Warrington, Arthur E.

Abstract Background Imaging-based glioma monitoring is confounded by treatment-related changes. D-2-hydroxyglutarate (D-2-HG), produced by the isocitrate dehydrogenase (IDH) mutation, is detectable in cerebrospinal fluid (CSF), which can be sampled from cranial or lumbar compartments. We evaluated CSF D-2-HG as a serially accessible biomarker for IDH-mutant gliomas, including the optimal compartment for longitudinal sampling. Methods Lumbar and cranial CSF samples were collected from patients with IDH-mutant gliomas or IDH-wild-type central nervous system pathologies via surgical field collection, lumbar punctures, and CSF access devices. CSF D-2-HG was quantified via our CLIA-certified gas chromatography mass spectrometry assay. Results D-2-HG was significantly higher in cranial than lumbar CSF from IDH-mutant glioma patients. Consistent with low D-2-HG abundance in lumbar CSF, lumbar samples could not discriminate IDH-mutant gliomas from IDH-wild-type lesions. In contrast, cranial CSF D-2-HG was significantly higher in IDH-mutant gliomas than wild-type lesions, providing an adequate baseline for initial evaluations of monitoring capabilities. Across 75 samples from 7 consecutive patients with grade 4 IDH-mutant astrocytomas, serial cranial CSF D-2-HG decreased with cytoreduction, remained unchanged with stable disease, and increased with disease progression, but not pseudoprogression. Conclusions Serial cranial CSF D-2-HG shows promise as a monitoring biomarker for IDH-mutant gliomas.

Abstract Polyamines are small molecules that play a critical role in tumor progression and in the regulation of immune responses within the tumor microenvironment. Elevated levels of polyamines in cerebrospinal fluid and tumor tissue have been reported in multiple studies and are shown to correlate with glioma grade. These findings suggest that the polyamine metabolic pathway may represent a therapeutic target in gliomas. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, expected to exert antitumor effects through polyamine depletion. Although the impact of DFMO against glioblastoma as a monotherapy appears limited, opportunities for synergy with other therapies remain understudied. In this study, we evaluated the therapeutic efficacy of DFMO using both in vitro and in vivo experiments with a murine glioma model (CT-2A) and patient-derived xenograft (PDX) cell lines. Among the models tested, CT-2A cells exhibited the highest expression of ODC1 and demonstrated strong sensitivity to DFMO in vitro. We compared two CT-2A variants: a commercially available line responsive to immunotherapy, and a widely used subline resistant to PD-1 blockade. While DFMO did not improve survival in mice implanted with the immunotherapy-sensitive commercial CT-2A line, it extended median survival by 8 days in mice bearing tumors derived from the immunotherapy-resistant subline. Transcriptomic analysis revealed that the DFMO-responsive, immunotherapy-resistant tumors exhibited lower baseline expression of immune-related genes, including those involved in interferon-β, interferon-γ, and IL-12 signaling pathways, compared to the immunotherapy-sensitive tumors. The latter showed markedly higher expression of STAT1 and interferon-γ and were enriched for immune-related Gene Ontology terms. These findings suggest that polyamine-targeted therapy may be appropriate for gliomas with an immunosuppressive, “cold” phenotype. Ongoing RNA sequencing analyses aim to further elucidate the immunomodulatory mechanisms of DFMO, and other agents comprising an adaptive toolkit to optimize and sustain anti-tumoral immunity.

Abstract BACKGROUND Many cancers demonstrate upregulated amino acid transport. In glioblastoma (GBM), evidence for upregulated amino acid flux includes high signal on amino acid PET imaging, increased expression of LAT1 (L-type amino acid transporter 1), and elevated extracellular amino acid levels via microdialysis. LAT1-targeted therapies have shown antitumor effects in certain cancer models by restricting essential amino acid supply and inhibiting the mTOR pathway, but their utility for GBM remains unknown. In this study, we evaluated impacts of a competitive LAT1 inhibitor, nanvuranlat, and a non-competitive inhibitor, JPH034 against glioblastoma. METHODS Murine (CT-2A, GL261) and human (GBM6, GBM39) GBM cell lines were tested under varying concentrations of nanvuranlat or JPH034. The competitive impacts of added essential amino acids, serum, and albumin were evaluated in vitro. Survival impacts were evaluated in an intracranial CT-2A glioma model. RESULTS Supplementing essential amino acids in vitro significantly decreased tumor cell sensitivity to nanvuranlat but did not change the IC50 of JPH034. Conversely, supplementing serum or albumin significantly reduced tumor cell sensitivity to JPH034 but did not change the IC50 of nanvuranlat. These findings were reproducible in all human and murine cell lines tested. In the intracranial mouse model, JPH034 treatment prolonged median overall survival from 16 days in the control group to 18.5 days, representing a 2.5-day extension (p = 0.060). CONCLUSION The antitumor effects of nanvuranlat and JPH034 on glioma cells in vitro, are influenced by concentrations of essential amino acids and albumin. Specifically, amino acids decreased impact of the competitive inhibitor (nanvuranlat) while albumin decreased impact of the non-competitive inhibitor (JPH034). These results suggest that the composition of the tumor microenvironment, which is impacted by BBB-disruption within GBM may differentially attenuate the therapeutic efficacy of certain drugs including LAT1 inhibitors.

Abstract Cerebrospinal fluid (CSF) cell-free DNA (cfDNA) is under evaluation as a liquid biopsy tool across multiple neuro-oncologic indications, including diagnosing lesions of uncertain etiology, delineating progression from pseudoprogression, and confirming suspected leptomeningeal disease (LMD). We evaluated the real-world performance of CSF cfDNA sequencing for identification of variant alleles (VA) using targeted panel sequencing (PredicineCARE) and/or copy number alterations (CNA) using low-pass whole-genome sequencing (LPWGS; PredicineSCORE) in a diverse cohort across three aforementioned indications. From 82 adult patients, 93 samples (92.4% from lumbar puncture and 7.6% from ventriculoperitoneal shunt taps) were analyzed. For CNS progression, gliomas were most frequently evaluated (60%); for LMD, metastatic disease was most common (86.7%). Median CSF volume utilized for analysis was 4.60 mL (range: 0.40-7.50 mL). Median cfDNA yield was 0.29 ng/mL (range: 0.00–1037.74 ng/mL). For lesions of uncertain etiology (n=40 patients, 1 sample/patient), VAs were identified in 10 samples (25%); an additional 10 samples had sufficient cfDNA for LPWGS (25%). 50% of samples in this indication had insufficient cfDNA for testing. For suspected progression (n=26 patients, 35 samples), VAs were identified in 6 samples (17.1%); an additional 14 samples underwent LPWGS (40%); 15 had insufficient cfDNA for further testing (42.9%). For suspected LMD (n=16 patients, 18 samples), VAs were quantifiable in 4 samples (22.2%); LPWGS could be performed in an additional 9 (50%); 5 had insufficient cfDNA (27.8%). Across indications, identification of known tumor-associated alterations in CSF, including those confirmed previously by tissue, served as a non-surgical diagnostic adjunct. In some patients, targetable mutations could be identified via CSF. Conversely, absence of such alterations and/or low cfDNA yields may lower suspicion for active neuro-oncologic disease, when evaluated alongside clinical history. Despite modest cell-free DNA yield, CSF cfDNA evaluation provides valuable information to aid clinical decision-making across neuro-oncologic indications.

Abstract Imaging-based monitoring of gliomas is hampered by treatment-related changes, as well as the insidious growth of gliomas harboring the isocitrate dehydrogenase (IDH) mutation. The IDH mutation results in elevated D-2-hydroxyglutarate (D-2-HG), which is detectable in cerebrospinal fluid (CSF) that can be accessed longitudinally from multiple compartments, including cranially via CSF access devices. As such, we evaluated the early utility of CSF D-2-HG as a serially accessible biomarker for IDH-mutant gliomas. A CLIA-approved gas chromatography mass spectrometry assay was developed for CSF D- and L-2-HG. Lumbar and cranial CSF samples were collected from patients with IDH-mutant gliomas or IDH-wild-type brain tumors and non-tumor pathologies via surgical field collection, lumbar punctures, Ommaya reservoirs, and ventriculoperitoneal shunts. First, to confirm the ability of CSF D-2-HG to detect IDH-mutant gliomas, concentrations were compared between IDH-mutant gliomas and wild-type lesions. Cranial, but not lumbar, CSF D-2-HG distinguished primary IDH-mutant gliomas from IDH-wild type lesions (cranial AUC= 0.89, 95% confidence interval (CI)= 0.80-0.97); lumbar AUC= 0.52, 95% CI=0.28-0.76). Indeed, CSF D-2-HG was significantly higher in cranial than lumbar samples from IDH-mutant glioma patients (median lumbar=0.20 μM, cranial = 1.72 μM; p<0.0001), confirming a significantly elevated abundance of CSF D-2-HG only in cranial CSF from IDH-mutant gliomas. Then, response to cytoreduction was evaluated, demonstrating that cranial CSF D-2-HG levels decreased to 0.54x of baseline with resection in seventeen patients (p=0.0129). Changes in tumor-to-CSF contact impacted the serial evaluation of CSF D-2-HG prior to versus after chemoradiation in some patients. Nevertheless, in patients with grade 4 IDH-mutant astrocytomas, serial cranial CSF D-2-HG increased with disease progression and differentiated stability from pseudoprogression when tumor-CSF contact was sufficient. In conclusion, serial cranial CSF D-2-HG shows promise as a monitoring biomarker in patients with IDH-mutant gliomas, although anatomic variables must be considered in parallel to CSF D-2-HG evaluation.

Abstract Background Cerebrospinal fluid cell-free DNA (cfDNA) can detect and monitor leptomeningeal disease but has not been previously used to monitor parenchymal lesions. Methods Herein, we report our initial experience with CSF cfDNA monitoring for 2 patients with colorectal cancer (CRC) metastases to the thalamus, causing obstructive hydrocephalus. Results CSF samples were obtained during ventriculoperitoneal shunt placement, demonstrating high levels of cfDNA in both cases. Several genomic alterations detected in the cfDNA sequencing matched those in the tumor tissue biopsy. Follow-up CSF evaluations after subsequent therapy were used to help adjudicate pseudo-progression versus true progression. Conclusions Neither patient developed leptomeningeal disease, demonstrating CSF’s utility in evaluating solitary brain metastases in direct contact with a CSF compartment.

Abstract Background While serial sampling of glioma tissue is rarely performed prior to recurrence, cerebrospinal fluid (CSF) is an underutilized longitudinal source of candidate glioma biomarkers for understanding therapeutic impacts. However, the impact of key variables to consider in longitudinal CSF samples for monitoring biomarker discovery, including anatomical location and post-surgical changes, remains unknown. Methods Aptamer-based proteomics was performed on 147 CSF samples from 74 patients; 71 of whom had grade 2–4 astrocytomas or grade 2–3 oligodendrogliomas. This included pre- versus post-resection intracranial CSF samples obtained at early (1–16 days; n = 20 patients) or delayed (86–153 days; n = 11 patients) time points for patients with glioma. Paired lumbar versus intracranial glioma CSF samples were also obtained (n = 14 patients). Results Significant differences were identified in the CSF proteome between lumbar, subarachnoid, and ventricular CSF in patients with gliomas. Importantly, we found that resection had a significant, evolving longitudinal impact on the CSF proteome, with distinct sets of proteins present at different time points since resection. Our analysis of serial intracranial CSF samples suggests the early potential for disease monitoring and evaluation of pharmacodynamic impact of targeted therapies, such as bevacizumab and immunotherapies. Conclusions The intracranial glioma CSF proteome serves as a rich and dynamic reservoir of potential biomarkers that can be used to evaluate the effects of resection and other therapies over time. All data within this study, including detailed individual clinical annotations, are shared as a resource for the neuro-oncology community to collectively address these unanswered questions and further understand glioma biology through CSF proteomics.

Abstract Cerebrospinal fluid (CSF) has emerged as a valuable liquid biopsy source for glioma biomarker discovery and validation. CSF produced within the ventricles circulates through the subarachnoid space, where the composition of glioma-derived analytes is influenced by the proximity and anatomical location of sampling relative to tumor, in addition to underlying tumor biology. The substantial gradients observed between lumbar and intracranial CSF compartments for tumor-derived analytes underscore the importance of sampling site selection. Moreover, radiographic features, such as tumor-CSF contact and blood-brain barrier disruption, are critical covariates that may affect biomarker detection and the abundance of plasma-derived analytes in CSF, respectively. Longitudinal intracranial CSF sampling, enabled by access devices like Ommaya reservoirs, may offer a window into treatment response and disease progression, though variability in analyte yield, sample volumes, and the dynamic effects of surgical resection pose challenges. This review critically evaluates the anatomic, radiographic, and longitudinal factors, or “time-space continuum,” that impact glioma CSF biomarker abundance. Practical considerations for longitudinal CSF biobanking, including access device placement and collection, are also reviewed. Key takeaways and recommendations for CSF glioma biomarker discovery and validation are provided as a “hitchhiker’s guide” based on our collective experience, along with resources for investigators aiming to develop CSF biobanking at their institutions.

Abstract Purpose: Current methods for glioma response assessment are limited. This study aimed to assess the technical and clinical feasibility of molecular profiling using longitudinal intracranial cerebrospinal fluid (CSF) from patients with gliomas. Experimental Design: Adults with gliomas underwent longitudinal intracranial CSF collection via Ommaya reservoirs or ventriculoperitoneal shunts. Cell-free DNA (cfDNA) was extracted and analyzed using PredicineCARE for cancer variant profiling and/or PredicineSCORE for low-pass whole-genome sequencing. Results: Five patients (two females and three males; median age, 40 years; range, 32–64 years) underwent longitudinal intracranial CSF collection via Ommaya reservoirs (n = 4) or ventriculoperitoneal shunts (n = 1). In total, 47 CSF samples were obtained (median volume, 4.00 mL; 0.5–5 mL). Forty-one samples (87.2%) yielded sufficient cfDNA for testing. Patient-specific tumor-associated variant allelic frequencies (VAF), and thus tumor fraction, decreased in pre- versus postchemoradiation samples, including through pseudoprogression. These also increased with radiographic progression in three patients, although identifying the time of definitive disease progression from MRIs was a significant limitation. In two patients with isocitrate dehydrogenase (IDH)–mutant gliomas, decreasing IDH1 VAF after resection and chemoradiation correlated with decreased CSF D-2-hydroxyglutarate levels (0.64× and 0.62×, respectively, for the first patient and 0.01× and 0.07× for the other patient), although D-2-hydroxyglutarate and IDH1 VAF were not concordant in one patient thereafter. Moreover, the copy-number burden decreased below the limit of quantification during treatment and increased above the limit at progression. Conclusions: Longitudinal intracranial CSF cfDNA can be obtained in patients with gliomas during their disease course. However, before deploying this technique, numerous questions and challenges should be answered.