Chaichana, Kaisorn L.

BACKGROUND AND OBJECTIVES: Despite standard of care with maximal safe resection and chemoradiation, glioblastoma (GBM) is the most common and aggressive type of primary brain cancer. Surgical resection provides a window of opportunity to locally treat gliomas while the patient is recovering and before initiating concomitant chemoradiation. The objective was to assess the safety and establish the maximum tolerated dose of adipose-derived mesenchymal stem cells (AMSCs) for the treatment of recurrent GBM. Secondary objectives were to assess the toxicity profile and long-term survival outcomes of patients enrolled in the trial. In addition, biospecimens will be collected to explore the local and systemic responses to this therapy. METHODS: We will conduct a phase 1, dose-escalated, nonrandomized, open-label, clinical trial of patients with GBM who are undergoing surgical resection for recurrence. Up to 18 patients will receive intracavitary application of AMSCs encapsulated in fibrin glue during surgical resection. All patients will be followed for up to 5 years for safety and survival data. Adverse events will be recorded using the CTCAE V5.0. EXPECTED OUTCOMES: This study will explore the maximum tolerated dose of AMSCs along with the toxicity profile of this therapy in patients with recurrent GBM. In addition, preliminary long-term survival and progression-free survival outcome analysis will be used to power further randomized studies. Finally, cerebrospinal fluid and blood will be obtained throughout the treatment period to investigate circulating molecular and inflammatory tumoral/stem cell markers and explore the mechanism of action of the therapeutic intervention. DISCUSSION: This prospective translational study will determine the initial safety and toxicity profile of local delivery of AMSCs for recurrent GBM. It will also provide additional survival metrics for future randomized trials.

Abstract The extracellular matrix (ECM) plays a critical role in cancer progression, with its biophysical properties influencing cancer behavior. ECM stiffness can induce long-lasting adaptations in cancer cells, promoting sustained invasion through a process known as “mechanical memory” (MM). Glioblastoma (GBM) exhibits spatial heterogeneity in stiffness, with stiffer ECM found at the tumor periphery where invasive cells reside and promote recurrence. We hypothesize that increased stiffness at the tumor periphery primes GBM cells for sustained invasion through MM. METHODS Cell lines were established from image-guided human GBM samples from tumor margin and core (n=10 patients, 20 cell lines). Cells from random locations were conditioned in physiologically relevant extracellular matrices for two weeks. Human organotypic assay (n=3), Migration Assays, Atomic Force Microscopy (AFM), Immunocytochemistry, Western Blot, and RNA-sequencing (n=6) were performed. RESULTS GBM cells at higher stiff surfaces displayed increased migration (p<0.01). Magnetic Resonance Elastography (n=9) confirmed GBM heterogeneity, with softer cores (0.2-0.5kPa) transitioning to stiffer margins (>2kPa). Organotypic scaffolds mimicking the tumor margin promoted enhanced migration of GFP-labeled GBM cells. Margin cells and cells conditioned on stiffer matrices demonstrated higher migratory potential compared to their core and soft-conditioned counterparts (p<0.05). These trends persist over time and across multiple passages. ECM stiffness exposure causes nanomechanical, cytoskeletal, and transcriptional adaptations. AFM shows that cells from the margin (n=6) are softer (p<0.01) and more deformable (p<0.01). RNAseq identified upregulation of MM-specific genes in GBM margin cells. The mechanosensitive transcription factor YAP1 emerged as a key driver of MM. Notably, YAP1 is targetable by the FDA-approved drug Verteporfin. YAP1 expression was elevated in margin and stiff-conditioned cells (p<0.05). Stiff-conditioned cells and margin cells displayed increased sensitivity to Verteporfin treatment (p<0.05), with treatment significantly reducing margin migration compared to the core (p<0.001). CONCLUSION Stiffer GBM margins promote the formation of aggressive phenotypes through MM. Targeting YAP1 with Verteporfin, a readily available drug, presents a promising strategy to halt GBM invasion and improve patient outcomes.

Abstract BACKGROUND Mouse double minute 2 (MDM2) inhibits the tumor suppressor protein 53 (p53). Brigimadlin, a potent oral MDM2-p53 antagonist, restores wild-type (wt) p53 function and has shown early efficacy in patients with solid tumors. Glioblastoma (GBM) is an area of unmet need with 5-year survival at <10%. In p53 wt GBM patient-derived xenograft models, brigimadlin promotes tumor cell apoptosis and extends survival in combination with radiotherapy (RT). METHODS NCT05376800 is a Phase 0/Ia non-randomized, open-label, single-arm trial that aims to measure brigimadlin concentration in brain tumor tissue in patients with histologically or radiologically newly diagnosed GBM eligible for resection (Phase 0), and determine the maximum tolerated dose of brigimadlin plus RT in patients with TP53 wt, IDH wt, MGMT promoter unmethylated GBM (Phase Ia). In Phase 0, patients received one brigimadlin dose (30 mg or 45 mg) ~12-24 hours before resection. Phase 0 primary endpoints are the measured total concentration and the calculated unbound concentration of brigimadlin in brain tissue homogenate from non-contrast enhancing (NCE) and contrast enhancing (CE) regions. The predefined threshold for trial continuation is 0.5 nmol/L (corresponding to IC50 in GBM cell lines) unbound brigimadlin in CE samples in ≥50% of patients. Brigimadlin concentration was measured using liquid chromatography-mass spectrometry and corrected for amount of brigimadlin in residual blood. Unbound concentration was calculated using an in vitro estimate of unbound fraction (fu): 0.654% (rat brain slice). fu in human plasma was 0.22%. Ratio of unbound concentration in brain versus plasma (Kp,uu) was calculated. Biomarker testing was performed. RESULTS Data are available for 11 patients (brigimadlin 30 mg: n=6; 45 mg: n=5). In the 30 mg group, median total brigimadlin concentration in NCE samples was 267 nmol/L (4 samples, range 86-316 nmol/L) and 332 nmol/L (6 samples, range 272-952 nmol/L) in CE samples. In the 45 mg group, median total brigimadlin concentration in NCE samples was 197 nmol/L (5 samples, range 140-347 nmol/L) and 603 nmol/L (5 samples, range 441-905 nmol/L) in CE samples. Unbound concentration exceeded the 0.5 nmol/L threshold in most cases. Post-brigimadlin, an increase in selected p53 target gene expression was observed in CE versus NCE tissue. CONCLUSION Unbound brigimadlin concentrations in CE regions in all patients receiving the low dose of 30 mg brigimadlin exceeded the 0.5 nmol/L threshold. Kp,uu in most patients was close to 1 in CE regions. Biomarker data support target engagement in brain tissue. Our findings support continued investigation of brigimadlin in GBM. Recruitment is ongoing. Updated data will be presented.The trial is funded by Boehringer Ingelheim.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially because of subventricular zone contact. Despite this, cross-talk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. In addition, GBM brain tumor–initiating cells (BTICs) increase expression of cathepsin B (CTSB) upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal that both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Last, we show LV-proximal CTSB up-regulation in patients, showing the relevance of this cross-talk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM.

2017 Background: MDM2 inhibits tumor suppressor p53. Brigimadlin, a potent MDM2–p53 antagonist, restores wild-type (wt) p53 function and has shown early efficacy in pts with solid tumors (LoRusso et al Cancer Disc 2023). GBM is an area of unmet need with 5-year survival <10%. In p53 wt GBM pt-derived xenograft models, brigimadlin promotes tumor cell apoptosis and extends survival in combination with RT. Methods: NCT05376800 is a Ph 0/Ia open-label, single-arm trial that aims to measure brigimadlin concentration in brain tumor tissue in pts with histologically or radiologically newly diagnosed GBM eligible for resection (Ph 0) and determine the maximum tolerated dose of brigimadlin plus RT in pts with TP53wt, IDH wt, MGMT promoter unmethylated GBM (Ph Ia). In Ph 0, pts received one brigimadlin dose (30 mg or 45 mg) ~12–24 h before resection. Ph 0 primary endpoints are the measured total concentration and the calculated unbound concentration of brigimadlin in brain tissue homogenate from non-contrast enhancing (NCE) and contrast enhancing (CE) regions. The predefined threshold for trial continuation is 0.5 nmol/L (corresponding to IC50 in GBM cell lines) unbound brigimadlin in CE samples in ≥50% of pts. Brigimadlin concentration was measured using LC/MS and corrected for amount of brigimadlin in residual blood. Unbound concentration was calculated using an in vitro estimate of unbound fraction (fu): 0.654% (rat brain slice). fu in human plasma was 0.22%. Kp,uu (ratio of unbound concentration in brain vs plasma) was calculated. Biomarker testing was performed. Results: Data are available for 11 pts (brigimadlin 30 mg: n=6; 45 mg: n=5). In the 30 mg group, median total brigimadlin concentration in NCE samples was 267 nmol/L (4 samples, range 86–316 nmol/L) and 332 nmol/L (6 samples, range 272–952 nmol/L) in CE samples. In the 45 mg group, median total brigimadlin concentration in NCE samples was 197 nmol/L (5 samples, range 140–347 nmol/L) and 603 nmol/L (5 samples, range 441–905 nmol/L) in CE samples. Unbound concentration exceeded the 0.5 nmol/L threshold in most cases (Table). Post-brigimadlin, an increase in selected p53 target gene expression was observed in CE vs NCE tissue. Conclusions: Unbound brigimadlin concentrations in CE regions in all pts receiving the low dose of 30 mg brigimadlin exceeded the 0.5 nmol/L threshold. Kp,uu in most patients was close to 1 in CE regions. Biomarker data support target engagement in brain tissue. Our findings support continued investigation of brigimadlin in GBM. Recruitment is ongoing. Updated data will be presented. Clinical trial information: NCT05376800 . [Table: see text]

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma ( P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.