Glioblastoma Response Research Articles

Development of a T-cell activation-related module with predictive value for the prognosis and immune checkpoint blockade therapy response in glioblastoma.

BackgroundDespite the rise in the use of immune checkpoint blockade drugs (ICBs) in recent years, there are no ICB drugs that are currently approved or under large-scale clinical trials for glioblastoma (GBM). T-cells, which mainly mediate adaptive immunity, are an important part of the tumor immune microenvironment. The activation of T-cells in tumors plays a key role in evaluating the sensitivity of patients to immunotherapy. Therefore, we applied bioinformatics approaches to construct a T-cell activation related risk score to study the effect of the activation of T-cells on the prognosis and ICB response of patients with GBM.Materials and MethodsThis study collected TCGA, CGGA, and GSE16011 glioma cohorts, as well as the IMvigor210 immunotherapy dataset, with complete mRNA expression profiles and clinical information. GraphPad Prism 8 and R 3.6.3 were used for bioinformatics analysis and plotting.ResultsThe activation of T-cells in patients with GBM is characterized by obvious heterogeneity. We established a T-cell activation-related risk score based on five univariate Cox regression prognostic genes (CD276, IL15, SLC11A1, TNFSF4, and TREML2) in GBM. The risk score was an independent risk factor for poor prognosis. The overall survival time of patients in the high-risk group was significantly lower than in the low-risk group. Moreover, the high-risk score was accompanied by a stronger immune response and a more complex tumor immune microenvironment. “Hot tumors” were mainly enriched in the high-risk group, and high-risk group patients highly expressed inhibitory immune checkpoints (PD1, PD-L1, TIM3 etc.). By combining the risk and priming scores we obtained the immunotherapy score, which was shown to be a good evaluation index for sensitivity to GBM immunotherapy.ConclusionsAs an independent risk factor for poor prognosis, the T-cell activation-related risk score, combined with other clinical characteristics, could efficiently evaluate the survival of patients with GBM. The immunotherapy score obtained by combining the risk and priming scores could evaluate the ICB response of patients with GBM, providing treatment opportunities.

IMMU-16. CHARACTERISTICS OF EGFRVIII-DIRECTED CART CELL INFUSION PRODUCT ASSOCIATED WITH CLINICAL RESPONSE IN RECURRENT GLIOBLASTOMA

Abstract BACKGROUND A therapeutic approach for chimeric antigen receptor T (CART) cell therapy in glioblastoma is targeting the epidermal growth factor receptor variant III (EGFRvIII), present in approximately 30% of glioblastomas. While earlier research demonstrated that phenotypic and genotypic characteristics in patient T cells and CART product may predict therapeutic success for hematologic malignancies, no study has investigated such determinants for clinical response in glioblastoma. METHODS We analyzed apheresis and infusion products for patients in the first-in-human trial of EGFRvIII-directed CART for recurrent glioblastoma (NCT02209376, n=9). Inherent T cell phenotypes were characterized by flow cytometry assay using a panel of 27 markers. Clinical response was quantified via engraftment in peripheral circulation and time-on-trial (ToT). RESULTS Mean area under the curve (AUC) peripheral CART engraftment for the study period was 213 log10 copies/μg (SD=173) and mean ToT was 134 days (SD=173). For the CAR+/CD4+ population in the infusion product, PD1 positivity was positively associated with AUC engraftment (r=0.753, P=0.012) and ToT (r=0.800, P=0.010). On immune checkpoint inhibitor analysis, CTLA-4, TIM3, and LAG3 did not exhibit significant associations with AUC engraftment or ToT. For activation markers, PD1/GRZB (r=0.794, P=0.011) and PD1/HLA-DR (r=0.769, P=0.016) were directly associated with ToT. PD1/GRZB was also predictive of AUC engraftment (r=0.794, P=0.011). For the CAR+/CD8+ population, PD1 positivity correlated with elevated AUC engraftment (r=0.639, P=0.047). In the CAR-/CD4+ population, PD1 remained positively correlated with AUC engraftment (r=0.690, P=0.027) and ToT (r=0.726, P=0.027). No significant associations were observed for apheresis products. CONCLUSION PD1 in CART infusion products predicted peripheral engraftment and ToT in recurrent glioblastoma. Double-positive cells for PD1 and activation markers also displayed a positive correlation, suggesting PD1 highlights a population of activated CAR T cells. Further characterization of predictors of EGFRvIII-directed CART treatment efficacy may improve selection of patients and starting T cell populations for clinical expansion.

CTNI-45. RESPONSE EVALUATION OF AR-67 FROM A PHASE-2 RECURRENT GLIOBLASTOMA TRIAL BY ARTIFICIAL INTELLIGENCE ASSISTED TUMOR VOLUMETRIC ESTIMATION: COMPARISON WITH THE SUM OF THE PERPENDICULAR DIAMETERS PRODUCT

Abstract BACKGROUND Modified Radiographic Response Assessment in Neuro-Oncology (“mRANO”) criteria based on SPDP form the basis for assessing treatment response in Glioblastoma but are subject to sampling bias and difficulty in differentiating between pseudo- and true disease progression. Volumetric image analysis using AI may overcome these limitations of standard techniques and improve our ability to detect changes earlier and more accurately. METHODS Images from eight reGBM patients enrolled in a Phase-2 reGBM study of Vivacitas Oncology’s drug, AR-67, were re-assessed using IAG’s AI-assisted volumetric measurements. A median of five MRI time points from each patient were included. The mRANO response was determined by central reading and tumor volumetric measurement using IAG’s proprietary platform. Statistical significance was set at p< .0001. RESULTS Four patients showed responses, two patients showed stable disease, and two patients showed progressive disease. Tumor volume was correlated (r=0.97) with SPDP, but was driven by high coefficients in large lesions. Standard SPDP overestimated tumor size in larger tumors using the Bland-Altman analysis (mean difference: 829; 95% CI: 704 to -2362) leading to discrepancies in response rates. For example, the mean response rate based on IAG’s volumetric criteria was +22% (1.29) compared with +17% using SPDP (0.81). Eight out of 45 time-points also differed in the directionality of responses (e.g., increase vs. decrease) with SPDP underestimating the positive effects of AR-67 compared to AI analysis. CONCLUSIONS IAG’s AI-assisted tumor volumetric analysis is feasible for clinical trials and may be more sensitive for evaluating treatment-related response rates vs. SPDP methodology. This is particularly true for measuring large lesions, and may also allow for more accurately differentiating between pseudo- and true disease progression. The data included eight patients’ MRI images from a Phase-2 reGBM study, showing that five patients achieved the primary end-point of six months Progression-Free Survival, suggesting AR-67’s therapeutic potential.

CBIO-20. HIGH LEVELS OF TERT CONFER SENSITIVITY TO THE DNA HYPOMETHYLATING AGENT DECITABINE (DAC) IN GLIOMAS

Abstract BACKGROUND Decitabine (DAC)-incorporated DNA binds DNMT1 enzyme and subsequently triggers DNMT1 degradation. Previously, we showed that DAC can mediate the anti-tumor effect in a preclinical model of IDH-mutant gliomas. Here, we further investigate molecular determinants of response to DAC in gliomas. METHODS DAC response was assessed by soft agar anchorage independent growth assays and cell proliferation measurements. Patient-derived IDH-mutant chromosome 1p/19q codeleted (codel) and non-codel glioma lines upon vehicle and DAC treatment were used for RNA sequencing and Gene Set Enrichment Analysis (GSEA). RESULTS We found that DAC treatment is effective in high TERT-expressing gliomas including IDH-mutant and IDH-wildtype glioma lines. In contrast, pharmacological inhibition of TERT reduces DAC response in glioma lines. Interestingly, transcriptomic profiling showed that DAC reduces the expression of TERT, along with increased CDKN1A/p21 expression. We experimentally validated that TERT expression depends on CDKN1A/p21. Furthermore, p53 is required for DAC-mediated CDKN1A/p21 induction. Importantly, DAC-mediated proliferation defects in TERT-proficient glioma cells are abolished by DNMT1 knockdown, indicative of an expected DAC mechanism. CONCLUSIONS DAC could elicit the pronounced anti-tumor response in IDH-mutant codel oligodendroglioma and IDH-wildtype glioblastoma with TERT activating mutations.

EXTH-21. MECHANISMS OF SYNERGISTIC GROWTH SUPPRESSION BY RADIOTHERAPY AND C-MET INHIBITION IN EXPERIMENTAL GLIOBLASTOMA

Abstract Glioblastoma remains to be one of the most lethal solid cancers and novel therapies are urgently needed. There is increasing interest in the role of the HGF/MET pathway in the response of glioblastoma to radiotherapy. c-MET-mediated radioresistance may be partially induced via proinvasive and DNA damageresponse pathways and HGF may be involved in the regulation of immune responses. Here, we explored the role of the c-MET pathway in response to radiotherapy and investigated potential modes of action that mediate synergistic effects of MET pathway inhibition and irradiation in syngeneic murine glioma models in vitro and in vivo. Murine glioma cells express HGF and c-MET and respond with c-MET phosphorylation upon exposure to exogenous HGF. In vitro, glioma cell viability and proliferation are not affected by pharmacological or genetic c-MET pathway interference, and the c-MET inhibitor tepotinib fails to sensitize glioma cells to irradiation. Conversely, in vivo c-MET inhibition combined with focal radiotherapy synergistically prolongs survival in two syngeneic orthotopic glioma models compared with either treatment alone. Complementary studies demonstrated that synergy was lost when gliomas were established and treated in immunodeficient mice, and importantly, also when c-MET gene expression was disrupted in the tumor. Thus, synergistic suppression of experimental syngeneic glioma growth by irradiation and c-MET inhibition requires at least two mechanisms, expression of c-MET in the tumor and a functional immune system. In summary, our data suggest clinical evaluation of c-MET pathway inhibition in combination with radiotherapy in human glioblastoma.

Gasdermin D Is a Novel Prognostic Biomarker and Relates to TMZ Response in Glioblastoma.

Simple SummaryGSMD family were crucial regulators of pyroptosis. We used WB, IHC and bioinformatics to explore expression and potential role of GSDMs family in the progression of GBM. We found that only GSDMD expression was upregulated in glioma compared with nontumor brain tissues both in the public datasets and in-house cohort. High GSDMD expression was significantly associated with WHO grade IV, IDH 1/2 wild-type, mesenchymal subtypes and shorter overall survival.Moreover, GSDMD expression increased with after treating with TMZ in a time-dependent manner. We conculded that GSDMD was a novel prognostic biomarker, as well as TMZ-treatment response marker in glioma.The gasdermin (GSDM) family act as executioners during pyroptosis. However, its expression and biological role in glioma remain to be determined. This study carried out gene expression from six public datasets. Westerns blots and immunohistochemistry (IHC) staining were employed to examine GSDM expression in glioma in an in-house cohort. Kaplan–Meier and Cox regression analyses were performed to evaluate the prognostic role of GSDMs in glioma. Association between gene expression and immune infiltration was assessed by IHC and immunofluorescence (IF) staining of tissue sections. TMZ-induced pyroptosis was assessed by observation of morphological changes, WB and ELISA detection. Only GSDMD expression was upregulated in glioma compared with nontumor brain tissues both in the public datasets and in-house cohort. High GSDMD expression was significantly associated with WHO grade IV, IDH 1/2 wild-type and mesenchymal subtypes. Besides, high GSDMD expression was associated with shorter overall survival and could be used as an independent risk factor for poor outcomes in LGG and GBM. GO enrichment analysis and IHC validation revealed that GSDMD expression might participate in regulating macrophage infiltration and polarization. TMZ treatment induced the pyroptosis in GBM cells and GSDMD expression increased with after treating with TMZ in a time-dependent manner. Moreover, knocking down GSDMD obviously decreased IL-1β expression and reduced TMZ-induced pyroptosis in in vitro. GSDMD was a novel prognostic biomarker, as well as TMZ-treatment response marker in glioma.

Auranofin and Cold Atmospheric Plasma Synergize to Trigger Distinct Cell Death Mechanisms and Immunogenic Responses in Glioblastoma.

Targeting the redox balance of malignant cells via the delivery of high oxidative stress unlocks a potential therapeutic strategy against glioblastoma (GBM). We investigated a novel reactive oxygen species (ROS)-inducing combination treatment strategy, by increasing exogenous ROS via cold atmospheric plasma and inhibiting the endogenous protective antioxidant system via auranofin (AF), a thioredoxin reductase 1 (TrxR) inhibitor. The sequential combination treatment of AF and cold atmospheric plasma-treated PBS (pPBS), or AF and direct plasma application, resulted in a synergistic response in 2D and 3D GBM cell cultures, respectively. Differences in the baseline protein levels related to the antioxidant systems explained the cell-line-dependent sensitivity towards the combination treatment. The highest decrease of TrxR activity and GSH levels was observed after combination treatment of AF and pPBS when compared to AF and pPBS monotherapies. This combination also led to the highest accumulation of intracellular ROS. We confirmed a ROS-mediated response to the combination of AF and pPBS, which was able to induce distinct cell death mechanisms. On the one hand, an increase in caspase-3/7 activity, with an increase in the proportion of annexin V positive cells, indicates the induction of apoptosis in the GBM cells. On the other hand, lipid peroxidation and inhibition of cell death through an iron chelator suggest the involvement of ferroptosis in the GBM cell lines. Both cell death mechanisms induced by the combination of AF and pPBS resulted in a significant increase in danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation, indicating a potential increase in immunogenicity, although the phagocytotic capacity of dendritic cells was inhibited by AF. In vivo, sequential combination treatment of AF and cold atmospheric plasma both reduced tumor growth kinetics and prolonged survival in GBM-bearing mice. Thus, our study provides a novel therapeutic strategy for GBM to enhance the efficacy of oxidative stress-inducing therapy through a combination of AF and cold atmospheric plasma.

NSUN6, an RNA methyltransferase of 5-mC controls glioblastoma response to temozolomide (TMZ) via NELFB and RPS6KB2 interaction

ABSTRACT Nop2/Sun RNA methyltransferase (NSUN6) is an RNA 5-methyl cytosine (5mC) transferase with little information known of its function in cancer and response to cancer therapy. Here, we show that NSUN6 methylates both large and small RNA in glioblastoma and controls glioblastoma response to temozolomide with or without influence of the MGMT promoter status, with high NSUN6 expression conferring survival benefit to glioblastoma patients and in other cancers. Mechanistically, our results show that NSUN6 controls response to TMZ therapy via 5mC-mediated regulation of NELFB and RPS6BK2. Taken together, we present evidence that show that NSUN6-mediated 5mC deposition regulates transcriptional pause by accumulation of NELFB and the general transcription factor complexes (POLR2A, TBP, TFIIA, and TFIIE) on the preinitiation complex at the TATA binding site to control translation machinery in glioblastoma response to alkylating agents. Our findings open a new frontier into controlling of transcriptional regulation by RNA methyltransferase and 5mC.

Artificial intelligence for early prediction of treatment response in glioblastoma

Abstract Aims Treatment response assessment in glioblastoma is challenging. Patients routinely undergo conventional magnetic resonance imaging (MRI), but it has a low diagnostic accuracy for distinguishing between true progression (tPD) and pseudoprogression (psPD) in the early post-chemoradiotherapy time period due to similar imaging appearances. The aim of this study was to use artificial intelligence (AI) on imaging data, clinical characteristics and molecular information within machine learning models, to distinguish between and predict early tPD from psPD in patients with glioblastoma. Method The study involved retrospective analysis of patients with newly-diagnosed glioblastoma over a 3.5 year period (n=340), undergoing surgery and standard chemoradiotherapy treatment, with an increase in contrast-enhancing disease on the baseline MRI study 4-6 weeks post-chemoradiotherapy. Studies had contrast-enhanced T1-weighted imaging (CE-T1WI), T2-weighted imaging (T2WI) and apparent diffusion coefficient (ADC) sequences, acquired at 1.5 Tesla with 6-months follow-up to determine the reference standard outcome. 76 patients (mean age 55 years, range 18-76 years, 39% female, 46 tPD, 30 psPD) were included. Machine learning models utilised information from clinical characteristics (age, gender, resection extent, performance status), O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status and 307 quantitative imaging features; extracted from baseline study CE-T1WI/ADC and T2WI sequences using semi-automatically segmented enhancing disease and perilesional oedema masks respectively. Feature selection was performed within bootstrapped cross-validated recursive feature elimination with a random forest algorithm and Naïve Bayes five-fold cross-validation to validate the final model. Results Treatment response assessment based on the standard-of-care reports by clinical neuroradiologists showed an accuracy of 33% (sensitivity/specificity 52%/3%) to distinguish between tPD and psPD from the early post-treatment MRI study at 4-6 weeks. Machine learning-based models based on clinical and molecular features alone demonstrated an AUC of 0.66 and models using radiomic features alone from the early post-treatment MRI demonstrated an AUC of 0.46-0.69 depending on the feature and mask subset. A combined clinico-radiomic model utilising top common features demonstrated an AUC of 0.80 and an accuracy of 74% (sensitivity/specificity 78%/67%). The features in the final model were age, MGMT promoter methylation status, two shape-based features from the enhancing disease mask (elongation and sphericity), three radiomic features from the enhancing disease mask on ADC (kurtosis, correlation, contrast) and one radiomic feature from the perilesional oedema mask on T2WI (dependence entropy). Conclusion Current standard-of-care glioblastoma treatment response assessment imaging has limitations. In this study, the use of AI through a machine learning-based approach incorporating clinical characteristics and MGMT promoter methylation status with quantitative radiomic features from standard MRI sequences at early 4-6 weeks post-treatment imaging showed the best model performance and a higher accuracy to distinguish between tPD and psPD for early prediction of glioblastoma treatment response.

DNA Damage Response in Glioblastoma: Mechanism for Treatment Resistance and Emerging Therapeutic Strategies.

Glioblastoma (GBM) is an intrinsically treatment-resistant tumor and has been shown to upregulate DNA damage response (DDR) components after treatment. DNA damage response signaling mediates treatment resistance by promoting cell cycle arrest in order to allow for DNA damage repair and avoid mitotic catastrophe. Therefore, targeting the DDR pathway is an attractive strategy to combat treatment resistance in GBM. In this review, we discuss the different DDR pathways and then summarize the current preclinical evidence for DDR inhibitors in GBM, as well as completed and ongoing clinical trials.

Intratumoral Delivery of STING Agonist Results in Clinical Responses in Canine Glioblastoma.

Activation of STING (stimulator of interferon genes) can trigger a robust, innate antitumor immune response in immunologically "cold" tumors such as glioblastoma. A small-molecule STING agonist, IACS-8779, was stereotactically administered using intraoperative navigation intratumorally in dogs with spontaneously arising glioblastoma. The phase I trial used an escalating dose design, ascending through four dose levels (5-20 μg). Treatment was repeated every 4-6 weeks for a minimum of two cycles. Radiographic response to treatment was determined by response assessment in neuro-oncology (RANO) criteria applied to isovoxel postcontrast T1-weighted MR images obtained on a single 3T magnet. Six dogs were enrolled and completed ≥1 cycle of treatment. One dog was determined to have an abscess and was removed from further analysis. One procedure-related fatality was observed. Radiographic responses were dose dependent after the first cycle. The first subject had progressive disease, whereas there was 25% volumetric reduction in one subject and greater than 50% in the remaining surviving subjects. The median progression-free survival time was 14 weeks (range: 0-22 weeks), and the median overall survival time was 32 weeks (range: 11-39 weeks). Intratumoral STING agonist (IACS-8779) administration was well tolerated in dogs with glioblastoma to a dose of 15 μg. Higher doses of IACS-8779 were associated with radiographic responses.

Transcriptional control of brain tumor stem cells by a carbohydrate binding protein.

Brain tumor stem cells (BTSCs) and intratumoral heterogeneity represent major challenges in glioblastoma therapy. Here, we report that the LGALS1 gene, encoding the carbohydrate binding protein, galectin1, is a key regulator of BTSCs and glioblastoma resistance to therapy. Genetic deletion of LGALS1 alters BTSC gene expression profiles and results in downregulation of gene sets associated with the mesenchymal subtype of glioblastoma. Using a combination of pharmacological and genetic approaches, we establish that inhibition of LGALS1 signaling in BTSCs impairs self-renewal, suppresses tumorigenesis, prolongs lifespan, and improves glioblastoma response to ionizing radiation in preclinical animal models. Mechanistically, we show that LGALS1 is a direct transcriptional target of STAT3 with its expression robustly regulated by the ligand OSM. Importantly, we establish that galectin1 forms a complex with the transcription factor HOXA5 to reprogram the BTSC transcriptional landscape. Our data unravel an oncogenic signaling pathway by which the galectin1/HOXA5 complex maintains BTSCs and promotes glioblastoma.

Selected Articles from This Issue

Resistance to current lines of therap. in gastrointestinal stromal tumors (GIST) can occur through a variety of secondary mutations that occur in KIT. Novel therapeutics are still needed to address the mutational heterogeneity of KIT in GIST patients. In this study, Gupta and collaborators demonstrate the broad spectrum of ripretinib against primary and secondary drug-resistant mutations. Ripretinib is a “switch-control” kinase inhibitor designed to address even aggressively activated mutants to generate inactive conformations. Knowing that imatinib and MEK inhibition previously showed promise against imatinib-sensitive tumors, the authors also demonstrate the potency of combining ripretinib with MEKi in vitro and in vivo.KRAS mutations are present in up to 95% of pancreatic ductal adenocarcinoma (PDAC) cases. Inhibiting MEK (downstream of KRAS) would therefore appear to be a viable strategy for treating PDAC. Unfortunately, MEK inhibition as a monotherapy has been suboptimal in preclinical and clinical studies. Since CDKN2A mutations are also prevalent in a majority of PDAC tumors, Willobee and collaborators investigated the combination of CDK4/6 inhibitors with MEK inhibition. Combined CDK4/6 and MEK inhibition resulted in a significant survival benefit in an aggressive genetic mouse model of PDAC and provided a mechanistic rationale to explore the combination in clinical studies.For many cancers, the overexpression of P-cadherin is significantly correlated with lower overall and progression-free survival. Due to its limited expression in normal tissue and broad expression in tumors, Sheng and colleagues developed the P-cadherin specific antibody-drug conjugate PCA062. PCA062 contains a P-cadherin specific antibody bound to DM1 via a non-cleavable thioester linkage SMCC. The in vivo efficacy of PCA062 was demonstrated in cell- and patient-derived xenograft models of esophageal, triple-negative breast, and head and neck cancers. The authors note that limited efficacy signals were observed at dose escalation in Phase 1 trials. Taken together, the authors narrate the promise and challenges in targeting P-cadherin with the ADC modality.Inhibition of the programmed cell death ligand 1 (PD-1) axis has succeeded in many different solid tumors. The response of glioblastoma to PD-1 inhibition, however, remains limited due to its cold immune environment. Zhang and colleagues analyzed data from TCGA and clinical glioma samples and determined that Wnt/β-catenin expression was inversely related to the degree of immune cell infiltration and PD-L1 expression. They demonstrate that Wnt inhibition enhanced anti-PD-1 antibodies in a homograft mouse model by increasing lymphocyte infiltration and IFN-γ secretion. Their results demonstrate the potential for modifying the glioblastoma tumor microenvironment using Wnt inhibitors to potentiate immune checkpoint inhibition.

A Comparative Retrospective Study of Immunotherapy RANO Versus Standard RANO Criteria in Glioblastoma Patients Receiving Immune Checkpoint Inhibitor Therapy

ObjectivesReal-time assessment of treatment response in glioblastoma (GBM) patients on immune checkpoint blockade (ICB) remains challenging because inflammatory effects of therapy may mimic progressive disease, and the temporal evolution of these inflammatory findings is poorly understood. We compare GBM patient response during ICB as assessed with the Immunotherapy Response Assessment in Neuro-Oncology (iRANO) and the standard Response Assessment in Neuro-Oncology (RANO) radiological criteria.Methods49 GBM patients (seven newly diagnosed and 42 recurrent) treated with ICBs at a single institution were identified. Tumor burden was quantified on serial MR scans according to RANO criteria during ICB. Radiographic response assessment by iRANO and RANO were compared.Results82% (40/49) of patients received anti–PD-1, 16% (8/49) received anti-PD-L1, and 2% (1/49) received anti-PD-1 and anti-CTLA4 treatment. Change in tumor burden and best overall response ranged from −100 to +557% (median: +48%). 12% (6/49) of patients were classified as concordant non-progressors by both RANO and iRANO (best response: one CR, one PR, and four SD). Another12% (6/49) had discordant assessments: 15% (6/41) of RANO grade progressive disease (PD) patients had iRANO grade of progressive disease unconfirmed (PDU). The final classification of these discordant patients was pseudoprogression (PsP) in three of six, PD in two of six, and PDU in one of six who went off study before the iRANO assessment of PDU. iRANO delayed diagnosis of PD by 42 and 93 days in the two PD patients. 76% (37/49) patients were classified as concordant PD by both RANO and iRANO. 12% (6/49) of all patients were classified as PsP, starting at a median of 12 weeks (range, 4–30 weeks) after ICB initiation.ConclusionsStandard RANO and iRANO have high concordance for assessing PD in patients within 6 months of ICB initiation. iRANO was beneficial in 6% (3/49) cases later proven to be PsP, but delayed confirmation of PD by <3 months in 4% (2/49). PsP occurred in 12% of patients, starting at up to 7 months after initiation of ICB. Further study to define the utility of modified RANO compared with iRANO in ICB GBM patients is needed.

Anti-PD-L1 treatment to enhance the response of glioblastoma to radiation and produce long-term survival in mice.

e14048 Background: Glioblastoma (GBM) is the most aggressive and most common primary central nervous system cancer in adults. Blocking the interaction between Programmed Cell Death Protein-1 (PD-1) and its ligand (PD-L1) has shown remarkable success in the treatment of several cancers. However, many challenges remain in improving the efficacy of using monoclonal antibodies (Ab) against the receptor PD-1 in GBM, mainly due to the “non-immunogenic” tumor characteristics of GBM. PD-L1 has been found to be overexpressed on the surface of human GBM tumor cells and tumor-associated macrophages (TAM). Radiotherapy (RT), as one of the standard therapy of GBM, could alter the tumor microenvironment and promote an antitumor immune response. We hypothesize that anti-PD-L1 therapy can enhance the RT effects and improve the outcome of treatment when combined. Methods: Using a preclinical orthotropic syngeneic CT-2A mouse GBM tumor model, we studied the efficacy of combined therapy with anti-PD-L1 and RT. Mice were stratified into four treatment groups: control, RT, anti-PD-L1 Ab, and anti-PD-L1 Ab plus RT. RT(8 Gy) was given one time simultaneously with the first dose of anti-PD-L1, followed by systemic anti-PD-L1 maintenance treatment to the mice. Overall survival and tumor growth were monitored. Immunohistochemistry on resected tumors during treatment was performed to characterize the immune response. Single-cell RNA sequencing (scRNA-seq) was also performed to further study the immunologic parameters in the mouse brain. Results: Our results showed that anti-PD-L1 Ab in combination with RT provided a remarkable antitumor immune response and improved overall survival, with 25.5, 34, and 30 days of median survival in control (no-treatment), RT, and anti-PD-L1 groups, respectively, and achieving long-term survival and complete tumor response in 80% of the mice in the anti-PD-L1+RT treatment group (median survival not reached) in GBM tumor-bearing mice. The combined therapy promoted the recruitment of tumor-infiltrating immune cells, reversed the hostile tumor immune environment with a higher M1/TAM ratio, CD8+ /CD4+ T cell ratio, and CD8+ T cell /Treg cell ratio in the tumor area comparing with those parameters in single modality treatment groups. Furthermore, scRNA-seq data demonstrated that anti-PD-L1 combined with RT resulted in robust higher CD8 effector T cells, while lower CD4 and CD8 exhausted T cells in the tumor region compared to other groups. Increased CD4 central memory T cells and CD8 central memory T cells were seen only in tumors treated with anti-PD-L1+RT providing immunologic explanations on the durable control of GBM achieved only by the combined therapy. Conclusions: The anti-PD-L1 therapy synergizes with RT by reversing the hostile tumor immune environment resulting in improved tumor control and long-term survival in the syngeneic mouse GBM model.

An Oncolytic Virus Expressing IL15/IL15Rα Combined with Off-the-Shelf EGFR-CAR NK Cells Targets Glioblastoma.

IL15 is a pleiotropic cytokine with multiple roles that improve immune responses to tumor cells. Oncolytic viruses (OV) specifically lyse tumors and activate immune responses. Systemic administration of IL15 or its complex with the IL15Rα and chimeric antigen receptor (CAR) natural killer (NK) cells are currently being tested in the clinic. Here, we generated a herpes simplex 1-based OV-expressing human IL15/IL15Rα sushi domain fusion protein (named OV-IL15C), as well as off-the-shelf EGFR-CAR NK cells, and studied their monotherapy and combination efficacy in vitro and in multiple glioblastoma (GBM) mouse models. In vitro, soluble IL15/IL15Rα complex was secreted from OV-IL15C-infected GBM cells, which promoted GBM cytotoxicity and improved survival of NK and CD8+ T cells. Frozen, readily available off-the-shelf EGFR-CAR NK cells showed enhanced killing of tumor cells compared with empty vector-transduced NK cells. In vivo, OV-IL15C significantly inhibited tumor growth and prolonged survival of GBM-bearing mice in the presence of CD8+ T cells compared with parental OV. OV-IL15C plus EGFR-CAR NK cells synergistically suppressed tumor growth and significantly improved survival compared with either monotherapy, correlating with increased intracranial infiltration and activation of NK and CD8+ T cells and elevated persistence of CAR NK cells in an immunocompetent model. Collectively, OV-IL15C and off-the-shelf EGFR-CAR NK cells represent promising therapeutic strategies for GBM treatment to improve the clinical management of this devastating disease. SIGNIFICANCE: The combination of an oncolytic virus expressing the IL15/IL15Rα complex and frozen, ready-to-use EGFR-CAR NK cells elicits strong antitumor responses in glioblastoma.

A Drug Screening Pipeline Using 2D and 3D Patient-Derived In Vitro Models for Pre-Clinical Analysis of Therapy Response in Glioblastoma.

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6–9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microenvironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cultures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.

Low conductivity on electrical properties tomography demonstrates unique tumor habitats indicating progression in glioblastoma.

Tissue conductivity measurements made with electrical properties tomography (EPT) can be used to define temporal changes in tissue habitats on longitudinal multiparametric MRI. We aimed to demonstrate the added insights for identifying tumor habitats obtained by including EPT with diffusion- and perfusion-weighted MRI, and to evaluate the use of these tumor habitats for determining tumor treatment response in post-treatment glioblastoma. Tumor habitats were developed from EPT, diffusion-weighted, and perfusion-weighted MRI in 60 patients with glioblastoma who underwent concurrent chemoradiotherapy. Voxels from EPT, apparent diffusion coefficient (ADC), and cerebral blood volume (CBV) maps were clustered into habitats, and each habitat was serially examined to assess its temporal change. The usefulness of temporal changes in tumor habitats for diagnosing tumor progression and treatment-related change was investigated using logistic regression. The performance of significant predictors was measured using the area under the curve (AUC) from receiver-operating-characteristics analysis with 1000-fold bootstrapping. Five tumor habitats were identified, and of these, the hypervascular cellular habitat (odds ratio [OR] 5.45; 95% CI, 1.75-31.42; p = .02), hypovascular low conductivity habitat (OR 2.00; 95% CI, 1.45-3.05; p < .001), and hypovascular intermediate habitat (OR 1.57; 95% CI, 1.18-2.30; p = .006) were predictive of tumor progression. Low EPT and low CBV reflected a unique hypovascular low conductivity habitat that showed the highest diagnostic performance (AUC 0.86; 95% CI, 0.76-0.96). The combined habitats showed high performance (AUC 0.90; 95% CI, 0.82-0.98) in the differentiation of tumor progression from treatment-related change. EPT reveals low conductivity habitats that can improve the diagnosis of tumor progression in post-treatment glioblastoma. • Electrical properties tomography (EPT) demonstrated lower conductivity in tumor progression than in treatment-related change. • EPT allowed identification of a unique hypovascular low conductivity habitat when combined with cerebral blood volume mapping. • Tumor habitats with a hypovascular low conductivity habitat, hypervascular cellular habitat, and hypovascular intermediate habitat yielded high diagnostic performance for diagnosing tumor progression.

Abstract PR-007: Targeting FASN improves type I interferon responses in irradiated glioblastoma

Abstract Glioblastoma (GBM) is a devastating primary brain cancer with a median survival of 11-15 months. Radiotherapy (RT), the standard of care for GBM, increases adjuvanticity of cancer cells, by at least generating cancer cell intrinsic type I interferon (IFN-I) responses to jumpstart antitumor immunity. However, RT can also exacerbate potent immune inhibitory mechanisms to facilitate immune evasion. Among which, metabolic reprogramming of irradiated GBM represents an emerging mechanism of immune resistance. Notably, increased de novo lipid synthesis by the fatty acid synthase (FASN) is a hallmark of GBM that is emerging as a mechanisms of therapy resistance and immune escape. Because FASN was found to impair IFN-I in antiviral immunity, we hypothesize that FASN is preventing RT-induced IFN-I responses to promote GBM survival and evade immune recognition. To test this hypothesis, we first defined RT-induced metabolic changes by seahorse assay and metabolomics in the GL261 murine GBM model. We observed an increase in mitochondrial respiration, glycolysis and in lipid metabolism-related pathways in 10 gray (Gy) irradiated GL261 cells. Consistent with this last observation, we found upregulation of FASN expression by western blot and intracellular neutral lipid accumulation by BODIPY staining and transmission electron microscopy (TEM) in irradiated GL261 cells (10 Gy). Confirming the role of FASN, RT-induced accumulation of neutral lipids was reverted when GL261 cells were incubated with a pharmacologic FASN inhibitor. Next, to ask whether FASN was impairing IFN-I in irradiated GBM, GL261 cells were engineered to express an inducible shRNA silencing FASN (GL261shFASN) or its non-silencing control (GL261shNS). As expected, irradiation of GL261shNS cells enhanced the secretion of IFN-I cytokines. Strikingly, we found that FASN knockdown spontaneously induced IFN-beta and CXCL10 secretion, and this effect was even more pronounced when cells were irradiated. Finally, GL261shNS and GL261shFASN cells were orthotopically implanted in mice and IFN-I signaling was blocked using an antibody against the interferon alpha/beta receptor (IFNAR). FASN knockdown alone was sufficient to increase the median survival of mice bearing GL261shFASN tumors by 51 days vs. 35 days for GL261shNS tumors. Moreover, 33% of mice bearing GL261 tumors unable to express FASN remained tumor free for up to 100 days, while none of the GL261shNS-tumor bearing animal survived. This significant survival prolongation of mice bearing GL261shFASN tumor was completely abrogated when IFNAR was blocked. Overall, our findings suggest that FASN-mediated lipogenesis prevents RT-induced cancer cell intrinsic IFN-I to promote GBM survival. Consequently, it is possible that FASN acts as an immuno-metabolic checkpoint capable to regulate the immune system upon metabolic cues generated by RT. Citation Format: Mara De Martino, Camille Daviaud, Claire Vanpouille-Box. Targeting FASN improves type I interferon responses in irradiated glioblastoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-007.

DDRE-21. THE FUNCTION OF MITOCHONDRIAL OSMR IN GLIOMA STEM CELL RESPIRATION AND GLIOBLASTOMA PATHOGENESIS

Glioblastoma contains a rare population of self-renewing brain tumor stem cells (BTSCs) which are endowed with properties to proliferate, spur the growth of new tumors, and at the same time, evade ionizing radiation (IR) and chemotherapy. However, the drivers of BTSC resistance to therapy remain unknown. The cytokine receptor for oncostatin M (OSMR) regulates BTSC proliferation and glioblastoma tumorigenesis. We have discovered that OSMR translocates to the mitochondria and regulates oxidative phosphorylation, independent of its role in cell proliferation. Mechanistically, OSMR is targeted to the mitochondrial matrix via the presequence translocase-associated motor complex components, mtHSP70 and TIM44. OSMR interacts with NADH ubiquinone oxidoreductase 1/2 (NDUFS1/2) of complex I and promotes mitochondrial respiration. Deletion of OSMR impairs spare respiratory capacity, increases reactive oxygen species, and sensitizes BTSCs to IR-induced cell death. Importantly, suppression of OSMR improves glioblastoma response to IR and prolongs lifespan.