma (GBM). Epigenetic silencing of the MGMT gene by promoter methylation is associated with greater overall and progression free survival with alkylating agent regimens. To date, there is marked heterogeneity in how MGMT promoter methylation is tested and which CpG sites are interrogated.MethodsTo further elucidate which MGMT promoter CpG sites are of greatest interest, we performed comprehensive searches in PubMed, Web of Science, and Embase and reviewed 2,925 article abstracts. We followed the GRADE scoring system to assess risk of bias and the quality of the studies we included.ResultsWe included articles on adult glioblastoma that examined significant sites or regions within MGMT promoter for the outcomes: overall survival, progression free survival, and/or MGMT expression. We excluded systemic reviews and articles on lower grade glioma. fifteen articles met inclusion criteria with variable overlap in laboratory and statistical methods employed, as well as CpG sites interrogated. Pyrosequencing or BeadChip arrays were the most popular methods utilized, and CpG sites between CpG’s 70–90 were most frequently investigated. Overall, there was moderate concordance between the CpG sites that the studies reported to be highly predictive of prognosis. Combinations or means of sites between CpG’s 73–89 were associated with improved OS and PFS. Six studies identified CpG sites associated with prognosis that were closer to the transcription start site: CpG’s 8, 19, 22, 25, 27, 32,38, and CpG sites 21–37, as well as low methylation level of the enhancer regions.ConclusionThe following systematic review details a comprehensive investigation of the current literature and highlights several potential key CpG sites that demonstrate significant association with OS, PFS, and MGMT expression. However, the relationship between extent of MGMT promoter methylation and survival may be non-linear and could be influenced by potential CpG hotspots, the extent of methylation at each CpG site, and MGMT enhancer methylation status. There were several limitations within the studies such as smaller sample sizes, variance between methylation testing methods, and differences in the various statistical methods to test for association to outcome. Further studies of high impact CpG sites in MGMT methylation is warranted.

Bioinformatics Analysis and Experimental Validation for Exploring Key Molecular Markers for Glioblastoma.

Glioblastoma (GBM) is the most common primary intracranial malignancy with a very low survival rate. Exploring key molecular markers for GBM can help with early diagnosis, prognostic prediction, and recurrence monitoring. This study aims to explore novel biomarkers for GBM via bioinformatics analysis and experimental verification. Dataset GSE103229 was obtained from the GEO database to search differentially expressed lncRNA (DELs), mRNAs (DEMs), and miRNAs (DEMis). Hub genes were selected to establish competing endogenous RNA (ceRNA) networks. The GEPIA database was employed for the survival analysis and expression detection of hub genes. Hub gene expression in GBM tissue samples and cell lines was validated using RT-qPCR. Western blotting was employed for protein expression evaluation. SYT1 overexpression vector was transfected in GBM cells. CCK-8 assay and flow cytometry were performed to detect the malignant phenotypes of GBM cells. There were 901 upregulated and 1086 downregulated DEMs identified, which were prominently enriched in various malignancy-related functions and pathways. Twenty-two hub genes were selected from PPI networks. Survival analysis and experimental validation revealed that four hub genes were tightly associated with GBM prognosis and progression, including SYT1, GRIN2A, KCNA1, and SYNPR. The four genes were significantly downregulated in GBM tissues and cell lines. Overexpressing SYT1 alleviated the proliferation and promoted the apoptosis of GBM cells in vitro. We identify four genes that may be potential molecular markers of GBM, which may provide new ideas for improving early diagnosis and prediction of the disease.

CDKN2A promoter methylation enhances self-renewal of glioblastoma stem cells and confers resistance to carmustine

BackgroundGlioblastoma, a highly aggressive form of brain cancer, poses significant challenges due to its resistance to therapy and high recurrence rates. This study aimed to investigate the expression and functional implications of CDKN2A, a key tumor suppressor gene, in glioblastoma cells, building upon the existing background of knowledge in this field.MethodQuantitative reverse transcription PCR (qRT-PCR) analysis was performed to evaluate CDKN2A expression in U87 glioblastoma cells compared to normal human astrocytes (NHA). CDKN2A expression levels were manipulated using small interfering RNA (siRNA) and CDKN2A overexpression vector. Cell viability assays and carmustine sensitivity tests were conducted to assess the impact of CDKN2A modulation on glioblastoma cell viability and drug response. Sphere formation assays and western blot analysis were performed to investigate the role of CDKN2A in glioblastoma stem cell (GSC) self-renewal and pluripotency marker expression. Additionally, methylation-specific PCR (MSP) assays and demethylation treatment were employed to elucidate the mechanism of CDKN2A downregulation in U87 cells.ResultCDKN2A expression was significantly reduced in glioblastoma cells compared to NHA. CDKN2A overexpression resulted in decreased cell viability and enhanced sensitivity to carmustine treatment. CDKN2A inhibition promoted self-renewal capacity and increased pluripotency marker expression in U87 cells. CDKN2A upregulation led to elevated protein levels of p16INK4a, p14ARF, P53, and P21, which are involved in cell cycle regulation. CDKN2A downregulation in U87 cells was associated with high promoter methylation, which was reversed by treatment with a demethylating agent.ConclusionOur findings demonstrate that CDKN2A downregulation in glioblastoma cells is associated with decreased cell viability, enhanced drug resistance, increased self-renewal capacity, and altered expression of pluripotency markers. The observed CDKN2A expression changes are mediated by promoter methylation. These results highlight the potential role of CDKN2A as a therapeutic target and prognostic marker in glioblastoma.

Ursolic acid inhibits glioblastoma through suppressing TGFβ-mediated epithelial-mesenchymal transition (EMT) and angiogenesis

Found in many fruits and plants, Ursolic acid (UA), a pentacyclic triterpene that occurs naturally, is recognized for its anti-cancer effects, especially in combating glioblastoma. However, the intricate molecular mechanisms underpinning its anti-tumor actions are still not fully understood, despite the recognition of these effects. By examining the functions of epithelial-mesenchymal transition (EMT) and angiogenesis, crucial for glioblastoma progression, and their regulation through Transforming Growth Factor Beta (TGFβ) – a key marker for glioblastoma, our research aims to fill this knowledge gap. This study explores how ursolic acid can block the progression of glioblastoma by precisely targeting TGFβ-triggered EMT and angiogenesis. The findings show that UA successfully blocks the spread, movement, and invasion of glioblastoma cells. Accompanying this, there is a significant reduction in the expression of TGFβ and crucial EMT indicators like snail and vimentin. Furthermore, UA shows a reduction in angiogenesis that depends on the dosage, highlighted by decreased vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Interestingly, increased TGFβ expression in U87 and U251 glioblastoma cell lines was found to weaken UA's anti-tumor properties, shedding more light on TGFβ′s critical function in glioblastoma's pathology. Supporting these laboratory results, UA also showed considerable inhibition of tumor growth in a glioblastoma xenograft mouse model. Overall, our research emphasizes Ursolic acid's promise as a new treatment for glioblastoma and clarifies its action mechanism, mainly by inhibiting TGFβ signaling and thereby EMT and angiogenesis.

Clinical roles of EGFR amplification in diffuse gliomas: a real-world study using the 2021 WHO classification of CNS tumors.

The 2021 World Health Organization Classification of Central Nervous System Tumors updates glioma subtyping and grading system, and incorporates EGFR amplification (Amp) as one of diagnostic markers for glioblastoma (GBM). This study aimed to describe the frequency, clinical value and molecular correlation of EGFR Amp in diffuse gliomas based on the latest classification. We reviewed glioma patients between 2011 and 2022 at our hospital, and included 187 adult glioma patients with available tumor tissue for detection of EGFR Amp and other 59 molecular markers of interest. Clinical, radiological and pathological data was analyzed based on the status of EGFR Amp in different glioma subtypes. 163 gliomas were classified as adult-type diffuse gliomas, and the number of astrocytoma, oligodendroglioma and GBM was 41, 46, and 76. EGFR Amp was more common in IDH-wildtype diffuse gliomas (66.0%) and GBM (85.5%) than IDH-mutant diffuse gliomas (32.2%) and its subtypes (astrocytoma, 29.3%; oligodendroglioma, 34.8%). EGFR Amp did not stratify overall survival (OS) in IDH-mutant diffuse gliomas and astrocytoma, while was significantly associated with poorer OS in IDH-wildtype diffuse gliomas, histologic grade 2 and 3 IDH-wildtype diffuse astrocytic gliomas and GBM. Our study validated EGFR Amp as a diagnostic marker for GBM and still a useful predictor for shortened OS in this group.

DIAPH3 predicts survival of patients with MGMT-methylated glioblastoma.

Glioblastoma is one of the most aggressive primary brain tumors, with a poor outcome despite multimodal treatment. Methylation of the MGMT promoter, which predicts the response to temozolomide, is a well-established prognostic marker for glioblastoma. However, a difference in survival can still be detected within the MGMT methylated group, with some patients exhibiting a shorter survival than others, emphasizing the need for additional predictive factors. We analyzed DIAPH3 expression in glioblastoma samples from the cancer genome atlas (TCGA). We also retrospectively analyzed one hundred seventeen histological glioblastomas from patients operated on at Saint-Luc University Hospital between May 2013 and August 2019. We analyzed the DIAPH3 expression, explored the relationship between mRNA levels and Patient's survival after the surgical resection. Finally, we assessed the methylation pattern of the DIAPH3 promoter using a targeted deep bisulfite sequencing approach. We found that 36% and 1% of the TCGA glioblastoma samples exhibit copy number alterations and mutations in DIAPH3, respectively. We scrutinized the expression of DIAPH3 at single cell level and detected an overlap with MKI67 expression in glioblastoma proliferating cells, including neural progenitor-like, oligodendrocyte progenitor-like and astrocyte-like states. We quantitatively analyzed DIAPH3 expression in our cohort and uncovered a positive correlation between DIAPH3 mRNA level and patient's survival. The effect of DIAPH3 was prominent in MGMT-methylated glioblastoma. Finally, we report that the expression of DIAPH3 is at least partially regulated by the methylation of three CpG sites in the promoter region. We propose that combining the DIAPH3 expression with MGMT methylation could offer a better prediction of survival and more adapted postsurgical treatment for patients with MGMT-methylated glioblastoma.

Live Organoid Cyclic Imaging.

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Early growth response 1 transcription factor and its context-dependent functions in glioblastoma.

Glioblastoma is the most aggressive form of primary brain tumour in adults. This tumour employs numerous transcription factors to advance and sustain its progression. Current evidence suggest that early growth response 1 (EGR1) plays a dual role as both an oncogene and a tumour suppressor in glioblastoma. Early growth response 1 expression is prevalent in glioblastoma, affecting over 80% of cases. Early growth response 1 regulatory roles extend to angiogenesis, cell adhesion, and resistance to chemotherapy, notably influencing pathways like hypoxia-inducible factor 1α and vascular endothelial growth factor A. Early growth response 1 can also induce cell adhesion, migration, chemoresistance against temozolomide, stemness, and self-renewal in glioblastoma cells. Despite its oncogenic functions, EGR1 can also suppress tumours by upregulating non-steroidal anti-inflammatory drug-activated gene 1 and phosphatase and tensin homolog deleted on chromosome ten, and inhibiting invasion and metastasis. Additionally, EGR1 may have hypothetical implications in the viral hit-and-run theory, particularly regarding cytomegalovirus infection. The key findings of this review are the context- dependent nature of EGR1's actions and its potential as a prognostic marker in glioblastoma. Further research is needed to understand EGR1's role fully and exploit its potential in clinics.

Identification of potential glioma drug resistance target proteins based on ultra-performance liquid chromatography-mass spectrometry differential proteomics.

In this study, to screen for candidate markers of temozolomide (TMZ) resistance in glioblastoma, we artificially established TMZ drug-resistant glioblastoma (GBM) cell lines, U251-TMZ and U87-TMZ. In the U251-TMZ and U87-TMZ cell lines, we screened and analyzed differentially expressed proteins using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) differential proteomics. Compared with the U251 and U87 control cell lines, 95 differential proteins were screened in the U251-TMZ and U87-TMZ cell lines, of which 28 proteins were upregulated and 67 proteins were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the co-upregulated proteins showed that most of the differentially expressed proteins were located in the cytoplasm and were significantly upregulated in the biological processes related to vesicular transport in the intimal system and inflammatory response mediated by myeloid leukocytes. Seven candidates were identified as potential GBM markers of TMZ resistance. Combined with existing research findings, our study supports that UAP1L1 and BCKDK are promising potential markers of TMZ resistance in GBM. This is important for further understanding the molecular mechanisms that drive the development and enhancement of TMZ resistance.

NIMG-34. TEMPORAL MUSCLE THICKNESS AS A PROGNOSTIC MARKER IN A REAL-LIFE COHORT OF NEWLY DIAGNOSED MGMT PROMOTER METHYLATED GLIOBLASTOMA PATIENTS: A MULTICENTRIC IMAGING ANALYSIS

Abstract BACKGROUND In cranial MRI assessed temporal muscle thickness (TMT) correlates with lumbar skeletal muscle mass, which serves as surrogate marker of sarcopenia. Prior research has identified TMT as a prognostic marker in glioblastoma. However, incorporation into clinical practice is challenging due to the heterogeneity of previous studies and the absence of established cutoff values. To address this, we conducted a multicenter analysis to validate recently proposed sex-specific cutoff values in a homogeneous real-life cohort of newly diagnosed MGMT promoter-methylated glioblastoma patients uniformly treated with combined CCNU/temozolomide. We implemented a balanced control cohort for comparison. METHODS TMT was measured at baseline using preoperative or early postoperative MRI and in the disease course using the first MRI after completion of radiotherapy. Patients were TMT-based divided into “at risk of sarcopenia” or “normal muscle status”. Kaplan-Meier statistics and multivariable Cox regression analysis were used for correlation with survival. RESULTS In total, n = 126 patients were analyzed (n = 66 received CCNU/temozolomide; n = 60 received temozolomide). Patients with normal muscle mass at baseline (> 6,3 mm for men; > 5,2 mm for women) had significantly prolonged mOS (44.3 versus 16.7 months for CCNU/temozolomide; 29.5 versus 17.4 months for temozolomide) compared to those at risk of sarcopenia. In a multivariable Cox regression analysis, normal muscle mass (HR: 0.39, 95% CI: 0.23-0.66; p = 0.0003) and age at diagnosis of < 50 years (HR: 0.42, 95% CI: 0.22-0.79; p = 0.0067) were significant prognostic markers. Longitudinally, survival was best in patients with lack of TMT decline. CONCLUSIONS This analysis suggests TMT to serve as a prognostic marker in glioblastoma patients treated with CCNU/temozolomide or with temozolomide. However, for TMT assessment to be implemented in a generalizable manner, such as for patient selection for therapeutic measures, further validation in prospective controlled trials is necessary.

BIOM-10. KI-67 IS ASSOCIATED WITH PROGRESSION-FREE SURVIVAL IN PATIENTS WITH GLIOBLASTOMA

Abstract Identifying predictive and prognostic markers for glioblastoma (GBM) is an area of intense investigation. The objective of this study was to evaluate OLIG2 and Ki-67 as prognostic biomarkers. A tissue microarray with clinical data was created for 70 patients diagnosed with GBM between 2002-2007 at a single institution. Immunohistochemistry was performed to evaluate expression of OLIG2 and Ki-67. Overall survival and progression free survival (PFS) were calculated using the Kaplan-Meier method. Univariable (UVA) and multivariable (MVA) analysis was performed using Cox proportional hazards modeling. The median age of diagnosis was 63, 66% of patients were male, 80% had a KPS ≥70, 64% had a subtotal resection or biopsy, and 36% had a gross total resection. Median OS and PFS were 13.3 months and 9.5 months respectively. For patients who received resection plus chemoradiation, median OS and PFS were 16.7 months and 9.5 months respectively. 36.4% of patient samples had Ki-67 expression fraction > 20%. 18.0% had heavy OLIG2 staining. On MVA, lower KPS was associated with shorter OS and radiation therapy was associated with longer OS. OLIG2 was not associated with OS or PFS. Patients who had a Ki-67 < 20% had a shorter PFS, but not OS on MVA. Our data demonstrate higher Ki-67 levels correlate with improved PFS without an OS benefit. This association persisted for patients receiving standard of care treatment. OLIG2 levels did not correlate with PFS or OS in either cohort. Our study indicates that further investigation is needed to determine whether Ki-67 is useful in predicting recurrence risk and potentially changing treatment strategies for these patients, as well as the mechanism behind the associated improvement seen with higher Ki-67 levels. These data are currently limited by lack of IDH and MGMT status but suggest Ki-67 should be further investigated as a biomarker for progression.