Burns, Terry C.

BACKGROUND AND OBJECTIVES: No new drug has improved survival for glioblastoma since temozolomide in 2005, due in part to the relative inaccessibility of each patientʼs individualized tumor biology and its response to therapy. We have identified a conserved extracellular metabolic signature of enhancing high-grade gliomas enriched for guanidinoacetate (GAA). GAA is coproduced with ornithine, the precursor to protumorigenic polyamines through ornithine decarboxylase (ODC). AMXT-1501 is a polyamine transporter inhibitor that can overcome tumoral resistance to the ODC inhibitor, difluoromethylornithine (DFMO). We will use DFMO with or without AMXT-1501 to identify candidate pharmacodynamic biomarkers of polyamine depletion in patients with high-grade gliomas in situ . We aim to determine (1) how blocking polyamine production affects intratumoral extracellular guanidinoacetate abundance and (2) the impact of polyamine depletion on the global extracellular metabolome within live human gliomas in situ. METHODS: DFMO, with or without AMXT-1501, will be administered postoperatively in 15 patients after clinically indicated subtotal resection for high-grade glioma. High–molecular weight microdialysis catheters implanted into residual tumor and adjacent brain will be used for postoperative monitoring of extracellular GAA and polyamines throughout therapeutic intervention from postoperative day (POD) 1 to POD5. Catheters will be removed on POD5 before discharge. EXPECTED OUTCOMES: We anticipate that GAA will be elevated in tumor relative to adjacent brain although it will decrease within 24 hours of ODC inhibition with DFMO. If AMXT-1501 effectively increases the cytotoxic impact of ODC inhibition, we expect an increase in biomarkers of cytotoxicity including glutamate with DFMO + AMXT-1501 treatment when compared with DFMO alone. DISCUSSION: Limited mechanistic feedback from individual patientsʼ gliomas hampers clinical translation of novel therapies. This pilot Phase 0 study will provide in situ feedback during DFMO + AMXT-1501 treatment to determine how high-grade gliomas respond to polyamine depletion.

Abstract Glioblastoma (GBM), a lethal astrocytic brain tumor, has limited available treatment options. A major hindrance to the progress of new therapies is the selectivity of the blood brain barrier (BBB) which limits drug access to the tumor. Further, there are currently no minimally-invasive tests that can quickly and accurately determine whether novel therapeutics have crossed the BBB during clinical trials. Therefore, development of a non-invasive test that can identify drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase trials of a new compound’s pharmacological suitability and potential efficacy. This study proposes that cargo contained in GBM-derived extracellular vesicles (EVs), which are small membrane-bound particles released from cells, may serve as one such pharmacodynamic reporter. We analyzed RNA-seq data for EVs isolated from two GBM patient-derived xenograft models grown as short-term in vitro cultures, GBM10 and GBM120, following treatment with either temozolomide (TMZ) or pevonedistat (MLN). We assessed whether EV RNA payloads vary with different drug concentrations (EC50 vs EC80) and at different time points (4hr vs 24hr) post treatment. In the samples treated with MLN, a few long non-coding RNAs are commonly upregulated across the different time points and two models. Under different TMZ concentrations, differentially expressed genes are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis. We also found a negative correlation between the TMZ concentration and Y RNA expression level, which are functionally involved with DNA replication. Additionally, using CIRI2, we predicted the amount of circular RNAs in the EV cargo; we found that the ratio of circular RNAs to their linear counterparts (CLR) varies under different treatments. In GBM10, the percentage of circRNA with CLR > 1 decreases with increasing drug effect. Thus, our preliminary results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To apply such an assessment to patients, we have simultaneously pursued the development of an immunoprecipitation (IP) approach to enrich GBM-derived EVs that can then undergo cargo profiling. Potential biomarkers were screened across isolated GBM EVs from in vitro supernatant by western blot and ExoView analysis. B7H3 has emerged as a lead candidate for GBM-EV enrichment. To validate the ability of EV cargo to reflect drug distribution across the BBB in patients, current efforts are focused on applying IP enrichment of GBM-EVs in longitudinally-acquired cerebrospinal fluid samples from patients during their disease course. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts. Citation Format: Valerie DeLuca, Nanyun Tang, Yue Hao, Anjali Raju, Charles Shaffer, Cecile Riviere-Cazaux, Terry C. Burns, Jann Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Extracellular vesicles as a pharmacodynamic reporter in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2788.

High-grade gliomas are the most common and aggressive adult primary brain tumors with a median survival of only 12–15 months. Current standard therapy consists of maximal safe surgical resection followed by DNA-damaging agents, such as irradiation and chemotherapy that can delay but not prevent inevitable recurrence. Some have interpreted glioma recurrence as evidence of glioma stem cells which persist in a relatively quiescent state after irradiation and chemotherapy, before the ultimate cell cycle re-entry and glioma recurrence. Conversely, latent cancer cells with a therapy-induced senescent phenotype have been shown to escape senescence, giving rise to more aggressive stem-like tumor cells than those present in the original tumor. Therefore, approaches are needed to either eliminate or keep these glioma initiating cells in a senescent state for a longer time to prolong survival. In our current study, we demonstrate that the radiation-induced cell cycle inhibitor P21 can provide a powerful route to induce cell death in short-term explants of PDXs derived from three molecularly diverse human gliomas. Additionally, cells not killed by P21 overexpression were maintained in a stable senescent state for longer than control cells. Collectively, these data suggest that P21 activation may provide an attractive therapeutic target to improve therapeutic outcomes.

Abstract Gliomas are genomically heterogeneous tumors that may harness a convergent and therapeutically targetable set of metabolic pathways. At present, the metabolic landscape of in situ human gliomas remains incompletely characterized, hampering translational progress. To that end, we are leveraging the previously untapped potential of high molecular weight microdialysis to determine the global extracellular metabolic profiles of live human gliomas. Under an investigational device exemption, HMW microdialysis (< 100 kDa) was performed at 2.0 μL/min in an initial discovery cohort of five patients in glioma and adjacent brain during neurosurgical resection; a subsequent cohort of five patients was independently analyzed to critically evaluate results from the discovery group. Untargeted metabolomics via ultra-performance liquid chromatography-tandem mass spectrometry revealed over 300 named metabolites and five drugs from only 20 μL of microdialysate, representing a short and feasible 10 minutes of intraoperative collection time. Enrichment analysis of each patient’s tumor vs. brain ranked extracellular metabolome highlighted marked metabolic convergence within the most aggressive regions of molecular diverse tumors (FDR = 0). Pathway analysis revealed significant enrichment for large neutral amino acid pathways, including valine, leucine, and isoleucine biosynthesis (p=1.6E-9) and degradation (p=0.001) and glycine, serine, and threonine metabolism (p=4.7E-5). Notably, this amino acid signature was not as abundantly present in nonenhancing tumor when compared to enhancing tumor (Average tumor/brain: 1.9x vs. 4.3x, respectively), suggesting upregulation of neutral amino acid transporters in enhancing tumor or delivery from plasma into the CNS via a disrupted BBB. Interestingly, guanidinoacetate (GAA) was our most highly conserved and upregulated metabolite (128.9x in tumor vs. brain). Given its co-production with ornithine, the precursor to protumorigenic polyamines, we posit that GAA may serve as a biomarker of increased ODC activity in live human gliomas. In conclusion, intraoperative HMW microdialysis feasibly offers improved opportunities to perform glioma metabolic biomarker and therapeutic discovery.

Abstract BACKGROUND Glioblastoma diagnostic criteria have been redefined with the 5th edition of WHO classification of tumors of the central nervous system. Glioblastomas are defined as IDH wildtype diffuse astrocytic glioma tumors with one of the following features: microvascular proliferation, or necrosis, or TERT promoter mutation, or EGFR gene amplification, or +7/-10 chromosome copy number changes. The aim of this study is to establish the impact of extent of resection in overall survival (OS) and progression free survival (PFS) in glioblastoma, IDH-wildtype (WT), WHO grade 4 (WHO 2021). METHODS Systematic literature search was performed using the following databases: PubMed, Web of Science, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews and ClinicalTrials.gov to identify studies comparing OS and PFS after gross total resection (GTR) vs subtotal resection (STR) or biopsy for glioblastoma IDH-WT. Prognostic hazard ratios (HR) for OS and PFS were analyzed using a random-effects model. RESULTS We identified 1439 publications. Nine studies met inclusion/exclusion criteria. 788 patients underwent GTR out of 1818. The meta-analysis showed a significant increase in OS and PFS duration when undergoing GTR for glioblastoma IDH-WT with a median OS of 20 months 95% CI (17-25) compared to 12 months 95% CI (9-15) for STR or biopsy and a median PFS of 11 months 95% CI (9-12) for GTR compared to 7 months 95% CI (5-7) for STR or biopsy respectively. GTR showed a 49% significant reduction of mortality risk HR=0.51 95%CI (0.42-0.59) and a 44% significant reduction of progression risk HR=0.56 95%CI (0.41-0.71) compared to STR or biopsy. CONCLUSIONS This systematic review indicates that GTR may be associated with improved OS and PFS compared to STR or biopsy for Glioblastoma, IDH-WT, WHO grade 4 (WHO 2021). However, this is limited by variable study design and significant clinical and methodological heterogeneity among studies.

Abstract BACKGROUND Glioblastoma (GBM) has dismal outcome of 14-16 months. One of the challenges of drug development in GBM is lack of requisite circulating-free (cfDNA) in blood samples to develop biomarker driven targeted therapy trials. The Exablate focused ultrasound with circulating microbubble resonators uses the concept of real-time imaging to treat small target spots focusing energy in a non-invasive manner, thereby disrupting the Blood-Brain Barrier (BBBD) with potential to increase cfDNA in blood. METHODS LIBERATE is a prospective, multi-center, self-controlled pivotal ongoing clinical trial in subjects with suspected GBM evaluating the safety and effectiveness of Exablate Model 4000 to disrupt BBB to achieve a greater proportion of cfDNA in samples taken post-BBB disruption as compared to the sample taken before BBBD. Fifty eligible subjects with suspected GBM who are scheduled to undergo brain tumor resection or biopsy at 10 centers in the U.S. will participate in this study. The primary endpoint is defined for each subject as ratio between his/her cfDNA amount found in the blood sample 1-hour post-BBBD procedure compared to the cfDNA amount found in the blood sample collected pre-BBBD procedure. This study proposes to demonstrate that on average there is at least 2-fold increase in cfDNA (measured by central laboratory) at 1 hour post BBBD. Confirmatory secondary analysis will evaluate correlation between patterns obtained in panel of biomarkers evaluated in the resected tumor tissue and/or biopsy sample and blood sample collected 1-hour post-BBBD. Exploratory endpoints will include: (1) sensitivity of detection of known somatic mutations in the circulating blood samples (circulating tumor DNA) before and after BBBD, (2) time of greatest yield of cfDNA in the samples collected post BBBD blood samples (at 30 min, 1-hour, 2-hour and 3-hour) to determine time of greatest yield, (3) correlation of biomarkers between the imaging and the post-BBBD biomarker samples. (NCT05383872)

Abstract The relative inaccessibility of a patient’s glioma throughout their disease course precludes individualized insights into tumor biology and responses thereof to therapeutic challenge. We propose that serial cerebrospinal fluid (CSF) samples can be used to perform glioma espionage in the form of biomarker discovery for analytes that are sensitive to tumor burden (for monitoring) and candidate therapies (for biological impact). To that end, we are routinely leveraging neurosurgical access to the CNS to collect CSF samples intraoperatively and from chronically implanted Ommaya reservoirs for longitudinal CSF access (NCT04692337, NCT04692324). Global proteomic analysis of CSF was performed on the Somalogic platform – an aptamer-based technology for highly sensitive and specific analysis of over 7,000 proteins. Using each patient’s ranked protein list (glioma vs. 3 control patients), enrichment analysis was performed in a discovery cohort of five patients to identify conserved proteins of glioma. To reveal candidate glioma monitoring, further enrichment analysis was performed to identify proteins that decreased with resection and increased with recurrence, using two sets of paired patient samples. Forty-four proteins met these enrichment criteria; this candidate high-grade glioma proteomic signature was highly significantly enriched in an independent cohort of twelve glioma patients (FDR=0 for all patients). The HGG signature decreased in abundance following resection in serial samples from two other patients, confirming decreased tumor burden. Top proteins included LANC2, DPYL3, Sorcin, Integrin A1B1, and lactate dehydrogenase (FC of glioma vs. control: 145.3x, 113.9x, 78.0x, 71.6x, and 62.7x, respectively). Interestingly, despite having explicitly chosen samples with little-to-no hemoglobin in the discovery cohort, this HGG signature was highly enriched for plasma-like proteins (FDR=0), suggesting a potential biological impact of blood-brain barrier disruption on the glioma extracellular proteome. In conclusion, our data demonstrate that the glioma extracellular proteome can feasibly be accessed to perform biomarker discovery (“espionage”) via serially-acquired CSF samples.

Abstract Diffuse gliomas are aggressive brain tumors that lack effective therapies. Despite advances in the molecular characterization of gliomas, targeted treatments have failed to improve patient outcomes to date. Here, we employed a functional genomics approach to rapidly identify therapeutic vulnerabilities and provide a strategy to individualize patient treatment. Our process combined comprehensive genomic and transcriptomic tumor profiling, custom algorithms and visualization software for data integration, and preclinical 3-dimensional ex vivo models (mcancers) for drug screening to assess response to therapeutic agents targeting specific molecular alterations. A total of 32 cases, including 5 oligodendrogliomas (1 primary and 4 recurrent), 5 astrocytomas (1 primary and 4 recurrent), and 22 glioblastomas (5 primary and 17 recurrent), were included in the study. Immunohistochemistry and integrated genomics confirmed that mcancers maintain key molecular alterations, including mutations, chromosomal rearrangements and focal amplifications, cellularity, and heterogeneity, faithfully recapitulating the original tumor. A total of 119 single drugs and 183 combinations were tested, including a panel of 26-brain penetrant targeted agents (18 single drugs and 8 combinations) that was curated to target common glioma oncogenic pathways and was tested across all cases. A strong correlation was observed between therapeutic vulnerabilities of glioma mcancers to targeted agents (EGFR, MET, PDGFR, HDAC inhibitors, etc.) and clinical experience. Single agent activity of panel compounds in the ex vivo setting was generally poor, with only 35% mCancers exhibiting > 70% growth inhibition at Cmax. In contrast, combination strategies, especially ones including epigenetic drugs, were significantly more effective (affecting 22 out of 32 mCancers), underscoring the molecular complexity of gliomas and the need for clinical trials testing combination treatments. Our data identified a number of novel combination treatments with significant efficacy across glioma subtypes and molecular profiles, suggesting that our functional genomics pipeline can guide treatment selection and inform clinical trial enrollment for gliomas.