GB Patients Research Articles

SelK promotes glioblastoma cell proliferation by inhibiting β-TrCP1 mediated ubiquitin-dependent degradation of CDK4

BackgroundGlioblastoma (GB) is recognized as one of the most aggressive brain tumors, with a median survival of 14.6 months. However, there are still some patients whose survival time was greater than 3 years, and the biological reasons behind this clinical phenomenon arouse our research interests. By conducting proteomic analysis on tumor tissues obtained from GB patients who survived over 3 years compared to those who survived less than 1 year, we identified a significant upregulation of SelK in patients with shorter survival times. Therefore, we hypothesized that SelK may be an important indicator related to the occurrence and progression of GBM.MethodsProteomics and immunohistochemistry from GB patients were analyzed to investigate the correlation between SelK and clinical prognosis. Cellular phenotypes were evaluated by cell cycle analysis, cell viability assays, and xenograft models. Immunoblots and co-immunoprecipitation were conducted to verify SelK-mediated ubiquitin-dependent degradation of CDK4.ResultsSelK was found to be significantly upregulated in GB samples from short-term survivors (≤ 1 year) compared to those from long-term survivors (≥ 3 years), and its expression levels were negatively correlated with clinical prognosis. Knocking down of SelK expression reduced GB cell viability, induced G0/G1 phase arrest, and impaired the growth of transplanted glioma cells in nude mice. Down-regulation of SelK-induced ER stress leads to a reduction in the expression of SKP2 and an up-regulation of β-TrCP1 expression. Up-regulation of β-TrCP1, thereby accelerating the ubiquitin-dependent degradation of CDK4 and ultimately inhibiting the malignant proliferation of the GB cells.ConclusionThis study discovered a significant increase in SelK expression in GB patients with poor prognosis, revealing a negative correlation between SelK expression and patient outcomes. Further mechanistic investigations revealed that SelK enhances the proliferation of GB cells by targeting the endoplasmic reticulum stress/SKP2/β-TrCP1/CDK4 axis.

Clinical impact of IDH1 mutations and MGMT methylation in adult glioblastoma

BackgroundImpact of Isocitrate dehydrogenase1 (IDH1) and O6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma (GBM) have been of great interest due to their implications in prediction of prognosis of several types of cancer. It was aimed to investigate the clinical role of IDH1 mutation and MGMT methylation pattern among GBM patients versus non-neuro-oncological diseases (NND) patients and their impact on survival criteria.MethodsFormalin-fixed paraffin-embedded (FFPE) tissue sections of 58 GBM and 20 non-neuro-oncological diseases patients were recruited and IDH1 mutation and MGMT methylation was detected using Cast-PCR technology and Methyl II quantitative PCR approach, respectively. Results were assessed with other clinicopathological criteria and survival patterns.ResultsIDH1 mutation was detected among 15 GBM cases (15/58) and it was not reported among NND (P = 0.011). Receiver operating characteristic (ROC) curve was plotted to discriminate between MGMT methylation among studied groups. Patients with MGMT methylation ≥ 66% were reported as high methylation, which was recorded significantly in 51.7% and 100% of GBM cases and NND, respectively. Both showed significant difference with performance status, while MGMT methylation was significantly related with tumor size and tumor location. IDH1 mutation and MGMT methylation reported significant increase with GB patients revealed complete response to treatment. Survival pattern was better for IDH1 mutation and MGMT high methylation as compared to IDH1 wild type or MGMT low–moderate methylation, respectively, and favorable survival was detected when both were combined than using either of them alone.ConclusionDetection of IDH1 mutation and MGMT methylation among GB patients could aid in prediction of their response to treatment and their survival patterns, and their combination is better than using any of them alone.

Abstract 788: Leveraging multi omics approach to examine the potential role of causative viruses in glioblastoma

Abstract Glioblastoma (GB), a grade IV brain cancer, has the lowest survival rate. Despite aggressive and targeted therapies, patients remain to have the worst prognosis. More than 16% of cancer incidence worldwide has been attributed to infectious agents, raising uncertainties about the role of infections in the development of brain cancer. While numerous studies have suggested potential viral involvement in GB pathogenesis, a common limitation is the reliance on a singular technology, such as DNA/RNA sequencing or immunohistochemistry, within each study. This approach may restrict the comprehensive exploration of viral contributions, as different technologies offer distinct insights into the complex mechanisms underlying GB development. Utilizing a combination of techniques could provide a more holistic understanding, enhancing the robustness of findings and potentially uncovering nuanced aspects of viral influence that a single technology might overlook. To clarify the role of viruses in GB pathology, our study employs a comprehensive approach integrating three distinct research methods. We screened 178 publicly accessible raw mass spectrometry (MS) data of GB tumors from three independent publications (PMID: 31331834, 31154438, 36720864) by employing our internally developed data identification pipeline. The pipeline transforms raw MS data for downstream analysis, conducts database searches against the latest viral protein databases and undergoes peptide validation for enhanced robustness. Subsequent analysis filters duplicate hits and consolidates nested peptides. The final protein list includes only those identified with two or more peptides of over eight amino acids. The pipeline concludes by inferring and listing the corresponding viruses, ensuring a comprehensive and refined approach to the identification and analysis of viral proteins in the context of MS data. To enhance this dataset, we incorporated whole-genome metagenomics and MS-based proteomics, examining a preliminary cohort of15 GB tumor tissues. This approach explored a wide range of viral encoded proteins. Our comprehensive analysis, integrating bioinformatics, genomics, and proteomics, identified multiple herpesvirus species across our cohorts. This potential identification of viruses within GB has the capacity to redefine the stratification of tumor types in GB patients, signaling a significant development in our understanding of the disease. Citation Format: Bavani Gunasegaran, Aziz Abdullah A Alnakli, Gilles J. Guillemin4, Seong Beom Ahn, Benjamin Heng. Leveraging multi omics approach to examine the potential role of causative viruses in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 788.

Abstract 4031: P-selectin as an emerging target for the treatment of primary and secondary brain tumors

Abstract Glioblastoma (GB) is an aggressive type of brain cancer with a high mortality rate. It is a highly angiogenic tumor exhibiting an extremely invasive nature. As such, its brain microenvironment plays a crucial role in its progression. Microglia are the brain resident immune cells that have been shown to facilitate GB cell invasion and immune suppression. The mechanism by which GB cells alter microglia behavior is yet to be fully understood. One proposed mechanism involves adhesion molecules such as the Selectins family of proteins which are expressed on the surface of endothelial and immune cells and are involved in immune modulation and cancer immunity. We have previously shown that one member of the Selectin family, P-Selectin (SELP), is expressed by GB cells. Here, we investigated the functional role of SELP in GB-microglia interactions. First, we found that microglia cells facilitate the expression and secretion of SELP by GB cells, and that GB cells facilitate the expression of P-Selectin ligand-1 (PSGL-1) by Glioma-associated microglia/macrophages (GAMs). We then showed that SELP mediates GAMs-enhanced GB invasion and proliferation in our unique 3D-bioprinted ex vitro models and has a role in GAMs activation state. These findings were validated in vivo, showing that inhibition or downregulation of SELP leads to reduced tumor growth, increased overall survival, and improved immune response. Single-cell RNA-seq analysis of the tumors revealed an increase in pro-inflammatory GAMs signature, reduction in cancer cell tumorigenesis potential, and improved T cell activation. Thus, combining SELP inhibition with other immune checkpoint inhibitors, such as anti-PD-1, may have a synergistic effect by harnessing both the innate and the adaptive immune systems against the tumor. Furthermore, we found SELP/PSGL-1 axis to be involved in the progression of brain metastasis originating from melanoma, and breast, and lung cancer. Thus, we have begun an investigator-initiated clinical trial, testing the efficacy of the anti-SELP antibody, Crizanlizumab, alone or in combination with anti-PD-1 antibody, Nivolumab, for GB and melanoma brain metastasis patients (NCT05909618). Crizanlizumab, approved by the FDA and EMA for sickle cell pain crisis (VOC, Vaso-Occlusive Crisis) of sickle-cell anemia patients, was proven to be safe for human use, and efficient in inhibiting SELP function. As such, it has the potential to reduce tumor burden and improve patient outcomes. This work can improve our understanding of GAMs function, which may pave the way for new and effective treatments for primary and secondary brain tumors. Citation Format: Eilam Yeini, Ronnie Frommer-Shapira, Alisa Talianski, Paula Ofek, Sabina Pozzi, Nitzan Albeck, Dikla Ben-Shushan, Galia Tiram, Sapir Golan, Ron Kleiner, Ron Sheinin, Shlomit Reich-Zeliger, Rachel Grossman, Zvi Ram, Henry Brem, Thomas M. Hyde, Prerna Magod, Dinorah Friedmann-Morvinski, Asaf Madi, Ronit Satchi-Fainaro. P-selectin as an emerging target for the treatment of primary and secondary brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4031.

Deciphering glycosylation-driven prognostic insights and therapeutic prospects in glioblastoma through a comprehensive regulatory model.

The oncogenesis and development of glioblastoma multiforme have been linked to glycosylation modifications, which are common post-translational protein modifications. Abnormal glycosyltransferase development leads to irregular glycosylation patterns, which hold clinical significance for GB prognosis. By utilizing both single-cell and bulk data, we developed a scoring system to assess glycosylation levels in GB. Moreover, a glycosylation-based signature was created to predict GB outcomes and therapy responsiveness. The study led to the development of an glyco-model incorporating nine key genes. This risk assessment tool effectively stratified GB patients into two distinct groups. Extensive validation through ROC analysis, RMST, and Kaplan-Meier (KM) survival analysis emphasized the model's robust predictive capabilities. Additionally, a nomogram was constructed to predict survival rates at specific time intervals. The research revealed substantial disparities in immune cell infiltration between low-risk and high-risk groups, characterized by differences in immune cell abundance and elevated immune scores. Notably, the glyco-model predicted diverse responses to immune checkpoint inhibitors and drug therapies, with high-risk groups exhibiting a preference for immune checkpoint inhibitors and demonstrated superior responses to drug treatments. Furthermore, the study identified two potential drug targets and utilized Connectivity Map analysis to pinpoint promising therapeutic agents. Clofarabine and YM155 were identified as potent candidates for the treatment of high-risk GB. Our well-crafted glyco-model effectively discriminates patients by calculating the risk score, accurately predicting GB outcomes, and significantly enhancing prognostic assessment while identifying novel immunotherapeutic and chemotherapeutic strategies for GB treatment.

P11.30.A LONG-TERM SURVIVING GLIOBLASTOMA. CLINICAL, MOLECULAR AND SURVIVAL ANALYSES

Abstract BACKGROUND Glioblastoma (GB) has a poor prognosis with median overall survival of 12-14 months. According to literature, patients surviving more than two years has progressively increased since 2005 and represent approximately 18% of this population, being considered long-term survivors (LTS). The aim of this study is to retrospectively analyze GB LTS treated in our Neurooncology Unit. MATERIAL AND METHODS From December 2009 to March 2021, 49 GB LTS patients were identified in our oncology data base, representing 21,7% of GB patients electronically registered during the same period. Patients were diagnosed and treated for GB according to the current WHO CNS classification at the time of diagnosis. Clinical and molecular characteristics as well as treatment and survival data were analyzed. Classic prognostic factors were analyzed including contact with the subventricular zone (SVZ) which has been identified as prognostic factor in some previous studies. RESULTS Median age at diagnosis was 57 years (range: 30-75 years). Median overall survival (OS) and median time to first progression were 43.6 months and 20.7 months respectively, with 9 patients alive and 6 patients without recurrence at the time of analyses. Rate of patients surviving more than 3, 4, 5 and 7 years were 55,1%, 20,4%, 12,24% and 6,12% respectively. Patients received a median of 2 rescue treatments after first relapse (range: 0-6 lines), including systemic therapies, surgery and re-irradiation. Rates for gross total resection, subtotal resection and biopsy were 32,6%, 47% and 20,4% respectively. pMGMT methylated patients and lesions without SVZ contact rates were 73,5% and 42,8% respectively. IDH analyses was available for 61.22% of our GB LTS patients (90% were IDH wild type and 10% mutant IDH, which according to WHO CNS 2021 classification would actually be defined as Astrocytoma grade 4). No statistical differences for OS or time to first progression were found according to sex, age (<55 years versus ≥55 years), pMGMT methylation status; Karnofsky performance status (≤70% versus >70%) and contact or not with the SVZ. CONCLUSION Classical prognostic factors did not show statistical differences for OS and time to first recurrence in our GB LTS sample, which could be explained by the small number of patients in the sample and the absence of statistically comparable differentiated groups for some of the variables. Larger and prospective analyses of GB LTS are needed. Molecular analyses with next generation sequencing (NGS) technics and radiomics analyses could apport new information predicting GB LTS.

Abstract 5474: Combined use of MRI and TAM analysis in GB monitoring

Abstract Glioblastoma (GB) is the most frequent and aggressive primary brain tumor. It is a highly infiltrating tumor. Complete eradication by surgery is difficult. The probability of survival beyond two years is very low. Magnetic resonance imaging (MRI) is a routine procedure for diagnosing gliomas. However, morphological MRI often fails to identify and quantify the presence of infiltration in peritumoral non-enhanced areas, resulting in inaccuracy in the assessment of invasive margins. Up to 50% of the patients show pseudo-progression. Morphological MRI fails to distinguish the marked enhancement in the tumor bed caused by radionecrosis from actual tumor progression. Diagnosis of GB recurrence through MR morphological sequences has a sensitivity and specificity of about 68% and 77%. Such percentages rise thanks to the use of non-morphological sequences (PWI, DWI, MRS), which are used in routine tumor protocols only in large centers and interpreted by expert neuroradiologists. Biomarkers in conjunction with MRI may help improve the classification, prognosis, and treatment choice. GB is highly infiltrated with monocytic cells, tumor-associated macrophages (TAMs), that invade the peripheral blood. Our group has recently identified a splice isoform of FK506-binding protein 51 (FKBP51) as a reliable TAM marker. We found an expansion in circulating CD163/FKBP51s monocytes in a cohort of GB patients. In patients with an MRI diagnosis of complete surgical tumor removal, these monocytes dramatically decreased. In patients with an MRI diagnosis of residual tumor, most of the peripheral CD163 monocytes that in the preoperative stage were FKBP51s- had turned into FKBP51s+. CD163/FKBP51s+ monocytes helped to distinguish between radionecrosis and actual tumor progression. These results suggest that circulating CD163/FKBP51s+ monocytes are associated with GB and can be helpful in monitoring GB patients. Citation Format: Carolina Giordano, Giuseppe Maria Della Pepa, Simona Romano, Laura Marrone, Carlo Maria Donzelli, Maria Fiammetta Romano, Simona Gaudino. Combined use of MRI and TAM analysis in GB monitoring. [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 5474.

MRI-derived radiomics to guide post-operative management of glioblastoma: Implication for personalized radiation treatment volume delineation.

The glioblastoma's bad prognosis is primarily due to intra-tumor heterogeneity, demonstrated from several studies that collected molecular biology, cytogenetic data and more recently radiomic features for a better prognostic stratification. The GLIFA project (GLIoblastoma Feature Analysis) is a multicentric project planned to investigate the role of radiomic analysis in GB management, to verify if radiomic features in the tissue around the resection cavity may guide the radiation target volume delineation. We retrospectively analyze from three centers radiomic features extracted from 90 patients with total or near total resection, who completed the standard adjuvant treatment and for whom we had post-operative images available for features extraction. The Manual segmentation was performed on post gadolinium T1w MRI sequence by 2 radiation oncologists and reviewed by a neuroradiologist, both with at least 10 years of experience. The Regions of interest (ROI) considered for the analysis were: the surgical cavity ± post-surgical residual mass (CTV_cavity); the CTV a margin of 1.5 cm added to CTV_cavity and the volume resulting from subtracting the CTV_cavity from the CTV was defined as CTV_Ring. Radiomic analysis and modeling were conducted in RStudio. Z-score normalization was applied to each radiomic feature. A radiomic model was generated using features extracted from the Ring to perform a binary classification and predict the PFS at 6 months. A 3-fold cross-validation repeated five times was implemented for internal validation of the model. Two-hundred and seventy ROIs were contoured. The proposed radiomic model was given by the best fitting logistic regression model, and included the following 3 features: F_cm_merged.contrast, F_cm_merged.info.corr.2, F_rlm_merged.rlnu. A good agreement between model predicted probabilities and observed outcome probabilities was obtained (p-value of 0.49 by Hosmer and Lemeshow statistical test). The ROC curve of the model reported an AUC of 0.78 (95% CI: 0.68-0.88). This is the first hypothesis-generating study which applies a radiomic analysis focusing on healthy tissue ring around the surgical cavity on post-operative MRI. This study provides a preliminary model for a decision support tool for a customization of the radiation target volume in GB patients in order to achieve a margin reduction strategy.

QLTI-02. AUTOMATED INTRAOPERATIVE RESECTION TECHNOLOGY GENERATES INCREASED TISSUE YIELD AND IMPROVED BIOLOGICAL PRESERVATION OF BRAIN TUMOR SPECIMENS FOR NEURO-ONCOLOGY RESEARCH

Abstract Glioblastoma (GB) is an aggressive tumor showing extensive intertumoral and intratumoral heterogeneity. While preserving as much surrounding normal brain tissue as possible, neurosurgeons must aim to harvest maximal tumor tissue from a variety of tumor locations in an effort to capture heterogenic samples in high volume for molecular and pathologic diagnosis and translational research. A key challenge is the ability to consistently procure high-quality biologically active specimens. In this investigation, we implemented an automated intraoperative system to eliminate inconsistencies in the methodology of tissue collection, handling, and biological preservation immediately in the OR suite to establish a repeatable, standardized practice for obtaining high-quality tissue samples without the need for additional staff. Through this process, we were able to characterize matched specimens from GB patients or GB tumors from corresponding GB-allograft mice and compare the quality of traditional handling and collection processes of intraoperative tissue used in most neurosurgical operating rooms versus an automated resection, collection, and biological preservation system (APS) that captures, preserves, and biologically maintains tissue in a prescribed and controlled microenvironment. Matched specimens/or tissues were then processed in parallel at various time points and temperatures, evaluating viability, RNA and protein concentrations, and isolation of GB cell lines. We found that APS-derived GB slices stored in an APS modified medium remained viable and maintained high-quality RNA and protein concentration for up to 24 hours. Our results showed that primary GB cell cultures derived in this manner had improved growth over the widely used collection and preservation methods. Currently, we are continuing the investigation of collected samples in brain tumor animal models to further understand potential differences within the tumor region harvested and the cellular changes that occur over time. Our hope is this research will lead to new discoveries in the diagnosis and treatment of brain tumors.

Advances in Preclinical/Clinical Glioblastoma Treatment: Can Nanoparticles Be of Help?

Simple SummaryAs one of the most lethal human cancers, glioblastoma treatment is a real challenge because of several resistance mechanisms, including limited drug entry into the central nervous system through the blood–brain barrier and the vast heterogeneity of this family of tumors. In the development of precision medicine, various nanoconstructs are being proposed to cross the BBB, specifically target GB tumors, release the therapeutic cargo in a controlled manner, and reduce therapeutic resistance. This review summarizes the different families of nanoparticles and approaches followed so far pursuing these aims.Glioblastoma multiforme (GB) is the most aggressive and frequent primary malignant tumor in the central nervous system (CNS), with unsatisfactory and challenging treatment nowadays. Current standard of care includes surgical resection followed by chemotherapy and radiotherapy. However, these treatments do not much improve the overall survival of GB patients, which is still below two years (the 5-year survival rate is below 7%). Despite various approaches having been followed to increase the release of anticancer drugs into the brain, few of them demonstrated a significant success, as the blood brain barrier (BBB) still restricts its uptake, thus limiting the therapeutic options. Therefore, enormous efforts are being devoted to the development of novel nanomedicines with the ability to cross the BBB and specifically target the cancer cells. In this context, the use of nanoparticles represents a promising non-invasive route, allowing to evade BBB and reducing systemic concentration of drugs and, hence, side effects. In this review, we revise with a critical view the different families of nanoparticles and approaches followed so far with this aim.

Fatigue, cognition and inflammatory biomarkers changes in response to computer-based cognitive training in multiple sclerosis patients: A randomized controlled trial.

Fatigue, cognition problems and multiple sclerosis (MS) inflammatory processes became main quality of life indicators and points of clinical significance in MS practice. The purpose of the current study was to investigate the changes in primary fatigue level, degree of cognitive dysfunction and level of inflammatory biomarkers in response to computer-based cognitive training in patients with MS. A total of 40 remitting-relapse MS patients were divided into two groups, both groups were suffering cognitive decline, primary fatigue with elevated serum levels of the inflammatory biomarkers. Patients in the control group (GA) underwent conventional physical therapy program for MS including aerobic training, resistive training and a flexibility program in addition to placebo cognitive sessions, study group patients (GB) received an intensive computer-based cognitive program using the REHACOM software in addition to the same conventional physical therapy program as in GA. Significant decrease in the level of primary fatigue and in the serum levels of inflammatory biomarkers in GB patients compared to GA, as well as a remarkable improvement in the cognitive abilities in favor to the study group (GB) (p < 0.05). The addition of computer-based cognitive training in the rehabilitation program of MS patients is efficient in improving disease course via decreasing fatigue levels, enhancing cognitive abilities and reducing level of inflammatory biomarkers.

Metabolic-imaging of human glioblastoma live tumors: A new precision-medicine approach to predict tumor treatment response early.

BackgroundGlioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) treatment, and radiotherapy remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical approaches that do not fully respect the complexity of GB cell biology and fail to test efficiently anti-cancer treatments. Therefore, better treatment screening approaches are needed. In this study, we have developed a novel functional precision medicine approach to test the response to anticancer treatments in organoids derived from the resected tumors of glioblastoma patients.MethodsGB organoids were grown for a short period of time to prevent any genetic and morphological evolution and divergence from the tumor of origin. We chose metabolic imaging by NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to predict early and non-invasively ex-vivo anti-cancer treatment responses of GB organoids. TMZ was used as the benchmark drug to validate the approach. Whole-transcriptome and whole-exome analyses were performed to characterize tumor cases stratification.ResultsOur functional precision medicine approach was completed within one week after surgery and two groups of TMZ Responder and Non-Responder tumors were identified. FLIM-based metabolic tumor stratification was well reflected at the molecular level, confirming the validity of our approach, highlighting also new target genes associated with TMZ treatment and identifying a new 17-gene molecular signature associated with survival. The number of MGMT gene promoter methylated tumors was higher in the responsive group, as expected, however, some non-methylated tumor cases turned out to be nevertheless responsive to TMZ, suggesting that our procedure could be synergistic with the classical MGMT methylation biomarker.ConclusionsFor the first time, FLIM-based metabolic imaging was used on live glioblastoma organoids. Unlike other approaches, ex-vivo patient-tailored drug response is performed at an early stage of tumor culturing with no animal involvement and with minimal tampering with the original tumor cytoarchitecture. This functional precision medicine approach can be exploited in a range of clinical and laboratory settings to improve the clinical management of GB patients and implemented on other cancers as well.

P11.36.A Could a transcriptome profile predict local control for glioblastoma?

Abstract Background Glioblastoma (GB) is the most frequent and aggressive primary brain tumor in adults. The standard of care is surgery followed by radiotherapy in combination with temozolomide. GB molecular subtypes (Classical [CL], Mesenchimal [MES] and Proneural [PN]) have been correlated with prognosis. In the preclinical setting, CL and MES glioma stem-like initiating cells have also been shown to be more radioresistant than PN subtype. If confirmed in patients, the molecular subtype could predict the specific benefit of adjuvant treatment. The aim of this work is therefore to investigate whether GB molecular profiles are associated with treatment resistance in vivo. Material and Methods We analyzed the spatiotemporal pattern of recurrence (progression-free survival [PFS] and local control) in 37 GB patients treated with adjuvant radiotherapy (30 x 2 Gy, following EORTC contouring guidelines) plus temozolomide in the context of a multi-centric study. Relapse was defined following the RANO criteria (gadolinium enhanced area on MRI T1 sequence) and classified into 3 groups: in-field relapse (Dmean &amp;gt; 57 Gy), out-of-field relapse (Dmean &amp;lt; 48 Gy) and not-defined relapse (no follow-up MR data or marginal relapse). We used three different classification algorithms (SVM, K-Nearest Neighbor and single sample gene set enrichment analysis) for the transcriptomic classification of tumor samples, and we only assigned a molecular profile when all three classifications agreed, otherwise we called it Mixed profile. Patients with a not-defined spatial pattern of recurrence were included in the PFS analysis, but excluded from the local control analysis. Results The transcriptomic classification resulted in 14 CL, 1 MES, 9 PN and 13 Mixed. Median PFS after radiotherapy was 6 months, with a mean follow-up of 11 months. No differences in PFS were found between CL, PN and Mixed groups (the single MES case was excluded). Local control was analyzed in 24 patients (10 CL, 4 PN, 10 Mixed). Non- statistically significant differences were found between PN/Mixed and CL subtypes, with a median in-field relapse-free survival of 6.9 months (PN), 6.3 months (Mixed) and 4.7 months (CL). Conclusion Although there is no difference in PFS among GB molecular subtypes, these results suggest that the Classical subtypes seem to relapse faster inside the radiotherapy field. This would suggest a higher radioresistance compared to Proneural and Mixed subtypes, in agreement with preclinical data. Due to the low number of patients these differences are statistically non-significant, but they provide the rationale for further investigation in a larger patient cohort. Grant: Marató -TV3

BSCI-07 ACETYL-AMANTADINE AS A DIAGNOSTIC BIOMARKER IN PATIENTS WITH GLIOBLASTOMA

AIMGlioblastoma (GB) is the most common malignant primary brain tumor in adults, with a prognosis as poor as 12-15 months with standard treatment. Spermidine/spermine N1-acetyltransferase (SAT1) is a rate limiting enzyme in polyamine metabolism and has been reported to be upregulated in various cancers, including GB. Amantadine is a Health Canada approved drug that is acetylated by SAT1. We established a clinical trial in GB patients to determine if plasma and urine acetyl amantadine (Ac-Am) can be used to measure SAT1 activity and whether levels correlate with their tumor burden.METHODSA clinical trial was established that is currently active and recruiting patients with GB who receive care at CancerCare Manitoba. A total of n=8 participants have been recruited thus far. Participants’ blood and urine were collected two hours after ingesting amantadine (200 mg). Levels of serum and urine Ac-Am were measured using liquid chromatography-tandem mass spectrometry. Acetyl-amantadine levels were correlated with tumour bidimensional diameter and volume measured on MRI. In addition, expression of SAT1 was examined in various cultured GB cells (both cell lines and patient-derived cells) and correlated with Ac-Am.RESULTSPreliminary results indicate that the levels of plasma Ac-Am in study participants positively correlate with their initial tumor burden (r = 0.41). While transient increases in plasma and urine Ac-Am above baseline levels were observed, the clinical significance of these findings is undergoing further analysis. SAT1 was detected in all the GB tumor cells examined. The production of Ac-Am in cultured GB cells varied as a function of SAT1 expression.SIGNIFICANCEA diagnostic biomarker, such as Ac-Am, that could effectively and reliably detect tumor progression and recurrence would be an invaluable adjunct to MRI imaging and could significantly impact the timing of appropriate treatment and reduce patients’ morbidity and mortality.

Association between microRNAs 10b/21/34a and acute toxicity in glioblastoma patients treated with radiotherapy and temozolomide

A personalized approach to chemoradiation is important in reducing its potential side effects and identifying a group of patients prone to toxicity. MicroRNAs have been shown to have a predictive potential for radiotoxicity. The goal of the study was to test if levels of miRNA in peripheral blood mononuclear cells of glioblastoma patients are associated with toxicity and to identify the peak time point for toxicity. MicroRNA-10b/21/34a levels were measured in 43 patients with and without toxicity, at baseline, at the 15th, and at the 30th fraction by Real-Time quantitative Polymerase Chain Reaction. MicroRNA-10b/21 levels increased with toxicity grade (p = 0.014; p = 0.013); miR-21/34a levels were significantly different between patients with and without toxicity at the 15th fraction (p = 0.030; p = 0.045), while miR-34a levels significantly changed during treatment (p < 0.001). All three miRNAs showed a significantly high positive correlation with one another. MiR-34a might be considered as a predictive factor for toxicity due to its changes during treatment, and differences between the groups with and without toxicity; miR-10b might be used to predict toxicity; miR-10b/21 might be used for predicting the grade of toxicity in GB patients.

Autophagy-based unconventional secretion of HMGB1 in glioblastoma promotes chemosensitivity to temozolomide through macrophage M1-like polarization

BackgroundGlioblastoma (GB) is the most common and highly malignant brain tumor characterized by aggressive growth and resistance to alkylating chemotherapy. Autophagy induction is one of the hallmark effects of anti-GB therapies with temozolomide (TMZ). However, the non-classical form of autophagy, autophagy-based unconventional secretion, also called secretory autophagy and its role in regulating the sensitivity of GB to TMZ remains unclear. There is an urgent need to illuminate the mechanism and to develop novel therapeutic targets for GB.MethodsCancer genome databases and paired-GB patient samples with or without TMZ treatment were used to assess the relationship between HMGB1 mRNA levels and overall patient survival. The relationship between HMGB1 protein level and TMZ sensitivity was measured by immunohistochemistry, ELISA, Western blot and qRT-PCR. GB cells were engineered to express a chimeric autophagic flux reporter protein consisting of mCherry, GFP and LC3B. The role of secretory autophagy in tumor microenvironment (TME) was analyzed by intracranial implantation of GL261 cells. Coimmunoprecipitation (Co-IP) and Western blotting were performed to test the RAGE-NFκB-NLRP3 inflammasome pathway.ResultsThe exocytosis of HMGB1 induced by TMZ in GB is dependent on the secretory autophagy. HMGB1 contributed to M1-like polarization of tumor associated macrophages (TAMs) and enhanced the sensitivity of GB cells to TMZ. Mechanistically, RAGE acted as a receptor for HMGB1 in TAMs and through RAGE-NFκB-NLRP3 inflammasome pathway, HMGB1 enhanced M1-like polarization of TAMs. Clinically, the elevated level of HMGB1 in sera may serve as a beneficial therapeutic-predictor for GB patients under TMZ treatment.ConclusionsWe demonstrated that enhanced secretory autophagy in GB facilitates M1-like polarization of TAMs to enhance TMZ sensitivity of GB cells. HMGB1 acts as a key regulator in the crosstalk between GB cells and tumor-suppressive M1-like TAMs in GB microenvironment and may be considered as an adjuvant for the chemotherapeutic agent TMZ.

Hypoxia: The Cornerstone of Glioblastoma.

Glioblastoma is the most aggressive form of brain tumor in adults and is characterized by the presence of hypervascularization and necrosis, both caused by a hypoxic microenvironment. In this review, we highlight that hypoxia-induced factor 1 (HIF-1), the main factor activated by hypoxia, is an important driver of tumor progression in GB patients. HIF-1α is a transcription factor regulated by the presence or absence of O2. The expression of HIF-1 has been related to high-grade gliomas and aggressive tumor behavior. HIF-1 promotes tumor progression via the activation of angiogenesis, immunosuppression, and metabolic reprogramming, promoting cell invasion and survival. Moreover, in GB, HIF-1 is not solely modulated by oxygen but also by oncogenic signaling pathways, such as MAPK/ERK, p53, and PI3K/PTEN. Therefore, the inhibition of the hypoxia pathway could represent an important treatment alternative in a disease with very few therapy options. Here, we review the roles of HIF-1 in GB progression and the inhibitors that have been studied thus far, with the aim of shedding light on this devastating disease.

Mechanically matching the rheological properties of brain tissue for drug-delivery in human glioblastoma models

Peptide functionalized hyaluronic acid (HACF) cross-linked by cucurbit[8]uril (CB[8]), a new class of drug-delivery reservoirs, is used to enable improved drug bioavailability for glioblastoma tumors in patient-derived xenograft (PDX) models. The mechanical and viscoelastic properties of native human and mouse tissues are measured over 8 h via oscillatory rheology under physiological conditions. Treatment with drug–loaded hydrogels allowed for a significant survival impact of 45 % (55.5–80.5 days). A relationship between the type of PDX tumor formed—a consequence of the heterogeneic nature of GB tumors—and changes in the initial survival is observed owing to greater local pressure from stiffer tumors. These biocompatible and tailorable materials warrant use as drug delivery reservoirs in PDX resection models, where the mechanical properties can be readily adjusted to match the stiffness of local tissue and thus have potential to improve the survival of GB patients.

The Role of Hypoxia in Glioblastoma Radiotherapy Resistance.

Simple SummaryGlioblastoma (GB) is the deadliest type of primary brain tumor. Following diagnosis the patient´s median survival is only 16 months. There are currently around 450 clinical trials focused on the development of more effective therapies for GB. Nevertheless, radiotherapy remains the most clinically relevant and effective treatment for this devastating disease. Unfortunately, radiotherapy resistance (radioresistance) is frequently observed in GB patients. As a consequence tumor regrowth (recurrence) occurs and eventually the patient succumbs to the disease. It is crucial to fully understand the mechanisms by which GB cells become resistant to radiation in order to improve the sensitivity of these cells to radiotherapy and develop novel strategies to overcome this issue. In this review, we examined how low tumor oxygenation (known as hypoxia) which is a main feature of GB contributes to radioresistance to better understand the implications of this tumor microenvironment in GB treatment and recurrence.Glioblastoma (GB) (grade IV astrocytoma) is the most malignant type of primary brain tumor with a 16 months median survival time following diagnosis. Despite increasing attention regarding the development of targeted therapies for GB that resulted in around 450 clinical trials currently undergoing, radiotherapy still remains the most clinically effective treatment for these patients. Nevertheless, radiotherapy resistance (radioresistance) is commonly observed in GB patients leading to tumor recurrence and eventually patient death. It is therefore essential to unravel the molecular mechanisms underpinning GB cell radioresistance in order to develop novel strategies and combinational therapies focused on enhancing tumor cell sensitivity to radiotherapy. In this review, we present a comprehensive examination of the current literature regarding the role of hypoxia (O2 partial pressure less than 10 mmHg), a main GB microenvironmental factor, in radioresistance with the ultimate goal of identifying potential molecular markers and therapeutic targets to overcome this issue in the future.

Angiopoietin-2 Combined with Radiochemotherapy Impedes Glioblastoma Recurrence by Acting in an Autocrine and Paracrine Manner: A Preclinical Study.

Simple SummaryGlioblastoma (GB) is a highly aggressive brain tumor characterized by poor prognosis and high rate of recurrence in response to conventional treatments consisting of tumor resection and radiochemotherapy (RCT). The reasons for this therapeutic failure are mainly due to the complexity of GB biology and its environment. GB progression is highly dependent on its vascularization and inflammatory status. Besides, evidence showed that RCT also induces vascular change and inflammation. In GB patients, Angiopoietin-2 (Ang2), biomarker of poor prognosis is a crucial angiogenic factor also involved in inflammation. Our aim was to clarify the role of Ang2 in RCT-induced changes in the GB environment. To this end, we generated Ang2-overexpressing GL261 cells and characterized tumor progression, as well as inflammation and vascularization, in response to RCT. We showed that Ang2 delays tumor recurrence and makes a lasting improvement in animal survival when combined with conventional RCT.(1) We wanted to assess the impact of Ang2 in RCT-induced changes in the environment of glioblastoma. (2) The effect of Ang2 overexpression in tumor cells was studied in the GL261 syngeneic immunocompetent model of GB in response to fractionated RCT. (3) We showed that RCT combined with Ang2 led to tumor clearance for the GL261-Ang2 group by acting on the tumor cells as well as on both vascular and immune compartments. (4) In vitro, Ang2 overexpression in GL261 cells exposed to RCT promoted senescence and induced robust genomic instability, leading to mitotic death. (5) Coculture experiments of GL261-Ang2 cells with RAW 264.7 cells resulted in a significant increase in macrophage migration, which was abrogated by the addition of soluble Tie2 receptor. (6) Together, these preclinical results showed that, combined with RCT, Ang2 acted in an autocrine manner by increasing GB cell senescence and in a paracrine manner by acting on the innate immune system while modulating the vascular tumor compartment. On this preclinical model, we found that an ectopic expression of Ang2 combined with RCT impedes tumor recurrence.