Recurrent Glioblastoma Multiforme Research Articles

Efficacy of Anti-VEGF Drugs Based Combination Therapies in Recurrent Glioblastoma: Systematic Review and Meta-Analysis.

Recurrent glioblastoma multiforme (rGBM) has a grim prognosis, with current therapies offering no survival benefit. Several combination therapies involving anti-VEGF agents have been studied with mixed results. A systematic search was performed using five electronic databases: PubMed, Scopus, ISI, Embase, and the Cochrane Library, without language limitations. The primary outcome of interest was progression-free survival (PFS). Secondary outcomes were overall survival (OS), objective response ratio (ORR), and grade ≥ 3 adverse events. Estimates for PFS and OS were calculated as random effects hazard ratio (HR) with 95% confidence intervals (CIs) using the generic inverse variance method. Estimates for ORR and grade ≥ 3 adverse events were calculated using a random-effects risk ratio (RR) with 95% confidence intervals (CIs) using the Mantel-Haenszel method. Thirteen studies met the inclusion criteria and a total of 1994 patients were included in the analysis. There was no statistically significant improvement in PFS (HR 0.84; 95% CI (0.68, 1.03); I2=81%), OS (HR 0.99; 95% CI (0.88, 1.12); I2=0%), and ORR (RR 1.36; 95% CI (0.96, 1.92); I2=61%) in the combination therapy group when compared to the control group. Significantly higher grade ≥ 3 adverse events (RR 1.30; 95% CI (1.14, 1.48); I2=47%) were seen in the combination therapy when compared to the control group. Our analysis showed that the use of combination therapy with anti-VEGF agents did not offer any benefit in PFS, OS, or ORR. In contrast, it had significantly higher grade 3-5 adverse events. Further studies are needed to identify effective therapies in rGBM that can improve survival.

DIPG-40. LONG TERM SURVIVAL OF A 14 YEAR OLD GIRL WITH H3K27M MUTATED DIFFUSE MIDLINE GLIOMA FOLLOWING RADIOTHERAPY AND PROLONGED TUMOR TREATING FIELDS (TTFIELDS) -CASE REPORT

Abstract INTRODUCTION Pediatric diffuse midline H3K27M mutated glioma has a dismal prognosis. Most cases are treated by radiotherapy and oral chemotherapy, with surgical resection having a debatable role in some. Tumor-treating fields (TTFields) is a noninvasive modality in which alternating electrical fields exert biophysical force on charged and polarizable molecules known as dipoles. TTFields has been shown to extend survival for adult patients with newly diagnosed and recurrent GBM, and is FDA approved for these indications. There is sparse data about pediatric patients. CASE REPORT A 14-year-old girl presented with headache and vomiting. A large left thalamic tumor was diagnosed and underwent subtotal resection. Diagnosis was diffuse midline glioma, H3K27M positive, IDH negative. Molecular Panel: Tumor mutation burden low, microsatellite stable. Genomic findings: FGFR1, NF1, H3F3A -K28M, ATRX R666, MAP3K1 She was re-operated due to a fast tumor relapse within a few weeks, prior to radiotherapy, followed with radiotherapy with local field 59Gy. She then received temozolamide (12 months) concomitant with TTfields (Optune, Novocure). TTfields therapy was administered for 5 years without adverse effects and excellent compliance (above 85%). After 5 years, therapy was withheld, and she is now off therapy NED for another 15 months. DISCUSSION Diffuse midline H3K27M glioma harbors a poor prognosis in children. Children refrain from this treatment both for lack of evidence and because of the difficulty in adhering to a regimen that demands carrying a battery of 1.5-2kg all day and shaving all hair every 3 days. Our patient had a high compliance and as she wore a head scarf for religious customs, the device was not visible. To our knowledge this is the first pediatric reported case of long-term survival of H3K27mutatted midline glioma using TTFields. Further pediatric studies are warranted.

Assembled Embedded 3D Hydrogel System for Asynchronous Drug Delivery to Inhibit Postoperative Recurrence of Malignant Glioma and Promote Neurological Recovery

AbstractSurgical resection of glioblastoma multiforme (GBM) often results in tumor recurrence and mild neurologic deficits. Here, a 3D asynchronous drug delivery system is innovatively developed to address the dual challenges of GBM recurrence and postoperative neurological deficit. Based on transcriptome analysis of tumor cells and tumor microenvironment (TME) cells between primary and recurrent mouse GBM tissues, a novel dual‐targeting approach is developed to combine mTOR pathway inhibition with microglia/macrophage repolarization. Then, in situ injectable methacrylated gelatin (GelMA) is constructed to perfectly fit into the tumor resection cavity and achieve direct delivery of dual‐targeted drugs, exhibiting outstanding postoperative GBM inhibitory effects in vivo. At the same time, neurotrophic factor‐saturated 3D‐printed GelMA patches are used to construct a 3D asynchronous drug delivery system, allowing gradual penetration of the neurotrophic factors into the underlying hydrogel to promote axonal sprouting after GBM suppression. Notably, this 3D asynchronous drug delivery system promotes neurological recovery without weakening the efficacy of inhibiting tumor recurrence. Therefore, this study not only proposes a new dual‐targeted GBM treatment strategy but also pioneers the construction of a 3D asynchronous drug delivery system for the comprehensive treatment of GBM. This study is expected to improve the poor prognosis of patients with GBM.

Safety and efficacy of B7-H3 targeting CAR-T cell therapy for patients with recurrent GBM.

2062 Background: Glioblastoma Multiforme (GBM) is one of the most deadly cancers, and nearly all patients experience recurrence upon the current standard of care for newly diagnosed GBM. The recurrent GBM (rGBM) exhibits a median survival of less than 8 months with limited therapeutic options. We have previously cloned the B7-H3 gene, characterized its function, demonstrated altered B7-H3 expression in human solid tumors, and identified B7-H3 as a promising therapeutic target. We present herein the preliminary findings of an investigator-initiated trial (NCT05241392), where patients with rGBM were treated with autologous chimeric antigen receptor-T (CAR-T) cells targeting B7-H3 (TX103). Methods: This is an open, single-arm, "3+3" dose-escalation and multiple-dose study. The primary objective was to evaluate the safety and the maximal-tolerated dose. Secondary objectives included survival outcomes, tumor responses, immunological responses, and pharmacokinetic parameters. Patients aged 18 to 75 were enrolled with confirmed recurrence by image scan and B7-H3 expression >= 30% by immunohistochemistry analysis. TX103 was administered intracavity and/or intraventricularly to patients via an Ommaya reservoir. TX103 was given biweekly to patients each cycle at dose levels 1, 2, and 3 (20, 60, 150 million cells, respectively), with four cycles as one course. Results: Between March 2022 and January 2024, 13 patients received at least one intracranial infusion of TX103, with 3, 4, and 6 patients in dose levels 1, 2, and 3, respectively. The median number of infusion cycles was 4 (Min, Max 1,9). All patients were included in the safety analysis, revealing no dose-limiting toxicity or CAR-T treatment-related death. All patients experienced at least one adverse event (AE), and the treatment-related AEs (TRAEs) included cytokine release syndrome, increased intracranial pressure (ICP), headache, epilepsy, decreased level of consciousness, vomiting, and pyrexia. Most TRAEs were grade 1-2, while three grade-3 TRAEs were observed: one case of increased ICP in dose level 2, one case of epilepsy, and another case of decreased level of consciousness in dose level 3. As of January 2024, six patients from dose levels 1 and 2 were eligible for a 12-month survival assessment; the 12-month overall survival (OS) rate was 83.3% (95% CI: 58.3%~100%), and the median OS was 20.3 mo (95% CI: 20.3~not reach). Two of the three patients from dose level 2 achieved partial and complete responses, respectively. Following CAR-T administration, significant increases in cytokines, including IL-6 and IFN-γ, and elevated copy numbers of the CAR gene were observed in the cerebrospinal fluid, while only minimal elevations were noted in the peripheral blood. Conclusions: In this trial, treating rGBM with TX103 is deemed safe and tolerated. These interim results support further study using TX103 to enhance the survival outcomes in patients with rGBM.

Phase 1 study of anti–immunoglobulin-like transcript 3 (ILT3) monoclonal antibody (mAb) MK-0482 + pembrolizumab (pembro) in patients with recurrent inoperable glioblastoma (GBM).

2046 Background: For patients with GBM, treatment options are limited once the disease has recurred and become inoperable after standard first-line therapy. ILT3 is an inhibitory receptor expressed on monocytic myeloid cells, including tolerogenic dendritic cells and myeloid-derived suppressor cells (MDSCs). High expression of ILT3 on these immune cells has been associated with immune tolerance and suppression of T-cell function. Data have shown that GBMs have high ILT3 expression and high levels of MDSCs within the tumor microenvironment (TME). MK-0482 is a novel humanized IgG4 mAb targeting ILT3. Inhibition of ILT3 with MK-0482 may help relieve immunosuppression and improve T-cell function within the TME. In the expansion cohort of the first-in-human phase 1 study (MK-0482-001; NCT03918278), MK-0482 was evaluated in combination with pembro in a cohort of patients with recurrent inoperable GBM. The safety and efficacy data from this cohort are presented herein. Methods: Patients aged ≥18 y with their first recurrent inoperable GBM and a Karnofsky performance status (KPS) ≥80 were enrolled. Patients received MK-0482 750 mg Q3W + pembro 200 mg Q3W IV for up to a maximum of 35 cycles. The primary objective was to determine safety and tolerability of MK-0482 + pembro. Secondary objectives were to evaluate ORR, DCR (CR + PR + SD), and DOR by investigator assessment per Response Assessment in Neuro-Oncology (RANO). Exploratory objectives included evaluation of PFS per RANO and OS. Results: Overall, 25 patients were enrolled and received study treatment. At data cutoff (October 3, 2023), median follow-up duration was 9.3 mo (range, 5.6-14.5); treatment was ongoing in 1 patient. Median age was 56 y (range, 33-76). Most patients were male (72%), had a KPS of 80 (68%), and had IDH1 wild-type tumors (88%). All patients had previously received systemic therapy: 24 of 25 (96%) received prior temozolomide and 3 of 25 (12%) received prior glasdegib. Overall, 22 patients (88%) had an adverse event (AE); 10 patients (40%) had treatment-related AEs, most commonly (≥10%) arthralgia (12%), fatigue (12%), and increased ALT (12%). Grade 3 treatment-related AEs occurred in 2 patients (8%; increased ALT [n = 1] and fatigue [n = 1]). No grade 4 or 5 treatment-related AEs occurred. Four patients (16%) experienced immune-mediated AEs (hypothyroidism, n = 2; hepatitis, n = 1; pancreatitis, n = 1); all were grade 1 or 2. Confirmed ORR was 12% (3/25; 95% CI, 2.5-31.2), DCR was 32% (8/25; 14.9-53.5), and median DOR was 11.1 mo (range, 9.9-16.6+). Median PFS was 1.4 mo (95% CI, 1.3-2.9) and median OS was 9.3 mo (6.3-13.7); 6-/12-mo PFS and OS rates were 24%/16% and 72%/36%, respectively. Biomarker data will be included in the presentation. Conclusions: MK-0482 + pembro had a manageable AE profile with modest antitumor activity in patients with recurrent inoperable GBM. Clinical trial information: NCT03918278 .

Deep learning for 4D modeling of glioblastoma multiforme with tumor treating fields (TTFields) therapy.

e14032 Background: Prediction of future tumor growth and recurrence are critical in the management of patients with glioblastoma multiforme (GBM). This study develops a deep learning method for estimating future areas of GBM spread across multiple timepoints using MRI in patients receiving tumor treating fields (TTFields) therapy. Methods: A single institutional database was queried to identify adult patients with histologically confirmed newly diagnosed and/or recurrent GBM undergoing TTFields therapy. For newly diagnosed GBM, patients received TTFields with temozolomide after maximal debulking surgery and chemoradiation therapy. For recurrent GBM, patients received TTFields as monotherapy. For each patient, all serial follow-up MRI exams were obtained, including T1 (pre-/post-contrast), T2, and T2/FLAIR sequences. On each exam, all regions of enhancing tumor core (excluding peritumoral edema, necrotic core, or resection cavity) were delineated and aligned across time points using nonlinear deformable registration. For any given pair of serial exams, a convolutional neural network (CNN) was designed to predict future GBM tumor on the follow-up exam given the precursor exam and time interval between the studies. The model is implemented as a 3D encoder-decoder architecture yielding a dense per-voxel prediction of future tumor probability optimized using a binary cross-entropy loss function. Class weights were used to develop high sensitivity and high positive predictive value (PPV) model variants. To generate final logit scores, time information is concatenated to the penultimate feature map as an additional feature channel, allowing the model to calibrate each estimate based on elapsed time between any pair of exams. Upon convergence, a 4D learned representation allows for prediction of spatial distribution of GBM tumor at any future time point. Results: A total of 123 patients (1112 total MR exams) were identified. For any given single patient, a median of 6 follow-up exams (IQR 2.5-12.5) at a median interval follow-up time of 46 days (IQR 15.75-63 days) between exams was observed. Upon five-fold cross-validation, the model demonstrated a 0.44 Dice score overlap between predicted and true areas of future GBM tumor growth. The high sensitivity model yielded a per-voxel sensitivity of 0.91 (IQR 0.77-0.99) and PPV of 0.26 (IQR 0.17 to 0.32), while the high PPV model yielded a per-voxel sensitivity of 0.14 (IQR 0.00 to 0.46) and PPV of 0.75 (IQR 0.56 to 0.94). Upon visual confirmation, model predictions across incremental time values for any given exam yielded expected gradual growth of tumor over time. Conclusions: A deep learning model can accurately predict future areas of GBM tumor growth in patients receiving TTFields therapy, with optimal performance that may be calibrated for high sensitivity or high PPV based on clinical use case.

Impact of next generation sequencing in high grade glioma management.

e14031 Background: Recent advances in genomic profiling have improved our understanding of the molecular pathogenesis of high grade gliomas. Increased availability of high-throughput Next Generation Sequencing (NGS) allows the identification of genetic alterations which could direct personalized cancer treatments for HGG patients. Methods: All patients with high grade glioma (WHO Grade ≥3 glioma by 2016 or 2022 classification) in our institution, who underwent NGS testing between 2019-2023, were identified from a prospectively maintained electronic database. NGS results, from the Oncomine Precision Assay, a multi-gene panel which tests for 50 different mutations, were collated. Medical records were systematically reviewed to collect demographic, treatment and survival data. Alterations detected on NGS were characterized using the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Results: 53 patient samples underwent NGS testing (34 males [64%]; 19 females [36%]). Median age at diagnosis was 52 years (range: 20-72 years). Thirty patients (57%) had an ECOG PS 1 at first clinical review. 18 tumors (34%) were found to have MGMT promoter hypermethylation. 28 patients (53%) were found to have potentially actionable alterations according to the ESCAT scale; 19 (35%) EGFR mutations (ESCAT IIIA level evidence) were detected, 5 PIK3CA (9%) mutations (ESCAT IIIA), 3 FGFR3 (6%) alterations (ESCAT IIB), 2 patients with CDK 4 (4%) copy number gain (ESCAT IIIA), 2 PTPRZ1-MET (4%) fusions (ESCAT IIIA) and 1 BRAF V600E (2%) mutation (ESCAT IB). 4 patients were found to have multiple mutations on NGS. 1 sample had insufficient tissue for analysis. 2 patients (4%) were considered for targeted therapy based on NGS results (1 BRAF V600E mutation and 1 FGFR3 alteration) but both deteriorated in advance of commencing treatment. One patient received nivolumab for MSI-H recurrent GBM (ESCAT IIIB) but progressed within 3 months of commencing treatment. Conclusions: Despite advances in our understanding of the pathogenesis of these high grade gliomas and improved awareness of molecular targets, the delivery of targeted therapy to patients with HGG remains challenging. Rapid access to targeted therapy via compassionate access programmes and improved availability and access to clinical trials for patients with rare cancers and molecular alterations, is paramount.

Exploratory phase I study of HF1K16 for the treatment of patients with refractory/recurrent advanced glioma: Preliminary efficacy and mechanism as a monotherapy.

e14030 Background: The absence of I.O success in advanced glioma treatment suggests that a deeper understanding of the brain tumor and the immune microenvironment is critical. It is well-documented that refractory/recurrent glioma has an immunosuppressive nature. One frequent observation is the high level of circulating myeloid-derived suppressor cells (MDSCs). We conducted a exploratory study to assess an immunomodulatory approach for relieving immune suppression employing HF1K16, a liposome all-trans retinoic acid (ATRA) suspension. Methods: This Phase I study (NCT05388487) comprise a conventional 3+3 dose escalation plus an advanced glioma-specific expansion arm. Eligible patients had prior confirmed advanced solid tumor and failed standard treatment. HF1K16 infusions were administered in 21-day cycles (q.o.d days 1-14). Prior to and during treatment, peripheral blood mononuclear cells were collected and analyzed with flow cytometry to monitor the changes in myeloid cell phenotype and T cell composition. Survival data were calculated as it is difficult to determine the timing of progression for immunotherapies owing to pseudoprogression. Results: As of Feb 2th, 2024, 14 recurrent/refractory glioma patients including 6 males and 8 females had been enrolled in this study. 2 of them withdrew before finished 1st cycle. 7 had received treatment for at least 3 cycles and 1 had achieved CR after 15 cycles. For the 8 primary diagnosed grade IV glioma patients, the mOS is 7.8m, and 6 (75%) are alive. The mOS for the primary dignosed grade II/III is 13.7m and 100% patients are alive (n=4). There is 1 recurrent GBM patent who had received 5 cycles of treatment. MRI showed an enlargement of tumor lesion while KPS score remained >90. A surgical resection was performed and we subsequently studied the posttreatment tumor specimens (window of opportunity). IHC and flow cytometry analysis revealed prominent perivascular and intratumoral inflammatory infiltrate, containing CD8+CTL cells and HLA-DR expressing cells. Conclusions: While the interpretation of our data is limited by the small sample size, the results provide an encouraging signal and warrants further assessment. Patient recruitment continues with anticipated completion in 2024. Clinical trial information: NCT05388487 . [Table: see text]

Phase 2 study of bizaxofusp, an IL-4R targeted toxin payload, in nonresectable recurrent GBM: Comparison of overall survival with contemporaneous eligibility-matched and propensity score balanced external control arm.

2079 Background: Bizaxofusp (MDNA55) is designed to selectively deliver a potent toxin payload to tumor cells by targeting the IL-4 receptor (IL-4R) overexpressed by GBM, but not normal brain. Localized delivery of bizaxofusp minimizes risk of off-target toxicities and systemic exposure while eliciting effective tumor cell killing. Bizaxofusp was evaluated using convection-enhanced delivery in MDNA55-05 (NCT02858895), a single-arm, open-label, multi-center Ph2 study in nonresectable recurrent GBM (rGBM). Methods: The study population comprised of de novo IDH wild-type GBM patients with 1st or 2nd relapse that were non-resectable. Forty-four patients were administered a single treatment of bizaxofusp (range: 18-240 μg). The primary endpoint was OS; secondary endpoints were safety and tumor response. IL-4R expression in tumor biopsies was determined using a validated assay. OS was compared to an eligibility matched external control arm (emECA) of 81 contemporaneous rGBM subjects receiving standard of care. The emECA was further refined by propensity-score balancing (pbECA) to the bizaxofusp arm based on known prognostic factors, resulting in a weighted sample size of 40.8. OS was defined as time from relapse (to unify index date in both arms) or treatment start (for analysis of tumor response) to death/censor. Tumor response was assessed following RANO and mRANO criteria. Results: Bizaxofusp showed an acceptable safety profile at doses up to 240 μg. TRAEs were primarily neurological or aggravation of pre-existing neurological deficits associated with GBM and were manageable with standard measures. Results showed that IL-4R expression did not impact mOS except in patients receiving low doses of bizaxofusp. The bizaxofusp arm had significantly longer median OS (mOS) than contemporaneous emECA (12.4 vs 7.7 months; HR: 0.64, 95% CI 0.46-0.93; p = 0.02). Survival benefit was also evident when compared to the pbECA: (12.4 vs 7.2 months; HR: 0.72, 95% CI 0.46-1.1; p = 0.27). Patients with tumor control (SD or PR/CR, n=21) had significantly longer mOS than patients with tumor progression (n=23) (16.7 vs 8.5 months; HR = 0.5, 95% CI 0.27-0.9; p = 0.01). Among patients with tumor control who had pseudoprogression (PsP) per mRANO, mOS (22.8 months) was significantly longer than patients with tumor progression (HR: 0.49, 95% CI 0.25-0.98; p = 0.049). Conclusions: Bizaxofusp achieved significant OS benefit in patients with nonresectable rGBM compared to contemporaneous emECA. Patients who experienced tumor control, including those with PsP, showed significantly longer survival than patients with tumor progression. Phase 3 ready registrational trial will comprise a high dose bizaxofusp arm and a hybrid control arm with 1/3 randomized subjects and 2/3 pmECA. Clinical trial information: NCT02858895 .

Phase 1 dose expansion and biomarker study assessing first-in-class tumor microenvironment modulator VT1021 in patients with advanced solid tumors

BackgroundPreclinical studies have demonstrated that VT1021, a first-in-class therapeutic agent, inhibits tumor growth via stimulation of thrombospondin-1 (TSP-1) and reprograms the tumor microenvironment. We recently reported data from the dose escalation part of a phase I study of VT1021 in solid tumors. Here, we report findings from the dose expansion phase of the same study.MethodsWe analyzed the safety and tolerability, clinical response, and biomarker profile of VT1021 in the expansion portion of the phase I study (NCT03364400). Safety/tolerability is determined by adverse events related to the treatment. Clinical response is determined by RECIST v1.1 and iRECIST. Biomarkers are measured by multiplexed ion beam imaging and enzyme-linked immunoassay (ELISA).ResultsFirst, we report the safety and tolerability data as the primary outcome of this study. Adverse events (AE) suspected to be related to the study treatment (RTEAEs) are mostly grade 1–2. There are no grade 4 or 5 adverse events. VT1021 is safe and well tolerated in patients with solid tumors in this study. We report clinical responses as a secondary efficacy outcome. VT1021 demonstrates promising single-agent clinical activity in recurrent GBM (rGBM) in this study. Among 22 patients with rGBM, the overall disease control rate (DCR) is 45% (95% confidence interval, 0.24-0.67). Finally, we report the exploratory outcomes of this study. We show the clinical confirmation of TSP-1 induction and TME remodeling by VT1021. Our biomarker analysis identifies several plasmatic cytokines as potential biomarkers for future clinical studies.ConclusionsVT1021 is safe and well-tolerated in patients with solid tumors in a phase I expansion study. VT1021 has advanced to a phase II/III clinical study in glioblastoma (NCT03970447).

Advanced tumor electric fields therapy: A review of innovative research and development and prospect of application in glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive malignant tumor with a high mortality rate and is the most prevalent primary intracranial tumor that remains incurable. The current standard treatment, which involves surgery along with concurrent radiotherapy and chemotherapy, only yields a survival time of 14-16 months. However, the introduction of tumor electric fields therapy (TEFT) has provided a glimmer of hope for patients with newly diagnosed and recurrent GBM, as it has been shown to extend the median survival time to 20 months. The combination of TEFT and other advanced therapies is a promising trend in the field of GBM, facilitated by advancements in medical technology. In this review, we provide a concise overview of the mechanism and efficacy of TEFT. In addition, we mainly discussed the innovation of TEFT and our proposed blueprint for TEFT implementation. Tumor electric fields therapy is an effective and highly promising treatment modality for GBM. The full therapeutic potential of TEFT can be exploited by combined with other innovative technologies and treatments.

Evaluating the efficacy and clinical outcomes of minimally invasive laser interstitial thermal therapy in the treatment of recurrent glioblastoma multiforme: meta-analysis protocol

Evaluating the efficacy and clinical outcomes of minimally invasive laser interstitial thermal therapy in the treatment of recurrent glioblastoma multiforme: meta-analysis protocol

383 Epigenetic Potentiation of Anti-tumor Immune Responses Using Viral Mimicry for Gliomas

INTRODUCTION: Human endogenous retroviruses (HERVs) account for approximately 8% of our genome and are evolutionary relics of a germline retroviral infection. Although normally silenced by a master epigenetic regulator, np220, HERVs may be re-expressed in cellular states with marked epigenetic dysregulation and induce innate anti-viral inflammatory responses. This viral mimicry immune response sensitizes tumors to immunotherapy by eliciting double-stranded RNAs (dsRNA)-mediated interferon signaling cascades. Hence, viral mimicry could serve as a predictor of clinical responses to immunotherapy. METHODS: We conducted a preclinical study on the role of np220 in GBM and mediating viral mimicry responses. RESULTS: Using a tailored bulk transcriptional dataset from 71 newly diagnosed high-grade GBMs, we demonstrated significantly negative correlations between np220 expression and 48 HERV superfamilies. Additionally, using a transcriptional deconvolution pipeline of low-and high-grade gliomas, we demonstrated that np220 is negatively correlated with innate antiviral immune response signatures (TLR3) (RTLR3 = -0.300, pTLR3 = 0.00006). Our in vitro studies have shown that NP220 knockdown induces intracellular HERV-mediated dsRNA-signaling cascades via RIG-1 and TLR3. NP220 knockdown exponentially increases PD-L1 expression (p-value: <0.001), and, increases cytoplasmic accumulation of cellular endogenous dsRNA in established human and mouse glioma cell lines which may induce the interferon-mediated response. This response was recapitulated in recurrent GBM samples with radiographic responses to checkpoint inhibition (anti-PD1) with widely increased expression of dsRNA, PD-L1 and perivascular CD8 cell infiltration, suggesting dsRNA-signaling may mediate response to immunotherapy. Furthermore, we showed np220 is linked to reduced immune cell infiltration (CD8, Dendritic Cells) in the glioma microenvironment through deconvolution of RNA-seq data from TCGA (RCD8 = -.202, RDC = -0.198) (pCD8 = 8.79E-06, pDC = 1.34E-05). CONCLUSIONS: Our findings suggest that np220 could serve as a target to potentiate glioma ICI for immune response.

Adjuvant re-irradiation vs. no early re-irradiation of resected recurrent glioblastoma: pooled comparative cohort analysis from two tertiary centers

BackgroundThe optimal management strategy for recurrent glioblastoma (rGBM) remains uncertain, and the impact of re-irradiation (Re-RT) on overall survival (OS) is still a matter of debate. This study included patients who achieved gross total resection (GTR) after a second surgery after recurrence, following the GlioCave criteria.MethodsInclusion criteria include being 18 years or older, having histologically confirmed locally recurrent IDHwt or IDH unknown GBM, achieving MRI-proven GTR after the second surgery, having a Karnofsky performance status of at least 60% after the second surgery, having a minimum interval of 6 months between the first radiotherapy and the second surgery, and a maximum of 8 weeks from second surgery to the start of Re-RT.ResultsA total of 44 patients have met the inclusion criteria. The median OS after the second surgery was 14 months. All patients underwent standard treatment after initial diagnosis, including maximum safe resection, adjuvant radiochemotherapy and adjuvant chemotherapy. Re-RT did not significantly impact OS. However, MGMT promoter methylation status and a longer interval (> 12 months) between treatments were associated with better OS. Multivariate analysis revealed the MGMT status as the only significant predictor of OS.ConclusionFactors such as MGMT promoter methylation status and treatment interval play crucial roles in determining patient outcomes after second surgery. Personalized treatment strategies should consider these factors to optimize the management of rGBM. Prospective research is needed to define the value of re-RT after second surgery and to inform decision making in this situation.

Abstract 4638: Integrated proteogenomic characterization of longitudinal glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor, with no curative treatment options and median patient survival time of almost one year. Despite multi-modal therapy including surgery, irradiation and chemotherapy, all patients experience tumor progression and recurrence. Our group proposed and validated the first single cell guided functional classification of primary GBM in four tumor-intrinsic cell states which informed clinical outcome and delivered therapeutic options. However, recurrent GBM remains therapeutically unresolved due in part to the diffusely invasive nature and in part to marked cellular heterogeneity of the tumor.The evolutionary trajectory of glioblastoma after therapy is a multifaceted biological process that extends beyond discrete genetic alterations alone. Here, we profiled by multi-omics platforms the largest dataset of matched primary and recurrent GBM including 123 longitudinal glioblastoma pairs, temporally separated by standard-of-care treatment. Genomics, proteomics, and phosphoproteomics all independently captured the loss of proliferative-progenitor characteristics and a significant upregulation of specialized neuronal and synaptic signaling programs in recurrent GBM. Proteomic and phosphoproteomic analyses revealed that the molecular transition from proliferative to neuronal state at recurrence is marked by coherent post-translational activation of the WNT/PCP signaling pathway and the BRAF protein kinase. Multi-omic analysis of Patient-Derived Xenograft (PDX) models mimicked the patterns of evolutionary trajectory, consisting of marked activation of neuronal signaling programs in recurrent patients. Inhibition of the BRAF kinase with small molecule inhibitors impaired both neuronal transition and migration capability of recurrent glioblastoma cells, which are the phenotypic hallmarks of glioblastoma progression after therapy. Accordingly, combinatorial treatment of temozolomide with BRAF inhibitor, vemurafenib, significantly prolonged the survival of mouse PDX models. This work provides comprehensive insights into the biological mechanisms of glioblastoma evolution and treatment resistance and highlights new therapeutic opportunities to effectively counter them in the clinic. Citation Format: Simona Migliozzi, Kyung-Hee Kim, Harim Koo, Jun-Hee Hong, Seung Min Park, Hyung Joon Kwon, Luciano Garofano, Young Taek Oh, Fulvio D'Angelo, Chan Il Kim, Anna-Luisa Di Stefano, Franck Bielle, Jinlong Yin, Marc Sanson, Do-Hyun Nam, Jason K. Sa, Anna Lasorella, Jong Bae Park, Antonio Iavarone. Integrated proteogenomic characterization of longitudinal 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 4638.

Abstract 2781: GBM cells mimic regulatory T cell function to protect the CSC pool from immune surveillance in recurrent GBM

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer, with incredibly high recurrence rates and virtually no long-term survival. Despite our growing understanding of GBM at the molecular level, we still don’t fully comprehend the molecular mechanisms driving recurrence in GBM. Current knowledge indicates that Glioma Stem Cells (GSCs) drive a resistant cell phenotype in GBM. Understanding the molecular events coordinated by GSCs leading to therapy-resistance can provide key advancements on how we treat recurrent GBM (rGBM). Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. The cross talk between cancer cells and the immune system is now classified as a hallmark of cancer. The current dogma is that cancer cells, including GSCs, influence recruitment of immune-suppressive cell subsets to tumor tissue, however how subsets of GSCs mimic this immune-suppressive effect within the tumor microenvironment remains to be elucidated. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3− cell subpopulation with high expression of TGFβ1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with regulatory T cell (Tregs) and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with TGFβ type II receptor (TGFBR2) expression in clinical GBM. Mechanistically, we show that blocking TGFBR2 or XBP1 signaling, key intermediary of this process, counteracts the immune-suppressive phenotype of rGBM cells by restoring CD4 and CD8 tumor killing capacity and reversing exhaustion in co-culture experiments. siRNA-mediated knock-down of TGFBR2 in rGBM cells also decreased CD206 expression and reduced release of anti-inflammatory chemokines in THP1 cells. By combining miRNA-based network analysis and advanced nanoparticle formulation for miRNA delivery we show that miR-16/124-3p effectively target all nodes of this immunosuppressive axis successfully blocking the immunosuppressive nature of rGBM cells. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Bachchu Lal, Amanda Johnson, Stephany Tzeng, Jordan Green, John Laterra. GBM cells mimic regulatory T cell function to protect the CSC pool from immune surveillance in recurrent GBM [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 2781.

Abstract 387: Ionizing radiation induces lipid metabolism-associated vulnerabilities in glioblastoma multiforme

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to its current standard-of-care regimen, which includes surgical resection followed by adjuvant ionizing radiation and temozolomide. Therapy resistance can be attributed to the abundance of genomic alterations in GBM tumors that evolve over time, which contribute to intra-and inter-tumoral heterogeneities that render targeted therapies as inadequate treatment options. Thus, there is an urgent unmet need for effective therapies targeting primary and recurrent GBM. Here, we investigated the impact of ionizing radiation on the transcriptome of patient derived GBM tumor spheres to identify key alterations and biological pathways involved in therapy resistance. Materials and methods: We acquired four independent human GBM tumor sphere cell lines, which were derived from patients and cultured in serum-free media. Tumor spheres were treated with either a sham radiation or a single dose of 10Gy. Using poly-A enrichment whole transcriptome sequencing, we evaluated transcriptional alterations occurring in GBM tumors spheres at 96h post-radiation. To do so, we performed differential expression analysis and gene set enrichment analysis (GSEA) to identify top differentially expressed genes and significant changes in biological phenomena. Additionally, we evaluated the cell viability response of these GBM tumor spheres to radiation. Results and discussion: PCA analysis of the four human GBM tumor sphere cell lines demonstrates that cell type has a substantial impact on variation among samples compared to non-irradiated and irradiated treatment conditions. Upon analysis of genes in common that are either upregulated or downregulated followed by GSEA, we identified enrichment of genes associated with inflammatory responses and ferroptosis repression (e.g., NUPR1, PTGS1, AOX1), and depletion of genes involved in fatty acid metabolism (e.g., INSIG1, PLA2G3, ACAT2). Furthermore, the GBM tumor sphere cell viability responses allowed us to stratify our models into radiosensitive and radioresistant subgroups. Conclusion: Radiosensitive human GBM tumor spheres exhibit transcriptional alterations in genes linked to inflammation and fatty acid metabolism to a greater extent compared to radioresistant tumor spheres. Our study characterizes the radiation response of patient derived GBM models, which is to be leveraged for combating therapeutic resistance. Citation Format: Arianna Richelle Izawa-Ishiguro, Subhiksha Nandakumar, Nicholas Carbone, Annalisa V. Ferrotta, Raashed Raziuddin, Kristen C. Vogt, Daniel A. Heller, Ingo K. Mellinghoff. Ionizing radiation induces lipid metabolism-associated vulnerabilities in glioblastoma multiforme [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 387.

Abstract 5496: Multi-omic spatial analysis of the tumor microenvironment in gliomas

Abstract Introduction: The diverse tumor environment of high-grade glioma, which remains refractory to treatment, demands new innovative, multi-omic approaches to characterize tumor heterogeneity and expression profiles associated with progression and therapeutic response. Approach: We have applied an integrated, multi-omic approach using paired spatial in situ sequencing (Xenium) and protein profiling (PhenoCycler® Fusion) of glioma tissue to identify distinct glioma cellular phenotypes, activation states and metabolic pathways at true single cell resolution. Our multi-omic approach used hundreds of phenotypic RNA target probes and >50 antibodies in an unbiased single-cell analysis of primary, recurrent, and IDH1 mutant/wild type tumors with a range of heterogeneity. Summary: We were able to distinguish differentially regulated genes and pathways between aggregated primary and recurrent GBM, including cell cycle pathways being upregulated in primary vs recurrent GBM glial cell clusters and a down regulation of ERBB4 signaling in vascular cell clusters in primary GBMs. Our data also suggests that SOX11, known to be involved in tumorigenesis, is >2-fold upregulated in annotated tumor cells in primary compared to recurrent tumors. We were also able to quantitatively localize the expression of Epidermal Growth Factor Receptor, variant III (EGFRvIII) to specific tumor-cell sub-types, which is important given its association with therapeutic resistance. Further, we have been able to establish the cellular neighborhoods and cell-cell and receptor-ligand relationships within the tumors. Our spatial protein analysis identified distinct tumor and immune phenotypes with varying abundance of myeloid and lymphoid populations in IDH1 wt and mt tumors. Moreover, spatial proximity analyses and cellular neighborhood analyses revealed differences in higher order organizational landscapes that may contribute to the differential outcomes across wt and mt tumor subtypes. Conclusion: Multiomic spatial analysis enables deeper characterization of the glioma cellular and functional landscape to broaden our understanding of the key TME features that contribute to disease pathogenesis and prognoses. Our study provides an analytical framework to combine RNA and protein-based spatial data for a holistic investigation into a variety of glioma subtypes and aid in the identification of novel biomarkers, spatial neighborhoods, and functional states that drive glioma progression. Citation Format: Dmytro Klymyshyn, Vaibhav Jain, Lauren Whaley, Emily Hocke, Bassem Ben Cheikh, Alan Smith, Karen Abramson, Nadine Nelson, Diane Satterfield, Elizabeth Thomas, Giselle Lopez, Seetha Hariharan, Michael Brown, Niyati Jhaveri, Roger McLendon, David Ashley, Matthew Waitkus, Simon Gregory. Multi-omic spatial analysis of the tumor microenvironment in gliomas [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 5496.

Abstract 5238: uPAR as a novel immunotherapeutic target in recurrent glioblastoma

Abstract Glioblastoma (GBM) is the common malignant brain tumor in adults, accounting for approximately 15% of all CNS tumors, and 48.6% of malignant brain tumors, with a median survival of approximately 15 months, and minimal clinical progress having been made in the past two decades. GBM is characterized by extensive inter- and intra-tumoral heterogeneity as well as an extremely immunosuppressive tumor microenvironment. Following Standard-of-Care (SoC) surgical resection and chemoradiotherapy at primary diagnosis, few therapeutic avenues exist at recurrence, owing in part due to a lack of clinically relevant targets. Data from our multi-institutional target pipeline shows that the extracellular urokinase plasminogen activator receptor (uPAR) is significantly upregulated at recurrence on putative GBM brain tumor initiating cells (BTICs), which are believed to drive de novo tumor formation, tumor recurrence, and therapeutic resistance. uPAR plays an important role in the plasminogen activation system, and in the context of cancer, has been implicated in numerous pro-tumorigenic processes such as invasion, proliferation, epithelial-to-mesenchymal transition, and therapy resistance. We used CRISPR-Cas9 to genetically delete uPAR from our in-house patient-derived GBM cell lines, and found that in vitro, knockout of uPAR expression in recurrent GBM cells significantly reduces proliferation and sphere formation capacity, two stem-like traits associated with BTICs. Additionally, we found that uPAR knockout significantly reduced the frequency of BTICs in a given population. Additionally, uPAR knockout cells display increased sensitivity to standard-of-care chemoradiotherapy, implicating uPAR expression in therapy resistance, as seen in recurrent disease. We orthotopically injected immunocompromised mice to generate patient-derived xenograft models of GBM, and found that knockout of uPAR significantly increases survival, and decreases tumor burden. Further, we generated novel anti-uPAR single domain antibodies (sdAbs) and found that they specifically bind uPAR at low nanomolar concentrations in vitro. Using these sdAbs, we created an anti-uPAR CAR T which showed potent cytotoxicity in vitro, and drastically reduced tumor burden and extended survival in vivo. From this work we believe uPAR to be a clinically relevant target in recurrent GBM, and further investigation into therapeutic strategies to target uPAR-positive GBMs should be investigated further. Citation Format: William T. Maich, Muhammad Vaseem Shaikh, Anish Puri, Alisha Anand, Chirayu Chokshi, Sabra Salim, Neil Savage, Chitra Venugopal, Martin Rossotti, Thomas Kislinger, Kevin Henry, Sheila Singh. uPAR as a novel immunotherapeutic target in recurrent 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 5238.

Abstract 1151: Reconstruction of the spatial ecosystem of glioblastoma reveals relationships between tumor cell states and microenvironment

Abstract Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor, with no curative treatment options. Multiple studies have characterized at single cell resolution the GBM as being composed of transcriptional cell states interconnected with components in the tumor immune microenvironment (TME). Our group proposed and validated the first single cell guided functional classification of GBM in four tumor-intrinsic cell states which informed clinical outcome and delivered therapeutic options. However, single cell technologies are unable to unravel the spatial relationships among the cell states of GBM and between GBM cell states and TME. Spatially resolved transcriptomic technologies are emerging as powerful tools to reconstruct the spatial architecture of a tissue. We performed spatial transcriptomics of multicellular regions of interest (ROI) in 6 primary IDH wild-type GBM and 2 recurrent GBM with both CosMx Spatial Molecular Imager, which analyzes 1,000 RNA probes and 64 proteins at single cell resolution, and GeoMx Digital Spatial Profiler which profiles the whole transcriptome (~18,000 genes) at ROI resolution. The development of computational tools aimed to integrate spatial proximity and CosMx derived single-cell transcriptomics revealed spatial segregation of the tumor cell clones and cellular states and highlighted recurrent patterns of cell states, distinct TME cell types associated with coherent histopathological features across multiple samples. The development of a spatial informed intercellular communication algorithm and the reconstruction of ligand-receptor-target networks will allow the discovery of tumor cell states-TME cross-talks and the biological signaling regulated by these interactions that are driving the heterogeneity of GBM and therefore potentially therapeutically targetable. Analysis of matched regions of interest profiled by GeoMx and spatial proteomics with CosMx further cross-validated the spatial ecosystem of glioblastoma as reconstructed at single-cell resolution. Our studies established a scalable approach to resolve the transcriptional heterogeneity of GBM and reconstruct the architecture of GBM cell states and tumor microenvironment. Citation Format: Bruno Adabbo, Simona Migliozzi, Luciano Garofano, Fulvio D'Angelo, Pedro Davila, Sakir H. Gultekin, Daniel Bilbao Cortes, Benjamin Currall, Sion L. Williams, Marc Sanson, Franck Bielle, Anna Luisa Di Stefano, Michele Ceccarelli, Anna Lasorella, Antonio Iavarone. Reconstruction of the spatial ecosystem of glioblastoma reveals relationships between tumor cell states and microenvironment [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 1151.