GBM Survival Research Articles

Effects of Reoperation Timing on Survival among Recurrent Glioblastoma Patients: A Retrospective Multicentric Descriptive Study.

Glioblastoma inevitably recurs, but no standard regimen has been established for treating this recurrent disease. Several reports claim that reoperative surgery can improve survival, but the effects of reoperation timing on survival have rarely been investigated. We, therefore, evaluated the relationship between reoperation timing and survival in recurrent GBM. A consecutive cohort of unselected patients (real-world data) from three neuro-oncology cancer centers was analyzed (a total of 109 patients). All patients underwent initial maximal safe resection followed by treatment according to the Stupp protocol. Those meeting the following criteria during progression were indicated for reoperation and were further analyzed in this study: (1) The tumor volume increased by >20-30% or a tumor was rediscovered after radiological disappearance; (2) The patient's clinical status was satisfactory (KS ≥ 70% and PS WHO ≤ gr. 2); (3) The tumor was localized without multifocality; (4) The minimum expected tumor volume reduction was above 80%. A univariate Cox regression analysis of postsurgical survival (PSS) revealed a statistically significant effect of reoperation on PSS from a threshold of 16 months after the first surgery. Cox regression models that stratified the Karnofsky score with age adjustment confirmed a statistically significant improvement in PSS for time-to-progression (TTP) thresholds of 22 and 24 months. The patient groups exhibiting the first recurrence at 22 and 24 months had better survival rates than those exhibiting earlier recurrences. For the 22-month group, the HR was 0.5 with a 95% CI of (0.27, 0.96) and a p-value of 0.036. For the 24-month group, the HR was 0.5 with a 95% CI of (0.25, 0.96) and a p-value of 0.039. Patients with the longest survival were also the best candidates for repeated surgery. Later recurrence of glioblastoma was associated with higher survival rates after reoperation.

Abstract 5025: Reduced level of protein kinase C iota renders human glioblastoma less invasive and induces apoptosis via modulating Tak1/NF-κB pathway

Abstract Glioblastoma (GBM) is characterized by poor therapeutic response and overall survival. It is the most aggressive form of primary brain tumor with a tendency to invade surrounding healthy brain tissues, rendering them largely incurable. In this study, small-interference RNA was used to knock down the expression of protein kinase C PKC-ι. The reduced PKC-ι expression impaired the phosphorylation of LIM kinase 1/2 (LIMK), which is a critical step in cofilin recycling and actin polymerization. Additionally, there was a down-regulation of matrix metalloprotease-9 expression in LN-229 and U87G cells, which coincided with decreased invasion. Therefore, PKC-ι regulates both cytoskeleton rearrangement and cell adhesion, which contributed to cell migration. Silencing of PKC-ι also dampened Transforming growth factor-β (TGF-β)-activated kinase 1 (Tak1) level. TAK1 is an essential component in genotoxic stresses-induced NF-κB-activation and mitogen-activated protein kinase (MAPK)-pathways in GBM. TAK1, when activated, forms a complex with TAB1-3 (Tak1 binding proteins). Our data reveal, reduced Tak1 level after silencing PKC-ι interferes with complex formation and reduces NF-κB-activation. Diminished PKC-ι level also reduces activation of Tak1/JNK axis. This deactivation exerts an anti-oncogenic effect on glioblastoma via suppression of Akt1, c-Myc, and STAT3. This implies PKC-ι may have a crucial role in the activation of Tak1. Reduced migration and invasion in glioblastoma cells upon inhibition of PKC-ι was further confirmed by Scratch assay and Boyden assay. These results indicate that PKC-ι is involved in the control of glioblastoma cell migration and invasion. PKC-ι is a central component of multiple converging and bifurcating pathways and is a potential therapeutic target against GBM survival and infiltration. Citation Format: Khandker Mohammad Khalid, Wishrawana S. Ratnayake, Avijit Dey, Nuzhat N. Oishee, Mahfuza Marzan, Mildred Acevedo-Duncan. Reduced level of protein kinase C iota renders human glioblastoma less invasive and induces apoptosis via modulating Tak1/NF-κB pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5025.

Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors.

Brain tumors are an important cause of suffering and death. Glioblastoma are the most frequent primary tumors of the central nervous system in adults. They are associated with a very poor prognosis, since only 10% of GBM patients survive 5 years after diagnosis. Medulloblastoma are the most frequent brain malignancies in childhood; they affect the cerebellum in children under 10 years of age in 75% of cases. The current multimodal treatment comes at the expense of serious and often long-lasting side effects. Herein, we propose the synthesis of a library of novel alkoxyamines as anticancer drug candidates. The most efficient molecule, ALK4, was selected based on its ability to inhibit both survival and migration of GBM and MB cells in 2D cultures and in 3D tumor spheroids. A fluorescent derivative was used to show the early cytosolic accumulation of ALK4 in tumor cells. Spontaneous homolysis of ALK4 led to the release of alkyl radicals, which triggered the generation of reactive oxygen species, fragmentation of the mitochondrial network and ultimately apoptosis. To control its homolytic process, the selected alkoxyamine was bioconjugated to a peptide selectively recognized by matrix metalloproteases. This bioconjugate, named ALK4-MMPp, successfully inhibited survival, proliferation, and invasion of GBM and MB tumor micromasses. We further developed innovative brain and cerebellum organotypic models to monitor treatment response over time. It confirmed that ALK4-MMPp significantly impaired tumor progression, while no significant damage was observed on normal brain tissue. Lastly, we showed that ALK4-MMPp was well-tolerated in vivo by zebrafish embryos. This study provides a new strategy to control the activation of alkoxyamines, and revealed the bioconjugate ALK4-MMPp bioconjugate as a good anticancer drug candidate.

DISP-06. POPULATION-LEVEL DETERMINANTS OF SOCIO-DEMOGRAPHIC, TUMOR, AND TREATMENT FACTORS IN GLIOBLASTOMA USING SEER DATABASE (1997-2016)

Abstract BACKGROUND Although socio-demographic and treatment factors have been separately linked to impact on prognosis in several GBM studies, no studies to date have collectively evaluated many potential individual patient and treatment-related factors and their impact on survival in GBM in a comprehensive, multi-institutional, population-based manner. The purpose of this study is to identify sociodemographic, tumor, treatment, and clinical characteristics affecting survival in GBM using a large cancer registry database, the Surveillance Epidemiology End Results (SEER) registry, and identify strategies to reduce risk and disparities for patients with GBM. METHODS Individual-level data were obtained from SEER 18 program (1997-2016) using SEER*Stat software. New variables on urban-rural area and treatment combination among surgery, radiotherapy, and chemotherapy were created to comprehensively identify determinants affecting survival in GBM. Univariate and multivariate survival analyses were performed to estimate median survival time using Kaplan-Meier methods and to evaluate the relative risks of potential determinants using Cox hazard methods. RESULTS (1) The important determinants include age, gender, race, rural-urban area, diagnostic confirmation, tumor size, resection type, metastases, laterality, treatment effects and combined effects. (2) In addition to the standard treatment on GBM, the socio-demographic and clinical factors like rural-urban area (access to hospital), diagnostic conformation, and report type were important to reduce the risk to GBM patients. (3) Coming to modern times, survival time and survival rate have improved, especially in terms of socio-economic factors of people living in metro areas and married people (HR = 0.897 to 0.813 for metro area and HR = 0.999 to 0.918 for married status). CONCLUSIONS Our findings of higher risk determinants in GBM might lead to better prognosis in GBM with socio-demographic and clinical factors affecting the survival in GBM. The future investigation including MGMT promoter methylation might be beneficial with better understanding of determinants affecting risk in GBM.

DNAR-15. POLYMORPHISM AT P21 CODON 31 PROVIDED A PREDICTION FOR BEVACIZUMAB THERAPY IN GLIOBLASTOMA MULTIFORME

Abstract Glioblastoma (GBM) is a common, malignant brain tumor. While it rarely metastasizes to distant organs, the diffuse invasion of glioma cells within the brain makes it difficult to resect. P21 (Waf-1) is a cyclin-dependent kinase inhibitor that plays an essential role in cell growth arrest, terminal differentiation, and apoptosis. Thus, the existence of natural variants of p21 could be linked to specific cancer. The purpose of this study was to identify the polymorphism variants of p21 codon 31 (Ser31Arg) and to explore its role in the therapy of glioblastoma multiforme. A total of unrelated 99 GBM subjects, were enrolled in the study, and genomic DNA was obtained from each tumor sample for genotyping. Genotyping was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism method (PCR-RFLP analysis. Odds ratios (Ors) and 95% confidence intervals (CIs) were estimated by unconditional logistic regression analysis. We found that among these GBM patients, the distribution of codon 31 polymorphisms was 20.2% for Serine homozygotes (Ser/Ser), 32.3% for arginine homozygotes (Arg/Arg), and 47.5% for Serine/arginine heterozygotes (Ser/Arg). Although the polymorphisms of p21 codon 31 were not directly associated with the overall survival of GBM, both the Arg/Arg and Arg/Ser genotypes were associated with significant benefits in terms of overall survival in patients treated with CCRT plus bevacizumab compared to patients treated with CCRT alone. Our findings suggest that the p21 Ser31Arg polymorphism may play an important role in the pathogenesis and initiation of GBM. Moreover, the polymorphism seems to contribute to a significant prediction at early diagnosis for bevacizumab therapy.

IMMU-05. THE INFLUENCE OF THE KETOGENIC DIET ON THE IMMUNE TOLERANT MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) represents a particularly aggressive and immune-resistant cancer. Preclinical investigations have identified the anti-tumor activity of a ketogenic diet (KD) in GBM, potentially being used as a tool to target its glycolytic phenotype. Since immune cells in the tumor have a similar reliance upon nutrients to perform their individual functions, we sought to determine if the KD influenced the immune landscape of GBM. Utilizing genetically-engineered murine GBM tumor cells orthotopically implanted in immune-competent mice, we demonstrate that KD improved survival in GBM. Immunophenotyping of tumors identified a novel role KD plays in macrophage polarization, with a paradoxical 50% increase in immune-suppressive M2-macrophages and a decrease in pro-inflammatory M1-macrophages. We recapitulated KD in vitro using a modified cell culture based on comprehensive metabolomic profiling of serum in KD-fed mice. Consistent with in vivo studies, murine macrophages cultured in these conditions skewed polarization towards the M2-phenotype with immune-suppressive properties. We went on to mechanistically link these findings to the activation of transcription factor PPARg. Although anti-tumor activity was observed in mice fed a KD, we hypothesized this parallel increase in M2 macrophage polarization tempered its potential therapeutic benefit. Colony-stimulating factor 1 (CSF-1) plays a central role in macrophage differentiation, and CSF-1R inhibition is actively being investigated as a strategy to skew their polarization towards an M1-phenotype. Therefore, we tested a combination of KD with the brain-penetrant CSF-1R inhibitor BLZ945. Consistent with our hypothesis, this combination demonstrated a striking improvement in survival (p = 0.0004), with 50% of mice achieving long-term survival ( > 50 days). Correlative studies confirmed the capacity of BLZ945 to normalize KD-induced increases in M2s, and the combination induced an increase of anti-tumor iNOS+M1s. Combinatorial strategies using agents designed to modulate macrophage polarization represent a rational approach to improve the anti-tumor activity of KD in GBM.

TMIC-37. SINGLE-CELL CHARACTERIZATION OF THE IMMUNE LANDSCAPE OF EXTREME LONG-TERM SURVIVORS WITH MALIGNANT GLIOMA

Abstract Glioblastoma Multiforme (GBM) remains the most common malignant primary brain tumor with a dismal prognosis that rarely exceeds beyond two years despite extensive therapy, which consists of maximal safe surgical resection, radiotherapy and/or chemotherapy. Recently, it has become clear that GBM is not one homogeneous entity and that both intra-and intertumoral heterogeneity contribute significantly to differences in tumoral behavior which may consequently be responsible for differences in survival. Strikingly and despite its dismal prognosis, small fractions of GBM patients seem to display extreme extended survival compared to the large majority of patients. The underlying mechanisms for this peculiarity remain largely unknown however, even though emerging data suggest that both cancer cell-autonomous and microenvironmental factors and their interplay probably play an important role. We used high-dimensional, multiplexed immunohistochemistry to spatially, and cytometry by time-of-flight to quantitively, characterize the cell constitution and interactions within the tumor microenvironment (TME) in 21 extreme long-term survivors (living over 10 year) and 42 deeply matched controls and therefore short-term survivors (living under 1.5 year) on a single cell level. For all tumors (epi)genetic data was also collected. We identified a high level of both inter-and intrapatient heterogeneity defined by several distinct tumoral niches, as well as described interactions within these niches and with the surrounding infiltrating immune cells of the TME in GBM. Finally, by linking patient characteristics with the heterogeneous immune composition we are able to create an immune stratification that can be linked to patient survival in GBM. Therefore, this study is an essential initial step towards strategies to alter the TME in a favorable way with a personalized modulation strategy.

QOL-01. RISK FACTORS FOR AND CONSEQUENCES OF PROLONGED DEXAMETHASONE USE IN PATIENTS WITH GLIOBLASTOMA (GBM)

Abstract BACKGROUND Dexamethasone is the primary treatment for tumor-associated edema. Prolonged use can have significant side effects and may negatively impact survival in GBM. Factors associated with steroid dependence and steroid associated side effects (SASEs) are not well established. METHODS We performed a retrospective study of patients with newly diagnosed, IDH-wildtype GBM at our center. Demographics, clinical and tumor characteristics, dexamethasone usage, and SASEs (infections, myopathy, hypertension, fractures, mood symptoms, weight gain, and hyperglycemia) were extracted through chart review. We compared survival outcomes (OS) between patients with and without steroid dependence. Risk factors for steroid dependence were identified through logistic regression analysis. RESULTS 39/92 (35.9%) patients developed steroid dependence. Of these, 26/39 (66.7%) were on steroids at the start of initial treatment. Patients who developed steroid dependence were older, more likely to have STR/biopsy, bilateral disease, KPS < 90, baseline neurological deficits, and to be obese (BMI ≥ 30). 94.9% of patients with steroid dependence experienced ≥ 1 SASE (vs. 34%) and 46.2% experienced ≥ 3 SASEs (vs. 3.8%). Mood symptoms (69.2% vs. 26.4%), hyperglycemia (53.8% vs. 7.5%) and infections (51.3% vs. 7.5%) were the most common SASEs. Median OS was inferior in steroid-dependent patients (18 vs 25 mo). Baseline obesity was identified as a risk factor for developing steroid dependence (OR 5.9). DISCUSSION SASEs are common in patients on prolonged steroids. Obesity may increase the risk of developing steroid dependence. Identifying patients at highest risk for steroid dependence may help guide future interventions to mitigate the negative effects of prolonged dexamethasone use.

CTIM-05. FINAL RESULTS OF 2-THE-TOP: A PILOT PHASE 2 STUDY OF TTFIELDS (OPTUNE) PLUS PEMBROLIZUMAB PLUS MAINTENANCE TEMOZOLOMIDE (TMZ) IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA (NDGBM)

Abstract INTRODUCTION TTFields induce anti-tumor immunity via type-1 interferon (T1IFN) pathways of the STING and AIM2 inflammasomes. Thus, TTFields may synergize with immune checkpoint inhibitors to prolong survival in GBM. METHODS We enrolled 26 ndGBM patients in a pilot phase 2 study combining TTFields, pembrolizumab and maintenance TMZ. Primary endpoints were PFS versus case-matched controls of TTFields plus TMZ in the EF-14 study and immune signatures by multiomics of PBMCs and tumors. Secondary endpoints included toxicity and OS. RESULTS The median age was 60.5 years. Fourteen (54%) had biopsy only or partial resection. Nineteen (73%) had unmethylated MGMT and 3 (11.5%) had an IDH mutation. As of 2/16/22, 7 (27%) were progression-free and 14 (54%) were alive with median follow-up of 16.8 months. Median PFS was 12.1 months versus 7.9 months in a case-matched control cohort of 26 patients (HR = 0.456; 95% CI: 0.222-0.937; P = 0.015). Twelve-month PFS was 52.4% (95% CI: 31.4-69.6%) versus 20.4% (95% CI: 5.5-41.7%) in controls; P = 0.012. Six of 15 (40%) patients with measurable disease achieved partial or complete response. Median OS was 25.2 months versus 15.9 months in controls (HR = 0.382; 95% CI: 0.168-0.861; P = 0.078). Two-year OS was 57.8% (95% CI: 33.7-75.9%) versus 19.2% (95% CI: 7.0-36.0%) in controls; P = 0.002. In a Cox regression analysis adjusting for key prognostic factors, P-value reached 0.033 for PFS and 0.020 for OS. Molecular analyses confirmed robust T cell activation via the T1IFN trajectory, which highly correlated with TCRαβ clonal expansion (Spearman coefficient = -0.8; P = 0.014), and defined a T cell-based gene signature of TTFields effects. The most common serious adverse events were thromboses, seizures, and metabolic disturbances in 4 (15%), 3 (11.5%), and 2 (8%) patients, respectively. CONCLUSIONS The triple combination demonstrated acceptable toxicity and promising efficacy in ndGBM. Survival and molecular data will be updated.

STEM-11. POLYAMINE METABOLISM AND INFLAMMATORY STATE CONTROL A VIRAL MIMICRY-DRIVEN QUIESCENT PHENOTYPE IN GLIOMA STEM CELLS (GSCS)

Abstract The rate limiting enzyme of the polyamine pathway, SAT1, is significantly upregulated in non-cycling GSCs. However, higher SAT1 expression predicts poorer survival in GBM suggesting a role in quiescence and recurrence post-treatment. Treating GSCs with N1N11-Diethylnorspermine (DENSpm) recapitulates SAT1 upregulation and triggers a chemoprotective, long-term reversible growth arrest, while not affecting expression of stem, differentiation, nor senescence-associated markers. Remarkably, DENSpm strongly induces double-stranded RNA in GSCs. Interestingly, S-Adenosyl methionine (SAM) is an essential aminopropyl donor for polyamine biosynthesis. We found a significant correlation between DNA methylation, SAM, and polyamines across GSCs suggesting that SAM depletion may cause epigenetic and post-transcriptional alterations leading to re-activation of endogenous retroviral elements (ERVs) and viral mimicry-driven quiescence. However, while we confirmed DENSpm-induced SAM depletion, we did not detect DNA, histone, nor RNA demethylation. Noteworthy, ERVs are the most accessible transposable elements in GSCs indicative of a poised state. Furthermore, the correlation between expression of ERVs and interferon stimulated genes reveals a spectrum of viral mimicry amongst GSCs. Using a larger cohort of GSCs and hfNSCs, we identified three DENSpm response subgroups. A resistant subgroup comprised of cells able to proliferate through treatment, a reversible one whereby cells become quiescent and readily re-enter cell cycle post-treatment, and an irreversible consisting of cells that enter a deep quiescent state which they cannot escape. Importantly, we found that DENSpm response correlates with the cell's basal inflammatory state and the activity of viral defense pathways. These results indicate that a viral mimicry-driven quiescence may be specific to slower-growing neurodevelopmental GSCs, while more proliferative, inflammatory-poised GSCs are impervious to these additional cues. We propose that polyamine metabolism, glioma stem cell hierarchy and tumour inflammatory context play a central role in regulating quiescence and have important clinical implications in the initial response to treatment, aggressiveness, and ultimately recurrence of GBM.

EPCO-05. DIFFERENTIAL MOLECULAR PROFILING OF IDH WILD TYPE GLIOBLASTOMA SURVIVORS REVEALS THE ASSOCIATION OF NEOANTIGEN QUALITY WITH EXCEPTIONAL LONG SURVIVAL

Abstract Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor. Despite intensive multimodal treatment, the prognosis of GBM patients is poor with the median overall survival under 15 months. Defining the genetic profiles of rare long-term GBM survivors may provide insights into the molecular basis of GBM progression. IDH mutations have been identified as a favorable prognostic factors, and define a clinically and molecularly distinct group of GBM. Additional molecular mechanisms underlying the prolonged survival in IDHwt GBM patients remained unclear. Therefore, we explored the molecular profiles of 71 IDHwt GBM homogeneously treated patients exhibiting disproportionate survival. The cohort included 36 short-term survivors that deceased within 1 year after diagnosis (median survival of 7 months) and 35 long-term survivors exhibiting an exceptional survival above 4 years (median survival of 88 months). The differential genetic landscape of short- and long-term IDHwt GBM survivors outlined similar mutation load and few differences in the frequency of recurrent alterations, including chromosomes 19 and 20 duplication significantly overrepresented in long-term survivors. To assess the potential impact of T lymphocyte neoantigen-mediated activation towards extended GBM survival, we inferred the restricted HLA binding affinity of the somatic mutations and applied the immune fitness model to accurately predict high-quality neoantigens. Although the general neoantigen burden remained unvaried between short- and long-term survivors, we found that the immune quality of the predicted neoantigens is significantly higher in the patients with exceptional long survival. Moreover, the immune deconvolution of the tumor microenvironment from the gene expression data indicated the presence of CD8+ T lymphocytes and pro-inflammatory macrophages in samples harboring high-quality neoantigens from long-term survivors. This study identified the neoantigen quality as a main distinctive feature of exceptionally long-term survivors supporting the key role of cellular-mediated immune response to restrict tumor progression in IDHwt GBM.

A Subregion-Based Survival Prediction Based on Multi-Parametric MRIs for Glioblastoma

<h3>Purpose/Objective(s)</h3> To predict the survival of GBM patients by using multi-parametric MR images, we developed a novel framework of subregion-based radiomic analysis. This study aims to search for the best combinations among tumor subregions and various MRI sequences for GBM survival prediction. <h3>Materials/Methods</h3> 119 GBM patients provided by BraTS 2020 were used in the study. 100 cases were used for training and evaluating under 5-fold cross-validation and the external 19 cases were used for independent testing. For each tumor subregion, we used open-source software to extract 680 subregional radiomic features from one MRI sequence for subsequent analysis. We proposed a novel algorithm to uncover an optimal MRIs combination from multiple MRI sequences. By a one-to-one matching relation between 4 sequences MRIs (T1/T1ce/T2/FLAIR) and 3 tumor subregions (gadolinium-enhancing tumor (ET), peritumoral edema (ED), and non-enhancing tumor core (NET)), we evaluated the 64 (4³) kinds of subregional radiomic features combinations (680 × 3) based on the evaluation of maximizing the performance of survival stage classification between long-stage (>365 days) and short-stage (<365 days) groups using the SVC (Support Vector Classification) classifier. The best combination with a quarter size compared to the initial imaging data (680 × 12) can be determined and achieved precisely prediction. <h3>Results</h3> As shown in table, the specific matching relation of NET-FLAIR, ED-T1ce, ET-T2 which used 3-sequence resulted in the highest AUCs of 0.998 and 0.983 on the training and independent test cohort respectively. The model generated from optimized 3 MR sequences of features was superior to those models using one single-sequence, 2-sequence, other combinations of 3-sequence and 4-sequence MRIs. <h3>Conclusion</h3> We demonstrated the efficacy of a novel subregion-based framework for survival prediction of GBM by using multi-parametric MRIs. The proposed approach effectively combined multiple MR images and achieved survival prediction precisely.

The prognostic significance of PD-L1 expression in patients with glioblastoma: A meta-analysis.

BackgroundGlioblastoma (GBM) is a malignant brain tumor associated with high morbidity and mortality rates with a poor prognosis. In recent years, studies on prognostic markers such as programmed death ligand 1 (PD-L1) have increased; however, their conclusions remain controversial. Here, relevant literature was reviewed and a meta-analysis was performed to clarify the correlation between PD-L1 expression and overall survival (OS) in GBM.MethodsThe non-foundational literature on PD-L1 expression associated with OS in GBM up to February 2022 was searched in the PubMed, Metstr, Cochrane, and Web of Science databases. Literature was rigorously screened according to inclusion and exclusion criteria, the total hazard ratio (HR), and corresponding 95% confidence intervals (CIs).ResultsCalculating the combined HR value and corresponding 95% CI of HR=1.124 (95% CI: 1.047–1.201, P=0.000, I2 (I-squared)=48.8%), it was shown that PD-L1 expression was significantly associated with low OS in GBM patients. Although I2 = 48.8% < 50%, to make the results more credible, in the cutoff values ≥10% subgroup HR=1.37 (95% CI: 1.07–1.67, P=0.000, I2 = 0%), which was also the result found in the first meta-analysis. In contrast, in the cutoff value ≥5% subgroup HR=1.14 (95% CI: 0.98–1.30, P=0.000, I2 = 59.8%) and in the cutoff value median PD-L1 expression levels subgroup HR=1.05 (95% CI: 0.92–1.18, P=0.000, I2 = 0%), indicating that PD-L1 expression was not associated with low OS in GBM. Furthermore, in four studies, we found no significant correlation between PD-L1 expression and the progression-free survival of GBM (HR=1.14, 95% CI:0.40–1.88, P=0.03, I2 = 29.3%).ConclusionPD-L1 expression was significantly associated with low OS in GBM patients; however, this result needs to be interpreted with caution and requires a large, multicenter clinical study in patients with similar baseline data for further evaluation.

TTC7B Is a Novel Prognostic-Related Biomarker in Glioma Correlating with Immune Infiltrates and Response to Oxidative Stress by Temozolomide.

Background Gliomas are one of the most prevalent malignant brain tumors. Hence, identifying biological markers for glioma is imperative. TTC7B (Tetratricopeptide Repeat Domain 7B) is a gene whose role in cancer in currently identified. To this end, we examined the TTC7B expression as well as its prognostic significance, biological roles, and immune system impacts in patients with glioma. Methods We evaluated the function of TTC7B in GBM and LGG through the published CGGA (Chinese Glioma Genome Atlas) and TCGA (The Cancer Genome Atlas) databases. CIBERSORT and TIMER were used to analyze the link between TTC7B and immune cells, while R was used for statistical analysis. In addition, Transwell analysis, including migration and invasion assays, was performed to identify the relationship between TTC7B and temozolomide. Results Low expression of TTC7B was observed in GBM and LGG. 1p/19q codeletion, IDH mutation, chemotherapy, and grade were found to have a significant correlation with TTC7B. Besides, low TTC7B expression was linked with low overall survival (OS) in both GBM and LGG. In the Cox analysis, TTC7B was found to independently function as a risk element for OS of patients with glioma. Furthermore, CIBERSORT analysis demonstrated a positive link between TTC7B and multiple immune cells, especially activated NK cells. Transwell analysis, including migration and invasion assays, revealed that temozolomide reduced the migration and invasion capacity of glioma cells and increased the expression of TTC7B. Conclusion In all, TTC7B could serve as a promising prognostic indicator of LGG and GBM, and is closely associated with immune infiltration and response to oxidative stress by temozolomide.

KS05.7.A Characterization of the immune composition of extreme long-term survivors with malignant glioma at single-cell level

Abstract Background Glioblastoma Multiforme (GBM) remains the most common malignant primary brain tumor with a dismal prognosis that rarely exceeds beyond two years despite extensive therapy, which consists of maximal safe surgical resection, radiotherapy and/or chemotherapy. Recently, it has become clear that GBM is not one homogeneous entity and that both intra-and intertumoral heterogeneity contribute significantly to differences in tumoral behavior which may consequently be responsible for differences in survival. Strikingly and in spite of its dismal prognosis, small fractions of GBM patients seem to display extended survival compared to the large majority of patients. The underlying mechanisms for this peculiarity remain largely unknown however, even though emerging data suggest that both cancer cell-autonomous and microenvironmental factors and their interplay probably play an important role. Material and Methods We used high-dimensional, multiplexed immunohistochemistry to spatially, and cytometry by time-of-flight to quantitively characterize the cell constitution and interactions within the tumor microenvironment (TME) in 21 extreme long-term survivors (living over ten years since primary diagnosis or five years after recurrence) and 42 deeply matched short-term controls (living under 1.5 year) on a single cell level. For all tumors (epi-)genetic data was also collected. Results We identified a high level of both inter-and intrapatient heterogeneity defined by several distinct tumor niches, as well as described interactions within these niches and with the surrounding infiltrating immune cells of the TME. By linking patient characteristics with the heterogeneous immune composition we are building an immune stratification that can be linked to patient survival in GBM. Conclusion Generating an immune stratification for GBM will allow us to identify immune characteristics responsible for longer or even exceptional survival, as well as thoroughly identify tumor components that may serve as a potential target for personalized treatment strategies. Therefore, this study is also an essential initial step towards such clinical trials which alter the TME in a favorable way with a personalized modulation strategy.

A novel prognostic 7-methylguanosine signature reflects immune microenvironment and alternative splicing in glioma based on multi-omics analysis.

Glioma is the most common type of central nervous system tumor with increasing incidence. 7-methylguanosine (m7G) is one of the diverse RNA modifications that is known to regulate RNA metabolism and its dysregulation was associated with various cancers. However, the expression pattern of m7G regulators and their roles in regulating tumor immune microenvironments (TIMEs) as well as alternative splicing events (ASEs) in glioma has not been reported. In this study, we showed that m7G regulators displayed a close correlation with each other and most of them were differentially expressed between normal and glioma tissues. Two m7G signatures were then constructed to predict the overall survival of both GBM and LGG patients with moderate predictive performance. The risk score calculated from the regression coefficient and expression level of signature genes was proved to be an independent prognostic factor for patients with LGG, thus, a nomogram was established on the risk score and other independent clinical parameters to predict the survival probability of LGG patients. We also investigated the correlation of m7G signatures with TIMEs in terms of immune scores, expression levels of HLA and immune checkpoint genes, immune cell composition, and immune-related functions. While exploring the correlation between signature genes and the ASEs in glioma, we found that EIF4E1B was a key regulator and might play dual roles depending on glioma grade. By incorporating spatial transcriptomic data, we found a cluster of cells featured by high expression of PTN exhibited the highest m7G score and may communicate with adjacent cancer cells via SPP1 and PTN signaling pathways. In conclusion, our work brought novel insights into the roles of m7G modification in TIMEs and ASEs in glioma, suggesting that evaluation of m7G in glioma could predict prognosis. Moreover, our data suggested that blocking SPP1 and PTN pathways might be a strategy for combating glioma.

Abstract 2991: Survival reliability of Glioblastoma on CD38

Abstract Glioblastoma (GBM) is a fatal malignancy of the brain. Unfortunately, the current multimodality management approach does not lead to long term survival benefits. Several prognostic factors such as Blood Brain Barrier (BBB) &amp; Blood Tumor Barrier (BTB), Glioma Stem-like Cells (GSCs), an immunosuppressive tumor microenvironment (TME) and genetic heterogeneity promotes GBM progression. To improve treatment efficacy, these factors should be addressed in a rational manner. Our pre-clinical investigations indicate that maintenance of CD38 expression, a type II transmembrane glycoprotein, is vital to GBM survival. CD38 could be an ideal biologically relevant target as it is homogeneously expressed on tumor cells and its TME elements. Furthermore, accessibility of FDA approved anti-CD38 therapies, non-overlapping toxicities and potential to cross the BBB has granted approval of our phase I study using Radiation/Temozolomide and Immunotherapy with Daratumumab to Improve Antitumor Efficacy in newly diagnosed Glioblastoma (PRIDE) (NCT04922723). In brief, we performed Western blot (WB), RT-qPCR, and flow cytometry to examine CD38 expression on human GBM cell lines and microglia (LN229, T98G, DI-321, SA21 and HMC3) including CD38 transfected cells. We used trans well assays to analyze changes in CD38 expression in several environments. MTS viability assay was used to study the viability of Temozolomide on GBM cells. Basal expression of CD38 on GBM cells was assessed, showing that CD38 expression varies amongst cell lines and components of its TME. CD38 negative (flow sorted) cells regain CD38 expression around Day 4 after sorting, indicating that CD38 is crucial for GBM survival. We then sorted cells into CD38 negative and positive and performed trans well experiments with overexpressing and knock down cells, revealing that CD38 expression changes based upon surrounding cells in its environment. Cells transfected with shRNA against CD38 have a slower proliferative rate compared to wild-type and CD38 overexpressing cells. We also saw that LN229 Temozolomide (TMZ) resistant (LN229-TR) cells also have a slower proliferative rate and show an increase in CD38 expression. We also observed that CD133, SOX2 and Nestin are significantly over expressed in LN229-TR compared to LN229 CD38 WT, while sharing similar CD38 expression as GSCs and malignant GBM cells. IC50 of TMZ on CD38 overexpressing cells was significantly higher than CD38 WT GBM cells suggesting that CD38 expression contributes towards treatment resistance. All experiments were performed in triplicate and p values less than 0.05 were considered significant. Our preliminary data suggests that CD38 is regulating the proliferation rate as well as stem cell like features of GBM cells. Moreover, overexpression of CD38 is leading to TMZ resistance in pre-clinical models. Our ongoing in vitro and in vivo gene deletion experiments will further solidify the biological importance of CD38 in GBM. Citation Format: Leland Earp, Srishti Kala, Sonikpreet Aulakh. Survival reliability of Glioblastoma on CD38 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2991.

Abstract 5498: Plinabulin, a novel tubulin targeting agent, collapses the proinvasion tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival

Abstract Glioblastoma multiforme (GBM) is the most common adult primary brain malignancy. Aggressive, diffuse invasion is regulated by a dynamic cytoskeleton and significantly contributes to poor survival. Intraparenchymal tumor cell dissemination prevents complete surgical resection and compromises essential brain functions. Invasive cells are inherently less sensitive to traditional cytotoxic therapies. Recent investigation into targeted therapies has failed to address invasion/cytoskeleton based GBM therapy resistance. Furthermore, established GBM cell line modeling in 2 or 3 dimensions (3D) does not capture the genetic diversity or clinical relevance of patient derived primary (1') GBM cell lines, which may uncover/predict differential responses in tumors to novel therapies. Cytoskeleton remodeling and cell invasion in patient-derived primary GBM cells generates unique structures called tumor microtubes (TMs) not seen in established, cultured GBM cell lines. TMs are ultralong, pro-invasive, tubulin-enriched protrusions that promote chemo- and radioresistance in GBM patient derived primary cells. Using our extensive GBM cell line biobank derived from 1' and/or therapy resistant recurrent (2') tumors, we investigated the effects of a novel drug targeting the microtubule cytoskeleton. Plinabulin is a tubulin targeting agent that crosses the blood brain barrier and demonstrated anticancer efficacy in Phase III trial patients with non-small cell lung carcinoma. Plinabulin also has antiproliferative properties in 2D GBM cells. Here, we evaluate the efficacy of plinabulin treatment upon 3D neurosphere viability and invasion in 5 GBM 1' and 2' recurrent patient derived cell lines. Within 24h of matrigel embedding, 3D GBM patient neurospheres generate robust pro-invasive TMs. Plinabulin treatment (10-30 nM) at embedding followed by drug washout after 24h eliminated existing TMs and inhibited single cell invasion (&amp;gt;80% relative to controls) in both 1' and 2' GBM patient neurospheres. Cell survival was significantly reduced in plinabulin treated neurospheres (&amp;gt;30 nM). Plinabulin efficacy was evaluated in a clinically relevant therapeutic ITR model: GBM patient neurospheres invaded (I) for 48h to establish TM networks; were treated (T) with 1-50 nM plinabulin for 24h prior to drug washout; and recovered (R) for 96h. In 1', 2', or matched 1' and 2' cell line pairs from the same patient, 10-40 nM plinabulin sustained invasion inhibition (&amp;gt;90%) through 96h post washout and completely blocked TM extension relative to controls, consistent with established clinical concentrations of plinabulin. This study reveals plinabulin as a potent GBM therapeutic targeting pro-invasion TM networks and survival in 1' and therapy resistant 2' GBM patient cell lines. Citation Format: Kathryn M. Eisenmann, Kenneth Lloyd, Krista M. Pettee, Kathryn N. Becker, Jason Schroeder, Kevin Reinard, James R. Tonra, Ramon Mohanlal, Lan Huang. Plinabulin, a novel tubulin targeting agent, collapses the proinvasion tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5498.

Abstract 101: JAK-independent STAT5 signaling as a node of therapeutic vulnerability in glioblastoma

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults, comprises all WHO Grade IV gliomas of astrocytic origin. Despite extensive characterization of the molecular and genetic features of GBM, there remains a paucity of viable treatment options, and this disease confers a dismal 5% 5-year survival rate. Genomic amplification of epidermal growth factor receptor (EGFR) occurs in 40-60% of primary GBM. Half of all EGFR-amplified cases of GBM also harbor EGFRvIII, a constitutively active, truncated variant of EGFR. EGFR and EGFRvIII transduce oncogenic signals via cytoplasmic mediators, including the JAK/STAT signal transduction pathway. The oncogenic role of STAT3 has been well-studied in CNS malignancies; STAT3 has been found to induce the expression of target genes involved in the proliferative and invasive phenotypes of GBM. However, little is known about the mechanism and role of STAT5 activation in GBM progression. Microenvironmental cues and EGFRvIII signaling, lost in adherent cell culture, are required for STAT5 activation, making STAT5 difficult to study by conventional cell culture methods. Our recent data from patient-derived xenograft (PDX) cultures demonstrates that activation of STAT5 is important in GBM cell invasion and survival. Further, STAT5 activation is present predominantly at the invasive periphery of the tumor. In this study, we seek to characterize the mechanism and role of STAT5 activation downstream of EGFRvIII in GBM. RNA sequencing of PDX tumors expressing stable shRNA knockdown of STAT3 or STAT5 indicate both redundant and distinct transcriptional changes induced by STAT3 and STAT5 in GBM. We also find that while STAT3 activation is dependent upon JAK1/2, STAT5 activation is JAK-independent downstream of EGFRvIII in GBM. Depletion of JAK1/2 expression by siRNA oligonucleotides and pharmacologic inhibition of JAK1/2 suppressed STAT3 activation but not STAT5. In fact, targeting JAK2 with kinase inhibitor WP1066 suppressed STAT3 activation, but increased activation of STAT5 in GBM cells expressing EGFR/EGFRvIII. Inhibition of Src family kinases (SFK), a family of upstream signaling effectors of the JAK/STAT pathway, attenuates both STAT3 and STAT5 activation. These results suggest that activation of STAT5 may provide a therapeutic escape mechanism to JAK-STAT3 inhibition in GBM, necessitating dual STAT3 and STAT5 inhibition to successfully inhibit the distinct oncogenic signals transmitted by STAT3 and STAT5 to promote GBM cell survival and resistance. Citation Format: Mylan Blomquist, Serdar Tuncali, Christopher Sereduk, Jean Kloss, Joseph Loftus, Nhan Tran. JAK-independent STAT5 signaling as a node of therapeutic vulnerability in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 101.

Molecular Docking Analysis of Bacoside A with Selected Signalling Factors Involved in Glioblastoma

Glioblastoma is the malignant tumor affecting the central nervous system. Despite the advancement in treatment modalities, presence of blood-brain barrier, recurrence after surgical removal, resistance to radiotherapy and chemotherapy remain the major obstacles for long term survival of the patients. In this context, finding suitable therapeutics which have anticancer potential and are nontoxic might be useful to improve the overall survival of GBM. Plant products are safer, nontoxic and cheaper when compared to the chemotherapeutic drugs in trend which are expensive and highly toxic, owing to their systemic effects. Bacoside A (BA) is one such plant constituent isolated from Bacopa monnieri which offers neuroprotection and possesses anticancer potential. The present investigation elucidates the specific interaction of BA with various cell surface receptors, signal transduction proteins, effector proteins and transcription factors involved in glioblastoma signalling such as EGFR/Ras/Raf/MAPK pathway, Notch signalling and Wnt-beta catenin signalling through molecular docking studies. The interaction between BA and the target proteins of glioblastoma were analysed through the Glide module (Version 6.5) of Schrodinger Suite (2015) software. According to the results of molecular docking, jagged-1 ligands interact with BA with stronger affinity and Frizzled receptors interact with least affinity in terms of glide score. The results indicate that BA interacts well with the polar amino acids such as Asn, Trp, Arg, Ser, Thr, Tyr, Gln, Asp, Lys and Glu. The ligand also showed interactions with specific hydrophobic amino acids such as Val, Ala and Leu in all the protein targets studied. The in vitro cytotoxicity studies reveal the cytotoxic potential of BA on the U87MG glioblastoma cell line. This study warrants further investigation to elucidate modulations on cell signalling pathways resulting from the specific BA-target interactions in glioblastoma.