Burns, Terry C.

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating-free DNA (cfDNA) blood levels in due to the blood-brain barrier (BBB). Real-time image-guided low-intensity focused ultrasound (LIFU) with intravenously administered microbubble oscillators non-invasively causes BBB disruption (BBBD). This clinical trial aimed to evaluate the utility of LIFU for increasing cfDNA in blood for liquid biopsy in GBM. METHODS LIBERATE is an ongoing, prospective, multi-center, self-controlled, ongoing, pivotal trial evaluating the safety and technical efficacy of LIFU for BBBD to increase cfDNA in blood for GBM. Patients aged 18-80 years with suspected GBM planned for tumor biopsy or resection at ten centers in the US are being included. Patients are administered intravenous oscillating microbubbles for enhancing sonication, after which MR-guided BBBD using 220 kHz LIFU device is performed, with real-time acoustic feedback for effective sonication. Pre- and post-procedure, phlebotomies, and MRI brain are performed. The primary study endpoint is defined, per subject, as the ratio between cfDNA levels in blood 1-hour post-LIFU procedure compared to cfDNA level in blood pre-procedure. The primary study hypothesis is that BBBD with LIFU leads to ≥2-fold increase in cfDNA in blood. The secondary hypothesis is that there exists ≥75% concordance between biomarker patterns in the cfDNA sample from 1-hour post-LIFU sample and tumor tissue obtained later. Exploratory endpoints include (1) sensitivity of detection of known somatic mutations in cfDNA from blood samples collected before and after LIFU, (2) estimation of cfDNA levels in samples collected at 30 minutes, 1 hour, 2 hours, and 3 hours post-LIFU to determine the time of greatest yield, (3) correlation of MRI parameters related to BBBD grading and biomarkers in cfDNA from post-LIFU samples. Patient enrollment commenced in 2022, and 13 patients have been recruited by 06/09/2023. Clinical trial information: NCT05383872.

Abstract OBJECTIVE This study aimed to analyze the hemorrhagic risk of melanoma brain metastases following SRS. METHODS A retrospective analysis was conducted using a prospective institutional database between 1990 and 2021. Lesional hemorrhage was categorized as definite or possible, and hemorrhage severity was graded using CTCAE. RESULTS The study identified 291 patients with 1083 lesions. The mean age of patients was 60 ± 15 years, with 61% being male. Median follow-up was 11 months, corresponding to 581 patient-years. Among the patients, 82 (28%) experienced 135 lesional hemorrhages (13%). Of these, 88 lesions in 61 patients were grade 1-2 hemorrhages, 42 lesions in 25 patients were grade 3, and five lesions in five patients progressed from grade 1 to 3. Surgical intervention was required for 17 lesions (2%) in 16 patients (5%). Lesional hemorrhage was associated with radiosurgery performed between 2015 and 2021 (OR, 0.373; 95% CI, 0.231-0.60; p < 0.001), increasing marginal dose (OR, 0.89; 95% CI, 0.82-0.98; p = 0.017), antiplatelet use (OR, 0.192; 95% CI, 0.082-0.45; p < 0.001), and WBRT (OR, 0.51; 95% CI, 0.330-0.77; p = 0.0017). The cumulative lesional hemorrhage risk reached 17–20% at 36 months following radiosurgery, with 95-96% of hemorrhages occurring within the initial year. Median OS was 11 months (95% CI, 9-13 months). Patients who received radiosurgery between 2015 and 2021 exhibited a median OS of 17 months (95% CI, 10-24 months; p < 0.001), while those without hemorrhage had a median OS of 12 months (95% CI, 10-14 months; p = 0.054). CONCLUSIONS Definitive hemorrhage risk was 17% for melanoma brain metastases after SRS within the first three years, with 95% of lesional hemorrhages occurring within the initial year. Surgical intervention was necessary in 5% of patients. Hemorrhage risk and overall survival improved after 2015.

Abstract Few methods exist via which to assess treatment response with high fidelity in gliomas. While CSF has previously been used as a source of cfDNA, it remains unknown how cfDNA abundance and sequencing changes throughout treatment and inevitable recurrence in gliomas. We utilized our biobank of longitudinal intracranial CSF specimens to determine the potential utility of CSF cfDNA as a monitoring tool for treatment response in patients with gliomas. Longitudinal CSF specimens were acquired via Ommaya reservoirs (NCT04692337) or ventriculoperitoneal shunts (NCT04692324) in patients with gliomas. cfDNA was extracted from 1-5 mL of CSF, and next-Generation sequencing or low-pass whole genome sequencing (LPWGS) was performed by Predicine, Inc. CSF acquired prior to versus after resection demonstrated an increase in quantified cfDNA (2.97x, range: 1.58-5.26x), consistent with the impact of increased parenchymal disruption and closer contact with CSF. Thereafter, CSF cfDNA continued to decrease throughout chemoradiation and adjuvant treatment and increased at recurrence, including in patients co-enrolled in immunotherapy trials for IL-7 agonists and pembrolizumab. Copy number burden (CNB) increased by the time of recurrence in three patients with known disease progression, and continued to decrease throughout immunotherapy treatment in one patient despite concerns of pseudoprogression. In one patient with a hypermutated glioblastoma, CSF cfDNA revealed over 59 genomic alterations, including MAP2K1, KIT, and PDGFRA, excluding even more variants of unknown significance, each of which had been detected in sequencing of the tissue acquired at resection and subsequently decreased with chemoradiation. In another patient with a known progressing EGFR-amplified GBM, over 200 new variants were identified by the patient’s final sample and EGFR copy number increased from 2 to over 30. In conclusion, cfDNA analysis over a patient’s disease course, including resection and treatment with standard-of-care and experimental therapies, may be of use for disease monitoring and treatment response.

Abstract Development of a non-invasive test that can measure drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase clinical trials of a new compound’s pharmacological suitability and potential efficacy. We present here data supporting that the cargo within extracellular vesicles (EV) can potentially serve as a liquid biopsy that reports on the pharmacodynamics of GBM therapeutics. We treated short-term PDX cultures with vehicle, temozolomide, MLN4924, or arsenic trioxide. EVs were isolated from culture supernatants using size exclusion chromatography and then analyzed by mass spectrometry (proteomics) and RNA sequencing to characterize and compare cargo profiles for each condition. We found that at the proteome level, FDFT1 and FARSB were detected only in temozolomide EVs across all cell lines. In addition, there were drug-agnostic indications of exposure, including decreased abundance in all treated EV conditions of SLC3A2, C3, PLG, and A1BG, and increased abundance of GSTP1. These cargo changes were further validated by western blotting and ExoView immunofluorescence on isolated EVs. At the EV-transcriptome level, depending on treatment, there were shared alterations in EV cargo between models in the expression of long non-coding RNAs, genes that are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis, and circular RNAs. Thus, these results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To allow immunoprecipitation of patient GBM-derived EVs with the selective interrogation, the potential biomarkers, including PTPRZ1, B7H3, IL13RA2, and EGFR were screened across isolated EVs from both pre-clinical and clinical patient samples. Future efforts are focused on evaluating immunoprecipitated GBM-EVs from longitudinally collected patient biofluids. 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.

OBJECTIVE The objective of this study was to analyze the hemorrhagic risk of melanoma brain metastases after Gamma Knife radiosurgery (GKRS). METHODS A prospective institutional database was retrospectively queried to identify patients who underwent GKRS for melanoma brain metastases between 1990 and 2021. Lesional hemorrhage was defined as definite or possible based on radiologists’ readings, and severity was graded according to Common Terminology Criteria for Adverse Events. RESULTS Two hundred ninety-one patients with 1083 lesions treated in 419 sessions were identified. The mean (± SD) patient age was 60 ± 15 years, and 61% were male. The median follow-up period for overall survival (OS) was 11 (range 0–214) months with 581 patient-years. Definite/possible lesional hemorrhages occurred in 13% of lesions, with grade 3 hemorrhages observed in 4% of lesions. Surgical intervention was required in 2% of cases (5% of patients), and all resected lesions were pathologically consistent with melanoma. A decreased risk of definite/possible lesional hemorrhage was associated with a later time period between 2015 and 2021 (OR 0.45, 95% CI 0.266–0.75, p = 0.0021), increased marginal dose (OR 0.91, 95% CI 0.83–0.99, p = 0.037), antiplatelet use post-GKRS (OR 0.195, 95% CI 0.083–0.46, p < 0.001), and whole-brain radiotherapy (WBRT; OR 0.53, 95% CI 0.344–0.82, p = 0.0042). After 2015, more patients received anticoagulation, B-Raf proto-oncogene inhibitors, and immune checkpoint inhibitors, and fewer received bevacizumab (p < 0.001). The cumulative risk of lesional hemorrhage was 17%–20% at 36 months from GKRS, with 95%–96% of cases occurring within 12 months. The median patient OS was 11 (95% CI 9–13) months, and multivariate Cox regression analysis revealed that antiplatelet agents (hazard ratio [HR] 0.66, 95% CI 0.45–0.96, p = 0.031) and immune checkpoint inhibitors (HR 0.35, 95% CI 0.26–0.48, p < 0.001) were associated with longer OS, while WBRT (HR 1.36, 95% CI 1.02–1.81, p = 0.037) and definite/possible hemorrhage (HR 1.39, 95% CI 1.04–1.85, p = 0.024) were associated with shorter OS. CONCLUSIONS The definite hemorrhage risk of melanoma brain metastases after GKRS was 17% in the first 3 years and 95% of the lesional hemorrhage occurred within the 1st year. Surgical intervention was needed in 5% of patients. Antiplatelet agents and immune checkpoint inhibitors were associated with improved OS, while definite/possible hemorrhage was associated with worse OS.

Abstract BACKGROUND Cerebrospinal fluid (CSF) is emerging as an abundant source of candidate biomarkers, including proteins, for gliomas. Longitudinal CSF access may enable insights into treatment response and resistance. To that end, we have established a growing biobank of CSF samples, many of which have been obtained longitudinally via CSF access devices, such as Ommaya reservoirs, through our ongoing biomarker discovery clinical trials (NCT04692337, 04692324). We have previously presented our initial data demonstrating a conserved proteomic signature of HGG that decreased with resection and increased with recurrence. Since then, samples have been acquired throughout individual patients’ treatments toward our goal of identifying CSF-derived biomarkers of treatment response. MATERIAL AND METHODS CSF samples obtained prior to, during, and throughout treatment underwent global proteomics analysis via the SomaScan® assay by Somalogic, which uses slow off-rate modified aptamers to sensitively detect over 7,000 proteins. Fold-changes for each protein were calculated between different timepoints for each patient and ranked based on the comparison of interest. Comparisons included prior to versus after resection, chemoradiation or adjuvant temozolomide (each with or without pembrolizumab), bevacizumab, and low versus high blood contamination. RESULTS Treatment with pembrolizumab alone resulted in increased IL-23 (5.01x), IL-12 (4.25x), lymphocyte activation gene 3 protein (LAG3) (3.26x), and granzyme A (2.93x). Chemoradiation, with or without pembrolizumab, consistently downregulated NAD kinase, acetyl CoA transferase, and Bcl-2 and upregulated proteins related to neuronal signaling, such as neurexins and cerebellins, in addition to LAG-3, suggesting potential for synergy between chemoradiation and anti-LAG-3 antibodies currently in clinical trials. Interestingly, pembrolizumab and chemoradiation decreased MMP-12 and MMP-9 abundance as compared to chemoradiation alone (0.25x vs. 18.5x; 0.54x vs. 4.99x). Finally, bevacizumab treatment consistently increased VEGF (7.32x), GFAP (3.38x), and MMP-3 (2.99x) abundance, while decreasing the presence of our previously reported plasma-associated protein signature in CSF, suggesting decreased blood-brain barrier disruption. CONCLUSION The global CSF proteome can be longitudinally accessed to evaluate response to standard-of-care and experimental therapies. Further studies are ongoing to identify signatures of therapeutic resistance and to assess the impact of novel immunotherapeutic combinations on the CSF proteome.

Abstract BACKGROUND D-2-hydroxyglutarate (D-2-HG) is a well-established oncometabolite of isocitrate dehydrogenase (IDH) mutant gliomas. While prior studies have demonstrated that D-2-HG is elevated in the cerebrospinal fluid (CSF) of patients with IDH-mutant gliomas, no study has evaluated the ability of CSF D-2-HG to quantitatively monitor tumor burden and therapeutic response. Toward this goal, we are routinely obtaining CSF samples from patients prior to, during, and after treatment via intra-operative collection and CSF-access devices. MATERIAL AND METHODS Patients were enrolled into our Ommaya reservoir study (NCT04692337), which enables longitudinal access to CSF. The catheter for the implanted reservoir was placed within the surgical resection cavity following a standard-of-care resection. Intra-operatively collected CSF samples were also biobanked through Mayo Clinic’s Neuro-Oncology biorepository. CSF samples were also acquired via clinically indicated ventriculoperitoneal shunts (VP shunts) or external ventricular drains via our ongoing CSF biomarkers study (NCT04692324). D-and-L-2-HG were assayed from each CSF sample via gas chromatography-mass spectrometry (GC-MS) at Mayo Clinic’s Biochemical Genetics Laboratory. RESULTS CSF was collected prior to and after resection in patients via Ommaya reservoirs or VP shunts, all of whom underwent near gross total resections (&gt;95% of tumor volume). D-2-HG and the D/L ratio fell on average by 57.9% (range: 15.1%-98.6%) and 68.8% (range: 49.3%-98.9%), respectively, following resection. In one patient with a largely intraventricular grade 4 IDH-mutant astrocytoma, D-and-L-2-HG fell by 99.1% and 99.3% by post-operative day 2, consistent with the impact of ventricular contact on baseline CSF D-2-HG levels. In patients for whom CSF was collected throughout chemoradiation, with or without immunotherapy, D-2-HG and the D/L ratio continued to decrease over time, despite concern for pseudoprogression versus progression on imaging. CONCLUSION Our early experience with this strategy demonstrates the potential for intracranial CSF D-2-HG as a monitoring biomarker for IDH-mutant gliomas. Work is ongoing to evaluate the reproducibility of these findings in a larger cohort of patients with low or high-grade gliomas, in addition to the impact of tumor recurrence on 2-HG. Future studies will evaluate the impact of IDH inhibitors and other experimental therapies on CSF 2-HG levels for pharmacodynamic monitoring of treatment response.

Abstract BACKGROUND Few methods exist via which to assess treatment response with high fidelity in gliomas. Cell-free DNA (cfDNA) is of growing interest as a liquid biomarker in plasma and CSF and has been used in other cancer types to optimize therapeutic regimens for individual patients. While CSF has previously been used as a source of cfDNA for gliomas, it remains unknown how cfDNA abundance and sequencing change throughout treatment and inevitable recurrence. To that end, we leveraged our growing biobank of longitudinal CSF specimens, acquired via CSF access devices, to determine the potential utility of CSF cfDNA as a monitoring tool for treatment response in patients with gliomas. MATERIAL AND METHODS Longitudinal CSF specimens were acquired via Ommaya reservoirs (NCT04692337) or ventriculoperitoneal shunts (NCT04692324) in patients with brain tumors, including gliomas. cfDNA was extracted from 1-5 mL of CSF based on available sample volumes and quantified via Qubit. Next-Generation sequencing (PredicineCARE) or low-pass whole genome sequencing (LPWGS) was performed by Predicine, Inc based on the amount of cfDNA available. RESULTS CSF acquired prior to versus after resection demonstrated an increase in quantified cfDNA (2.97x, range: 1.58-5.26x), consistent with the impact of increased parenchymal disruption and closer contact with CSF. Thereafter, CSF cfDNA continued to decrease throughout chemoradiation and adjuvant treatment and increased at recurrence, including in patients co-enrolled in immunotherapy trials for IL-7 agonists and pembrolizumab. Interestingly, copy number burden (CNB) increased by the time of recurrence in three patients with known disease progression, and continued to decrease throughout immunotherapy treatment in one patient despite concerns of pseudoprogression. In one patient with a hypermutated glioblastoma, CSF cfDNA revealed over 59 genomic alterations, including MAP2K1, KIT, and PDGFRA, and over 100 more variants of unknown significance, each of which had been detected in sequencing of the tissue acquired at resection and subsequently decreased with chemoradiation. In another patient with a known progressing EGFR-amplified GBM, over 200 new variants were identified by the patient’s final sample and EGFR copy number increased from 2 to over 30. CONCLUSION Analysis of cfDNA over a patient’s disease course, including resection and treatment with standard-of-care and experimental therapies, may be of use for disease monitoring and treatment response. Further work is needed to evaluate these findings in a larger cohort of patients and to determine the sensitivity of changes in mutations, CNB, and cfDNA quantity for glioma disease burden.

Abstract BACKGROUND The microenvironment of glioblastoma is complex and defined in part by protumorigenic metabolic changes. Within this microenvironment, blood-brain barrier disruption is often associated with the most aggressive portions of the tumor. To date, the metabolic tumor microenvironments of enhancing, blood-brain barrier (BBB) disrupted portions of gliomas, as compared to BBB-intact tumor, remain poorly understood. Specifically, it is unclear if the associated blood-brain barrier disruption merely reflects or functionally supports tumor aggressiveness. METHODS Intra-operative high-molecular weight microdialysis was utilized under an investigational device exemption (NCT NCT04047264) to sample the extracellular metabolome of radiographically diverse regions during fifteen neurosurgical resections. The global extracellular metabolome of recovered microdialysate from contrast-enhancing and non-enhancing tumor, as well as brain adjacent to tumor, was evaluated via ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) on the Metabolon platform and assessed using enrichment and correlation analyses. RESULTS Among 162 named metabolites identified via ultra-performance liquid chromatography tandem mass spectrometry, a novel glioma-associated metabolite, guanidinoacetate (GAA), was 126.32x higher in enhancing tumor than in adjacent brain and 13.63x higher in non-enhancing tumor than brain. Forty-eight additional metabolites were 2.05-10.18x more abundant in enhancing tumor than brain, including carnitine family members and all nine large neutral amino acids. Except for GAA and 2-hydroxyglutarate in IDH-mutant gliomas, differences between non-enhancing tumor and brain microdialysate were comparatively modest and less consistent, suggesting heterogeneity in the glioma metabolic microenvironment of blood-brain barrier (BBB) intact areas as compared to BBB-disrupted portions of gliomas. Interestingly, the enhancing but not the non-enhancing glioma metabolome was significantly enriched for plasma-associated metabolites, largely comprising amino acids and carnitines. CONCLUSION Our patient-derived data suggest that the BBB-disrupted metabolome of high-grade gliomas is characterized by metabolite diffusion from circulation into the tumor microenvironment. Further studies are ongoing to determine how this alteration in the glioma extracellular metabolome impacts glioma behavior and how it could be therapeutically targeted via BBB-modulating drugs.

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating-free DNA (cfDNA) levels in blood due to the blood-brain barrier (BBB). This results in challenges to the identification of blood-based biomarkers and the development of novel biomarker-driven systemic therapies. Real-time image-guided low intensity focused ultrasound (LIFU) combined with intravenously administered microbubble oscillators non-invasively causes BBB disruption (BBBD). This clinical trial aimed to evaluate the utility of LIFU for increasing cfDNA in blood for liquid biopsy in GBM. METHODS LIBERATE is a prospective, multi-center, self-controlled, ongoing, pivotal trial evaluating the safety and technical efficacy of LIFU for BBBD to increase cfDNA in blood for GBM. Patients aged 18-80 years with suspected GBM planned for tumor biopsy or resection at ten centers in the US are being included. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered intravenous oscillating microbubbles for enhancing sonication, after which MR-guided BBBD using a 220 kHz LIFU device is performed with real-time acoustic feedback for effective cavitation. Before and after the procedure, phlebotomies and MRI brain are performed to evaluate outcomes. The primary study endpoint is defined, per subject, as the ratio between their cfDNA level in blood 1-hour post-LIFU procedure compared to cfDNA level in blood pre-procedure. The primary study hypothesis is that BBBD with LIFU leads to a ≥2-fold increase in cfDNA in blood. The secondary hypothesis is that there exists ≥75% agreement between biomarker pattern in cfDNA sample from 1-hour post-LIFU sample and biomarker pattern in tumor tissue obtained later. The trial has been powered to evaluate both primary and secondary hypotheses. Based on an assumed true agreement rate of 91% and a one-sided alpha of 0.025, an exact test for binomial proportions provides a sample of N=50 with 84% power for the secondary hypothesis. Exploratory endpoints include (1) sensitivity of detection of known somatic mutations in cfDNA from blood samples collected before and after LIFU, (2) estimation of cfDNA levels post-LIFU in samples collected at 30 minutes, 1 hour, 2 hours, and 3 hours to determine the time of greatest yield, (3) correlation of MRI parameters related to grading of BBBD and biomarkers positive in cfDNA from post-LIFU blood samples. RESULTS Patient enrollment commenced in 2022, and 8 patients have been recruited by 04/11/2023. CONCLUSION Findings from this trial will be critical to liquid biopsy in GBM (Clinical trial information: NCT05383872).