Glioblastoma Multiforme Patients Research Articles

51. Discerning cell types and states in spatial transcriptomics data using topic modelling

Glioblastoma multiforme (GBM) is the most common adult brain tumour. GBM cells include a diverse collection of normal and tumour cells with distinct molecular capabilities and with differential levels of sensitivity to treatment that led to treatment resistance. Spatial transcriptomics technologies (e.g. 10X Visium) have enabled analyzing tumour-normal cells interactions in a physical tissue context. Using Visium, a well-annotated cohort of adult GBM patient samples grown as patient-derived xenograft (PDX) models were profiled at different time points of the disease. However, the identification of cell types or states of Visium spots is a challenging problem as each spot contains a mixture of cells. Current tools for cell-type deconvolution are limited in discovering new novel cell types because they require a well-annotated reference dataset. Moreover, the PDX data has a mixture of human and mouse cell types at the same spot. In this work, we have applied topic modelling, a well-known probabilistic approach in natural language processing to discover the distribution of topics in language documents. Using the analogy between documents and spatial transcriptomic data, we were able to identify the main cell types in spatial PDX data without gene expression references dataset of tumour states, normal mouse, and immune cell types. Finally, we compared various probabilistic topic modelling algorithms to fit the PDX data, and to understand the cell-to-cell interactions between tumour and normal cells.

Prognostic Factors of Survival in Glioblastoma Multiforme Patients-A Retrospective Study.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor that occurs in adults. In spite of prompt diagnosis and rapidly administered treatment, the survival expectancy is tremendously poor. Extensive research has been performed in order to establish factors to predict the outcome of GBM patients; however, worldwide accepted prognostic markers are still lacking. We retrospectively assessed all adult patients who were diagnosed with primary GBM and underwent surgical treatment during a three-year period (January 2017-December 2019) in the Neurosurgery Department of the Emergency Clinical County Hospital of Târgu Mureș, Romania. Our aim was to find any statistically relevant connections between clinical, imagistic, and histopathological characteristics and patients' survival. A total of 75 patients were eventually included in our statistical analysis: 40 males and 35 females, with a median age of 61 years. The mean tumor dimension was 45.28 ± 15.52 mm, while the mean survival rate was 4 ± 6.75 months. A univariate analysis demonstrated a statistically significant impact of tumor size, pre-, and postoperative KPSI on survival rate. In addition, a Cox multivariate assessment strengthened previous findings regarding postoperative KPSI (regression coefficient -0.03, HR 0.97, 95% CI (HR) 0.96-0.99, p = 0.002) as a favorable prognostic factor and GBM size (regression coefficient 0.03, HR 1.03, 95% CI (HR) 1.01-1.05, p = 0.005) as a poor prognostic marker for patients' survival. The results of our retrospective study are consistent with prior scientific results that provide evidence supporting the importance of clinical (quantified by KPSI) and imagistic (particularly tumor dimensions) features as reliable prognostic factors in GBM patients' survival.

Comparative analysis of the prognosis of external beam radiation therapy (EBRT) and EBRT plus brachytherapy for glioblastoma multiforme: a SEER population-based study

ObjectiveRadiotherapy is one of the effective ways to treat glioblastoma multiforme (GBM). We aimed to explore the prognostic difference between external beam radiotherapy (EBRT) and EBRT combined with brachytherapy (EBRT + BT).MethodsThe GBM patients from the Surveillance, Epidemiology, and End Results (SEER) database were divided into two cohorts: the EBRT cohort and the EBRT + BT cohort. Kaplan–Meier (KM) analysis and Cox proportional hazards regression were used to determine the underlying risk factors for overall survival (OS) and disease-specific survival (DSS). And the competing risk model and propensity score matching (PSM) was adopted to eliminate potential biases. We also conducted subgroup analyses and interaction tests as well.ResultsThere was a total of 41,010 eligible GBM patients. The median OS (15 months) and DSS (17 months) of the EBRT + BT cohort were significantly longer than that of the EBRT cohort (OS = 11 months, DSS = 12 months). After using the competing risk model and PSM, we found that only advanced age was the independent risk factor, while only EBRT + BT was the independent protective factor (HR = 0.84, 95%CI [0.74,0.96], p = 0.01). EBRT had universal effects in the treatment of GBM, and EBRT + BT had a more pronounced protective effect in the subgroups of males (HR = 0.81, 95%CI [0.68,0.97], p = 0.02) and local excision (HR = 0.82, 95%CI [0.34,0.95], p = 0.01).ConclusionsThe therapeutical effect of EBRT + BT treatment is better than that of EBRT alone, especially in male patients or patients who have undergone local resection. Our findings may provide novel evidence to develop a better radiotherapy strategy for GBM patients.

Development of a novel glycolysis-related genes signature for isocitrate dehydrogenase 1-associated glioblastoma multiforme.

The significant difference in prognosis between IDH1 wild-type and IDH1 mutant glioblastoma multiforme (GBM) may be attributed to their metabolic discrepancies. Hence, we try to construct a prognostic signature based on glycolysis-related genes (GRGs) for IDH1-associated GBM and further investigate its relationships with immunity. Differentially expressed GRGs between IDH1 wild-type and IDH1 mutant GBM were screened based on the TCGA database and the Molecular Signature Database (MSigDB). Consensus Cluster Plus analysis and KEGG pathway analyses were used to establish a new GRGs set. WGCNA, univariate Cox, and LASSO regression analyses were then performed to construct the prognostic signature. Then, we evaluated association of the prognostic signature with patients' survival, clinical characteristics, tumor immunogenicity, immune infiltration, and validated one hub gene. 956 differentially expressed genes (DEGs) between IDH1 wild-type and mutant GBM were screened out and six key prognostically related GRGs were rigorously selected to construct a prognostic signature. Further evaluation and validation showed that the signature independently predicted GBM patients' prognosis with moderate accuracy. In addition, the prognostic signature was also significantly correlated with clinical traits (sex and MGMT promoter status), tumor immunogenicity (mRNAsi, EREG-mRNAsi and HRD-TAI), and immune infiltration (stemness index, immune cells infiltration, immune score, and gene mutation). Among six key prognostically related GRGs, CLEC5A was selected and validated to potentially play oncogenic roles in GBM. Construction of GRGs prognostic signature and identification of close correlation between the signature and immune landscape would suggest its potential applicability in immunotherapy of GBM in the future.

LINC01711 promotes transforming growth factor-beta (TGF-β) induced invasion in glioblastoma multiforme (GBM) by acting as a competing endogenous RNA for miR-34a and promoting ZEB1 expression

GBM is the central nervous system's most aggressive and malignant tumor. TGF-β expression is elevated in GBM, and it promotes invasion and EMT. TGF-β regulates the expression of several lncRNAs, which promote glioma pathogenesis. Here we characterize the role of TGF-β-induced lncRNA- LINC01711 in glioma pathogenesis. We show that LINC01711 expression is significantly upregulated in GBM tissues and is associated with poor overall survival of GBM patients. Loss-of-function studies illustrate that LINC01711 promotes proliferation, migration, and invasion in GBM. In addition, LINC01711 depletion sensitizes glioma cells to Temozolomide (TMZ) induced apoptosis by inhibiting ZEB1 expression. LINC01711 functions as a competing endogenous RNA for miR-34a and promotes ZEB1 expression to regulate invasion. Our findings suggest that LINC01711 is an attractive therapeutic target for GBM.

A novel compound EPIC-0412 reverses temozolomide resistance via inhibiting DNA repair/MGMT in glioblastoma.

Temozolomide (TMZ) resistance has become an important obstacle affecting its therapeutic benefits. O6-methylguanine DNA methyltransferase (MGMT) is primarily responsible for the TMZ resistance in Glioblastoma multiforme (GBM) patients. In addition, active DNA damage repair pathways can also lead to TMZ resistance. Here, we reported a novel small-molecule inhibitor EPIC-0412 that improved the therapeutic efficacy of TMZ by inhibiting the DNA damage repair pathway and MGMT in GBM via epigenetic pathways. The small-molecule compound EPIC-0412 was obtained through high-throughput screening. RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), and chromatin immunoprecipitation (ChIP) assays were used to verify the effect of EPIC-0412. Co-immunoprecipitation (Co-IP) was used to elucidate the interactions of transcription factors at the MGMT promoter region. Animal experiments using a mouse model were performed to verify the efficacy of EPIC-0412 in sensitizing GBM cells to TMZ. EPIC-0412 physically interrupts the binding of HOTAIR and EZH2, leading to the upregulation of CDKN1A and BBC3, causing cell cycle arrest and apoptosis in GBM cells. EPIC-0412 inhibits DNA damage response in GBM cells through the p21-E2F1 DNA damage repair axis. EPIC-0412 epigenetically silences MGMT through its interaction with the ATF3-p-p65-HADC1 axis at the MGMT promoter region. The application of EPIC-0412 restored the TMZ sensitivity in GBM in vivo experiments. This study discovered a small-molecule inhibitor EPIC-0412, which enhanced the chemotherapeutic effect of TMZ by acting on the p21-E2F1 DNA damage repair axis and ATF3-p-p65-MGMT axis, providing evidence for combining epigenetic drugs to increase the sensitization toward TMZ in GBM patients.

Voxel-Wise GBM Recurrence Prediction Based on Sparse Attention Multi-Modal MR Image Fusion Coupling with Stem Cell Niches Proximity Estimation

<h3>Purpose/Objective(s)</h3> Nearly all glioblastoma multiforme (GBM) patients experience local recurrence despite aggressive therapies. The outcomes of patients with recurrence continue to be disappointing, emphasizing the importance of early and voxel-wise recurrence prediction for early treatment intervention. Emerging evidence has linked the tumorigenesis of recurrence to stem cell niches (SCN). For the first time, our group introduced a novel multidimensional support vector machine (SVM) method coupling with SCN proximity estimation to predict GBM recurrence on the voxel level. The current study aims to develop an L1-sparsity regularized attention fusion U-net (L1-AF U-net) to further distinguish the most salient and complementary features from multi-modal MR images, improving voxel-wise GBM recurrence prediction. <h3>Materials/Methods</h3> With IRB approval, fifty patients with pre-and post-surgery MR scans were retrospectively solicited, including T1, T2, T1_CE, ADC, and FLAIR. Post-surgery MR scans included two months before the clinical diagnosis of GBM recurrence and the day of the radiologically confirmed recurrence. The recurrences were manually annotated on the contrast-enhanced T1 MR, which were deformably registered to the 2-month pre-recurrence MR scans. The processing pipeline included a sparse attention learning module with L1 norm regularization for multi-modal feature fusion and a U-net for voxel-wise GBM recurrence prediction. The L1 sparsity regularized attention network embedded in the U-net architecture learns the coherent and mutual-complementary features across five MR modalities while eliminating the inherent redundancy and noisy artifacts. The high-risk recurrence (HRR) proximity map was determined by the weighted sum of inverse distances from two possible origins of recurrence: SCN and tumor cavity. The U-net was trained given the discriminative feature fusion in HRR for voxel-wise GBM recurrence prediction <h3>Results</h3> On the 2-month pre-recurrence testing MRs, the U-net segmentation with sparse multi-modal attention fusion achieved a recall of 0.83±0.15, a precision of 0.75±0.12, and an F1-score of 0.79±0.14. As a comparison, our previous GBM recurrence prediction using proximity estimation coupled SVM (SVM_PE) achieved a recall of 0.80±0.10, a precision of 0.69±0.14, and an F1-score of 0.74±0.10. A Student's t-test has also been performed to evaluate the significance of the proposed method over SVM_PE. The P-values are 0.0013 (for recall), 4.97 × 10⁻¹⁰ (for precision) and 7.94 × 10⁻⁸ (for F1-score), respectively. <h3>Conclusion</h3> We present an advanced U-net-based GBM recurrence prediction framework with sparse multi-modal MR fusion. This constrained attention fusion of the multi-modal MR images facilitated the U-net to learn more effective features from the multi-modal MR images and enhanced the prediction performance comparing to SVM method.

Dosimetric Evaluation of Physical Radiation Delivery Limits of a Weak Magnetic Field Generator on GBM Dose Distributions

<h3>Purpose/Objective(s)</h3> To demonstrate the feasibility of meeting dose prescription criteria when using a weak magnetic field generator to support glioblastoma multiforme (GBM) radiation therapy. <h3>Materials/Methods</h3> Weak magnetic fields extend free radical life, and recent experimental results have shown that adding weak magnetic fields (20G-40G) to radiation therapy delivery enhances free radical production, increases tumor cell death, and slows tumor growth. Based on these results, we have built the first clinical unit, consisting of two 63 cm ID Helmholtz coils, spaced 35 cm apart. The coil system is placed such that the patient and Linac couch are within the coils, and the Linac isocenter is coincident with the center of the coil system. We propose to use GBM for our first clinical trial. The limitation imposed by the coil system is a fixed Linac couch angle, constraining the gantry rotation to slightly more than 180°, from lateral to lateral. The impact on treatment plan quality of the corresponding reduction in available beam angles was unknown, so we conducted a treatment planning study to determine if we could provide clinically equivalent treatment plans with the coil in place. We evaluated the treatment plans for 10 GBM patients that had been treated with arc therapy. The prescription was 60 Gy (2 Gy x 30 fractions) for 6 patients, 55.8 Gy (1.8 Gy x 31 fractions) for 2 patients, 54 Gy (1.8 Gy x 30 fractions) for 1 patient, and 40.05 Gy (2.67 Gy x 15 fractions) for 1 patient. The dose was delivered to 95% of the planning target volume (PTV). The PTV margin was 1.3-2 cm surrounding the gross tumor volume. Critical structures, such as the brainstem, optic nerves, and optic chiasm, were contoured and assigned constraints of 62 Gy, 54 Gy, and 54 Gy, respectively. We repeated the treatment planning session using only coplanar arcs that spanned from 90° to 270° (half-arcs). <h3>Results</h3> All the treatment plans generated with the half-arcs met the clinical criteria. The minimum PTV doses for the half arc and clinical plans were 50.0 ± 6.1 Gy and 51.4 ± 7.1 Gy, and the maximum PTV doses were 61.9 ± 7.1 Gy and 62.7 ± 8.6 Gy, respectively. Table 1 summarizes the results of four reported critical structures. All the critical structures met the criteria, and the half-arc plans were neither better nor worse than the clinical plans. <h3>Conclusion</h3> The physical limits imposed by the magnetic field generator on the Linac beam angles do not degrade treatment planning for GBM radiation therapy. The prescribed dose can be adequately delivered while avoiding critical structures. With this groundwork, we can develop clinical trials to evaluate the impact of utilizing weak magnetic fields in radiation therapy.

The effect of anticonvulsants on survival among patients with GBM brain tumors undergoing radiation: A SEER-Medicare analysis

BackgroundPatients with glioblastoma multiforme (GBM) who undergo radiation often require anticonvulsants during treatment. The aim of this study was to determine the effects of anticonvulsants on GBM clinical outcomes. MethodsA retrospective analysis was performed using the SEER-Medicare database. All patients with GBM who were treated with radiation and concurrently taking an anticonvulsant were included in final analysis. Each class of medication was further subdivided by mechanism of action. Descriptive statistics were performed for all variables. Kaplan Meier survival curves were generated for each class of medication and Cox regression analysis was performed to assess the effect of each individual variable on survival. ResultsThere were 1561 patients available for final analysis. On multivariate Cox regression analysis, GBM patients taking sodium/calcium (Na/Ca) channel blocker anticonvulsants during radiation therapy demonstrated both improved overall survival (OS) (HR, 0.799; 95% CI [0.716, 0.891]; P < 0.001) and cancer specific survival (CSS) (HR, 0.814; 95% CI [0.727, 0.911]; P < 0.001). ConclusionOS was significantly better in patients taking NA/Ca channel blockers among patients with GBM who were concurrently undergoing radiation therapy.

ATF4-dependent fructolysis fuels growth of glioblastoma multiforme

Excessive consumption of fructose in the Western diet contributes to cancer development. However, it is still unclear how cancer cells coordinate glucose and fructose metabolism during tumor malignant progression. We demonstrate here that glioblastoma multiforme (GBM) cells switch their energy supply from glycolysis to fructolysis in response to glucose deprivation. Mechanistically, glucose deprivation induces expression of two essential fructolytic proteins GLUT5 and ALDOB through selectively activating translation of activating transcription factor 4 (ATF4). Functionally, genetic or pharmacological disruption of ATF4-dependent fructolysis significantly inhibits growth and colony formation of GBM cells in vitro and GBM growth in vivo. In addition, ATF4, GLUT5, and ALDOB levels positively correlate with each other in GBM specimens and are poor prognostic indicators in GBM patients. This work highlights ATF4-dependent fructolysis as a metabolic feature and a potential therapeutic target for GBM.

Feasibility and Safety of a Combined Metabolic Strategy in Glioblastoma Multiforme: A Prospective Case Series.

Background Glioblastoma multiforme (GBM) may be susceptible to metabolic strategies such as fasting and ketogenic diets, which lower blood glucose and elevate ketones. Combining these two strategies may be an ideal approach for sustaining a potentially therapeutic glucose ketone index (GKI). In this prospective case series, we observed whether a combined metabolic strategy was feasible, safe, and capable of sustaining a GKI <6 in patients with GBM. Methods We provided recommendations and guidelines to 10 GBM patients at various stages of tumour progression and treatment that enabled them to complete a 5–7-day fast every 1–2 months combined with a modified ketogenic diet during the intervening weeks. Patients monitored their blood glucose and ketone levels and body weight. Adverse effects were assessed. Results Patients completed a mean of 161 ± 74 days of the combined metabolic strategy, with 34 ± 18 (21%) days of prolonged fasting (mean fast duration: 6.0 ± 1.4 days) and 127 ± 59 (79%) days on the ketogenic diet. The mean GKI for all 10 patients was 3.22 (1.28 during the fasts, 5.10 during the ketogenic diet). Body weight decreased by 8.4 ± 6.9 kg (11.2% decrease in baseline weight). The most common adverse effects attributed to the fasts and ketogenic diet were fatigue, irritability, and feeling lightheaded. The metabolic strategy did not interfere with standard oncological treatments. Conclusion This is the first study to observe the feasibility and safety of repeated, prolonged fasting combined with a modified ketogenic diet in patients with GBM. Using minimal support, patients maintained the combined metabolic strategy for 5–6 months while sustaining a potentially therapeutic mean GKI of 3.22. Weight loss was considerable. Adverse effects attributed to the metabolic strategy were mild, and it did not interfere with standard oncological treatments. Study Registration: This study is registered on the Australia New Zealand Clinical Trials Registry, number ACTRN12620001310954. The study was registered on 4 December 2020.

Risk of Glioblastoma Multiforme in Patients Taking Ion Channel Blockers.

Background Ion channels play a role in the development and progression of glioblastoma multiforme. This study investigates the association between the risk of developing glioblastoma multiforme in patients taking these medications. Methods A retrospective propensity score-matched analysis was performed using the TriNetX multinational electronic health record database for patients taking verapamil, digoxin, amiodarone, or diltiazem versus those not taking these medications. The outcome of interest was the incidence of glioblastoma multiforme. Results Verapamil users had an OR of 0.494 (p < 0.0001) of developing glioblastoma versus verapamil non-users. Patients on digoxin had an OR of 0.793 (p = 0.2393), patients on amiodarone had an OR of 0.600 (p = 0.0035), patients on diltiazem had an OR of 0.584 (p < 0.0001), and patients on verapamil, digoxin, amiodarone, or diltiazem had an OR of 0.641 (p < 0.0001) of developing glioblastoma versus patients not taking these medications. Conclusion In patients taking the ion channel blockers diltiazem, amiodarone, or verapamil, the odds of developing glioblastoma multiforme were lower than in patients not taking these medications.

The miR-429 suppresses proliferation and migration in glioblastoma cells and induces cell-cycle arrest and apoptosis via modulating several target genes ofERBB signaling pathway.

Glioblastoma multiforme (GBM) is an aggressive and lethal brain cancer, which is incurable with standard cancer treatments. miRNAs have great potential to be used for gene therapy due to their ability to modulate several target genes simultaneously. We found miR-429 is downregulated in GBM and has several predicted target genes from the ERBB signaling pathway using bioinformatics tools. ERBB is the most over-activated genetic pathway in GBM patients, which is responsible for augmented cell proliferation and migration in GBM. Here, miR-429 was overexpressed using lentiviral vectors in U-251 and U-87 GBM cells and it was observed that the expression level of several oncogenes of the ERBB pathway, EGFR, PIK3CA, PIK3CB, KRAS, and MYC significantly decreased, as shown by real-time PCR and western blotting. Using the luciferase assay, we showed that miR-429 directly targets MYC, BCL2, and EGFR. In comparison to scrambled control, miR-429 had a significant inhibitory effect on cell proliferation and migration as deduced from MTT and scratch wound assays and induced cell-cycle arrest and apoptosis in flow cytometry. Altogether, miR-429 seems to be an efficient suppressor of the ERBB genetic signaling pathway and a potential therapeutic for GBM.

Can Extended Chemotherapy Improve Glioblastoma Outcomes? A Retrospective Analysis of Survival in Real-World Patients.

Standard treatment for glioblastoma multiforme (GBM) is surgery followed by radiotherapy plus concurrent chemotherapy with daily temozolomide (TMZ), and six subsequent TMZ 5/28-day cycles. Research has focused on identifying more effective alternatives to the current protocol, including extension of the number of adjuvant TMZ cycles. We performed a retrospective analysis of all GBM patients treated in our hospital (160 patients, 2011–2020). Median follow-up was 16.0 months. Analysis of prognostic factors was performed with a particular focus on the benefit of extending TMZ chemotherapy. Improved survival correlated with younger age, female gender, good performance status, absence of cognitive dysfunctions, no steroid use, and total tumor resection. Median progression-free survival (PFS) was 12 months and median overall survival (OS) was 20.0 months for the entire cohort. Median OS by adjuvant TMZ was 10.0 months if no adjuvant chemotherapy given (group 0), 15.0 months for patients that did not complete six TMZ cycles (group A), 24.0 months for those that did (group B), and 29.0 months for patients having received more than six cycles (group C) (p < 0.0001). At the three-year mark, 15.9% patients were alive in group A, 24.4% in group B and 38.1% in group C. Carefully selected GBM patients may derive benefit from extending the standard adjuvant chemotherapy beyond six TMZ cycles, but more data is required.

The GBM Tumor Microenvironment as a Modulator of Therapy Response: ADAM8 Causes Tumor Infiltration of Tams through HB-EGF/EGFR-Mediated CCL2 Expression and Overcomes TMZ Chemosensitization in Glioblastoma.

Simple SummaryResistance to standard therapies impose a huge challenge on the treatment for glioblastoma multiforme (GBM), which is often considered as a cell intrinsic property of either GBM or, more significantly, of GBM stem-like cells. Tumor-associated macrophages and microglia (TAMs) take up the majority of the immune population in the tumor microenvironment of GBM and potentially participating in modulating therapy responses. However, little is known about the mechanisms underlying the effect of TAMs on temozolomide (TMZ) induced chemoresistance. Members of the metzincin superfamily such as Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloprotease (ADAM) proteases are important participants in the process of intercellular communications in the tumor microenvironment. Herein, we revealed a novel concept of an intra-tumoral ADAM8 mediated malignant positive feedback loop constituted by the intimate interaction of tumor associated macrophages (TAMs) and GBM cells under TMZ treatment. These findings provide a convincing example and further support the notion that the tumor microenvironment, in addition to GBM cells and GBM stem-like cells, should be considered as an essential modulator of therapy in GBM. In conclusion, our study provides a rational basis for TAM sparing ADAM8-targeting in GBM to optimize standard chemotherapy.Standard chemotherapy of Glioblastoma multiforme (GBM) using temozolomide (TMZ) frequently fails due to acquired chemoresistance. Tumor-associated macrophages and microglia (TAMs) as major immune cell population in the tumor microenvironment are potential modulators of TMZ response. However; little is known about how TAMs participate in TMZ induced chemoresistance. Members of the metzincin superfamily such as Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloprotease (ADAM) proteases are important mediators of cellular communication in the tumor microenvironment. A qPCR screening was performed to identify potential targets within the ADAM and MMP family members in GBM cells. In co-culture with macrophages ADAM8 was the only signature gene up-regulated in GBM cells induced by macrophages under TMZ treatment. The relationship between ADAM8 expression and TAM infiltration in GBM was determined in a patient cohort by qPCR; IF; and IHC staining and TCGA data analysis. Moreover; RNA-seq was carried out to identify the potential targets regulated by ADAM8. CCL2 expression levels were determined by qPCR; Western blot; IF; and ELISA. Utilizing qPCR; IF; and IHC staining; we observed a positive relationship between ADAM8 expression and TAMs infiltration level in GBM patient tissues. Furthermore; ADAM8 induced TAMs recruitment in vitro and in vivo. Mechanistically; we revealed that ADAM8 activated HB-EGF/EGFR signaling and subsequently up-regulated production of CCL2 in GBM cells in the presence of TMZ treatment; promoting TAMs recruitment; which further induced ADAM8 expression in GBM cells to mediate TMZ chemoresistance. Thus; we revealed an ADAM8 dependent positive feedback loop between TAMs and GBM cells under TMZ treatment which involves CCL2 and EGFR signaling to cause TMZ resistance in GBM.

66 (PB056) - Anti-tumor activity of BDTX-1535, an irreversible CNS penetrant inhibitor of multiple EGFR extracellular domain alterations, in preclinical glioblastoma models

Background: EGFR genetic alterations, including mutation and amplification, are very common in glioblastoma multiforme (GBM), however, no targeted therapy has been approved for this disease. EGFR oncogenic mutations in GBM promote EGFR oncogenic covalent homodimer formation which leads to paradoxical activation and renders reversible inhibitors ineffective. An effective EGFR inhibitor in GBM must meet four conditions:1Highly potent and selective against a broad range of extracellular domain mutations and amplified EGFR,2Wild-type EGFR sparing,3Irreversible binding to circumvent paradoxical activation,4High CNS penetration. Our MAP discovery platform led to the identification of MasterKey inhibitor BDTX-1535, an irreversible 4th-generation CNS penetrant EGFR tyrosine kinase inhibitor (TKI) that potently and selectively targets a broad range of EGFR alterations in GBM. Materials and Methods: BDTX-1535 preclinical exposure was evaluated in multiple species in plasma and brain. Antitumor activity was assessed across a broad range of mouse PDX and allograft models, including an intracranial PDX. 2540 tumors were sequenced with the Tempus xT NGS assay. Mutational frequencies and co-expression status of oncogenic EGFR variants were assessed. The relationship between treatment history and oncogenic EGFR mutational frequency was analyzed. Results: BDTX-1535 is a small molecule that irreversibly inhibits a spectrum of EGFR alterations expressed in GBM together with EGFR amplification, while sparing inhibition of normally expressed wild type EGFR. BDTX-1535 avoids efficacy limiting paradoxical activation that can occur with reversible EGFR inhibitors. BDTX-1535 exhibits good oral bioavailability and a CNS Kpuu of 0.55 and 0.48 in rat and dog, respectively, comparing favorably with other CNS penetrant TKIs. Tumor regression and survival advantage were observed across all tumor models with EGFR amplification or mutation, including in an intracranial PDX model. EGFR mutations and amplification are commonly expressed in GBM. Consistent with published data, EGFR mutations, variants, and copy number gain were frequently co-expressed. For example, among tumors expressing EGFRvIII, ∼95% co-expressed at least one other variant, and among tumors expressing EGFRvVI, ∼45% co-expressed at least one other variant. Within a subset of GBM patients with longitudinal sequencing data, oncogenic EGFR mutations were expressed and persisted following front-line treatment. Conclusions: BDTX-1535 is a potent, CNS penetrant, irreversible small molecule EGFR inhibitor that shows efficacy against a broad range of extracellular domain mutations and EGFR amplification found in GBM patients. RWE findings demonstrate a heterogeneous EGFR mutational profile in GBM patients, with persistent prevalence over time. BDTX-1535 is currently in a phase I clinical study (NCT05256290). Conflict of interest: Ownership: Black Diamond Therapeutics Tempus Labs

High Expression of Fibronectin 1 Predicts a Poor Prognosis in Glioblastoma.

Glioblastoma multiforme (GBM), the most malignant intracranial neoplasm, is associated with a high mortality and recurrence rate due to the aggressive nature and heterogeneity of the tumor. Some of the molecular markers involved in the tumorigenesis of GBM are essential in prognosis, diagnosis, and treatment. Due to the limitations of therapeutic effects, this study aims to explore novel biomarkers with prognostic value and to provide new insights into therapeutic targets. The expression profile of mRNAs in GBM was detected by RNA-sequencing, and differentially expressed genes were identified by integrating the data from RNA-seq results and the GEPIA2 database. Of the total 40 hub genes, FN1, P4HB, and PPIB showed prognostic significance based on both GEPIA2 and CGGA databases. The validation of FN1, P4HB, and PPIB expression by qPCR and correlation analysis with clinicopathological features were performed in 41 GBM tissues from our institution. Kaplan-Meier analysis revealed that FN1 and P4HB expressions levels were related to the overall survival (OS) of GBM patients (P<0.05). Multivariate analysis showed that FN1 overexpression (HR=9.199, P=0.002) was an independent and unfavorable prognostic factor for GBM patients. The median survival time was 8.5 months and 21 months for high and low expressions of FN1, respectively. It was suggested that FN1 could be an ideal target for prognosis and a potential therapeutic target in GBM.

CONSENT - A Randomised Controlled Trial of Enhanced Informed Consent Compared to Standard Informed Consent to Improve Patient Understanding of Early Phase Oncology Clinical Trials – GBM Cohort (Nonrandomised) Analysis

Abstract AIMS Early phase cancer clinical trials have become more complicated and patients often misunderstand their nature and purpose. CONSENT (NCT04407676) is a randomised controlled trial testing whether enhanced informed consent for patient education can improve comprehension – since patients with glioblastoma multiforme (GBM) have a higher rate of baseline cognitive impairment, we studied this group separately. METHOD GBM patients (from the ICE-CAP Phase 1 study - NCT03673787), underwent the schedule for the standard CONSENT arm - full length trial PIS, Quality of Informed Consent Questionnaire Parts A and B (QuIC-A and QuIC-B), experimental intervention (2 page study aid and 10 educational videos), and a repeat QuIC-A and QuIC-B. The primary endpoint for this subgroup was the difference in QuIC-A scores before and after the intervention using a paired t-test. RESULTS 6 patients with GBM were recruited - 3 did not complete any study questionnaires. The three pre intervention QuIC-A scores were 70, 81, 88, with a mean of 75 (unit reference 76). The three pre intervention QuIC-B scores were 69, 62, 75 with a mean of 69 (unit reference is 91). Only one patient completed the post intervention questionnaire - their QuIC-A score moved from 88 to 100. CONCLUSION This study demonstrates the significant difficulties in studying comprehension in patients with GBM considering early phase trials. There is a need for creative multi-modality solutions to provide information to GBM patients considering clinical trials, and novel tools to assess the effectiveness of these solutions.

Atypical induction of HIF-1α expression by pericellular Notch1 signaling suffices for the malignancy of glioblastoma multiforme cells

Contact-based pericellular interactions play important roles in cancer progression via juxtacrine signaling pathways. The present study revealed that hypoxia-inducible factor-1α (HIF-1α), induced even in non-hypoxic conditions by cell-to-cell contact, was a critical cue responsible for the malignant characteristics of glioblastoma multiforme (GBM) cells through Notch1 signaling. Densely cultured GBM cells showed enhanced viability and resistance to temozolomide (TMZ) compared to GBM cells at a low density. Ablating Notch1 signaling by a γ-secretase inhibitor or siRNA transfection resensitized resistant GBM cells to TMZ treatment and decreased their viability under dense culture conditions. The expression of HIF-1α was significantly elevated in highly dense GBM cells even under non-hypoxic conditions. Atypical HIF-1α expression was associated with the Notch1 signaling pathway in both GBM and glioblastoma stem cells (GSC). Proteasomal degradation of HIF-1α was prevented by binding with Notch1 intracellular domain (NICD), which translocated to the nuclei of GBM cells. Silencing Notch1 signaling using a doxycycline-inducible Notch1 RNA-interfering system or treatment with chetomin, a HIF pathway inhibitor, retarded tumor development with a significant anti-cancer effect in a murine U251-xenograft model. Using GBM patient tissue microarray analysis, a significant increase in HIF-1α expression was identified in the group with Notch1 expression compared to the group without Notch1 expression among those with positive HIF-1α expression. Collectively, these findings highlight the critical role of cell-to-cell contact-dependent signaling in GBM progression. They provide a rationale for targeting HIF-1α signaling even in a non-hypoxic microenvironment.

Assessment of the Efficacy of an Alternative Regimen of Lomustine in Recurrent GBM – A Single Centre Experience

Abstract AIMS Lomustine (CCNU) based regimens are frequently used for recurrent glioblastoma multiforme (GBM). The usual CCNU regimen is 100-130mg/m2, day 1 x 6-weekly cycle for up to 6 cycles. Efficacy is limited, with median progression-free survival (PFS) approximating 1.0-3.0 months with higher haematological toxicities, particularly thrombocytopenia around 13-25%. The Clatterbridge Cancer Centre, UK, has adopted the regimen of 40mg once a day over 4 days x 4-weekly for up to 6 cycles. This study aims to identify the efficacy and toxicity profile of this regimen in recurrent GBM. METHOD A retrospective analysis on 113 recurrent GBM patients, treated with single-agent CCNU as a second-line, between June 2016 and January 2020. Kaplan-Meier survival estimates identified PFS, PFS at 6 months (PFS6) and Overall Survival (OS) using SPSS v.27. Overall Responses were based on imaging or clinical assessment in patients with at least 2 CCNU cycles. SACT assessments and blood test records, using CTCAE v5.0 grading, identified clinical adverse events. RESULTS We observed an 18.8% overall response rate with 2.4% partial response and 16.5% stable disease. There was a CCNU-specific 8-month median OS, 4-month median PFS, and 20.4%% PFS6. This regimen was well-tolerated. The most common toxicity was grade 1 fatigue (38.9%). The Grade 3 or 4 haematological toxicity was low with 12% thrombocytopenia and 2.7% neutropenia rates in patients. CONCLUSION This study suggests a reasonable alternative to the usual regimen, offering improved tolerance, lower toxicity rates, and equivalent efficacy. We propose prospective studies comparing differing CCNU regimens to mitigate retrospective studies’ limitations.