Riviere-Cazaux, Cécile

Abstract Purpose: Monitoring disease progression in patients with high-grade gliomas (HGG) is challenging due to treatment-related changes in imaging and the requirement for neurosurgical intervention to obtain diagnostic tissue. DNA junctions in HGG often amplify oncogenes, making these DNA fragments potentially more abundant in blood than monoallelic mutations. In this study, we piloted a cell-free DNA approach for disease detection in the plasma of patients with HGG by leveraging patient-specific DNA junctions associated with oncogene amplifications. Experimental Design: Whole-genome sequencing of grade 3 or 4 isocitrate dehydrogenase–mutant or wild-type astrocytomas was utilized to identify amplified junctions. Individualized qPCR assays were developed using patient-specific primers designed for the amplified junction. ctDNA levels containing these junctions were measured in patient plasma samples. Results: Unique amplified junctions were evaluated by individualized semi-qPCR assays in presurgical plasma of 18 patients, 15 with tumor-associated focal amplifications and three without tumor-associated focal amplifications. high copy-number junctions were robustly detected in the plasma of 14 of 15 (93.3%) patients with amplified junctions and none of the controls. Changes in junction abundance correlated with disease trajectory in serial plasma samples from five patients, including increased abundance of amplified junctions preceding radiographic disease progression. Conclusions: In patients with grade 3 or 4 astrocytomas who had tumor-associated amplifications, patient-specific amplified junctions were successfully detected in assayed plasma from most patients. Longitudinal analysis of plasma samples correlated with disease trajectory, including cytoreduction and progression.

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.

Abstract Glioblastoma (GBM) presents as a highly aggressive and malignant brain tumor with limited treatment options. Despite the availability of existing therapies, the invasive characteristics of GBM frequently result in tumor recurrence and poor prognosis. We previously developed a B-cell-based therapy (BVax) that has shown promising results in its ability to induce therapeutic responses in preclinical models of GBM. Our research revealed potency of BVax can be attributed to its robust antigen-presenting capability, cognate antigen specificity with T cells, and ability to generate tumor-reactive antibodies. The present study aims to characterize the unique antigenic reactivity of BVax-derived antibodies by evaluating their immunological effects and therapeutic potential in both murine models and GBM patient-specific samples. Leveraging CD45.1 and CD45.2 congenic mouse models, we first revealed that BVax preferentially migrated to glioma-bearing brains upon intravenous injection, resulting in a CD38+CD20-CD19+ plasmablast phenotype. To further characterize the reactivity of BVax-derived immunoglobulins (Ig), we utilized proteomic analysis and identified preferential targets of Gelsolin, Fibronectin, Versican, Fibrinogen, and collagen type IV alpha, commonly associated with cell motility and the extracellular matrix. Microdialysis data confirmed the increased presence of many of these BVax-derived Igs-recognized antigens in tumor regions compared to normal brain regions in GBM patients. Utilizing purified BVax-derived antibodies from patient specimen, in vitro cell motility assays demonstrated a significant prevention of the invasion and migration of cell lines from patient derived xenograft (PDX). In this study, we concluded that BVax elicits antitumor activity through targeted migration to tumor-bearing regions, differentiation into plasmablasts, and robust secretion of specific Igs that retain the ability to inhibit functionalities associated with cell invasion and migration. Here, we present the first in-depth study that characterizes GBM patients’ antibody reactivity, specifically a subset of inflammatory B cells (BVax) with therapeutic effects that offer an indication for a new direction of immunotherapies.

Abstract Improved response assessment methods are needed for patients with gliomas. Intracranial cerebrospinal fluid (CSF) is longitudinally accessible and provides an abundance source of candidate biomarkers, such as cell-free DNA (cfDNA), for disease monitoring. To evaluate the clinical feasibility of molecular profiling from longitudinal intracranial CSF, CSF was acquired from patients with CSF access devices, including Ommaya reservoirs and ventriculoperitoneal shunts. cfDNA was extracted and analyzed via PredicineCARE (Next Generation sequencing) or PredicineSCORE (low pass whole genome sequencing). Five patients (2 females, 3 males; median age: 40 years, range 32-64 years) underwent longitudinal intracranial CSF collection via Ommaya reservoirs (n=4) or VP shunt (n=1). In total, forty-eight CSF samples were obtained (median volume: 4.00 mL; 0.5-5 mL), with 39 samples (81.3%) yielding sufficient cfDNA for variant profiling. Our initial findings suggest that tumor fraction increased by 6.28x, 2.87x, and 8.31x with radiographic progression in three patients. Tumor fraction decreased by 0.08x and 0.04x in two patients with paired immediate pre-versus-post chemoradiation samples. Despite ongoing pseudoprogression in one patient, tumor fraction decreased to unmeasurable levels from a post-resection baseline of 0.26. Patient-specific tumor-associated variant allelic frequencies (VAFs), including TP53, PTEN, TERT, and CDKN2A/B, decreased within individual patients after resection and chemoradiation. In two patients with isocitrate dehydrogenase (IDH) mutant gliomas, decreasing IDH1 VAF after resection and chemoradiation correlated with decreased CSF D-2-hydroxyglutarate (D-2-HG) levels (0.64x and 0.62x, respectively, for the first patient, and 0.01x and 0.07x for the other patient). Both CSF IDH1 VAF and CSF D-2-HG increased a patient who had radiographic progression (2.56x and 9.21x, respectively). Moreover, CNB decreased below the limit of quantification during treatment and increased above the limit at progression. In conclusion, longitudinal CSF cfDNA can feasibly be obtained via CSF access devices in patients with gliomas during their disease course toward evaluating candidate monitoring biomarkers.

Abstract While serial sampling of glioma tissue is rarely performed before recurrence, cerebrospinal fluid (CSF) can be acquired serially to evaluate candidate monitoring and pharmacodynamic biomarkers in patients with gliomas. However, the impact of key variables to consider in longitudinal CSF, including anatomical location and post-surgical changes, remains unknown. To that end, pre- versus post-resection intracranial CSF samples from patients with gliomas were obtained at early (1-16 days; n=20) or delayed (86-153 days; n=11) timepoints, as well as lumbar-versus-intracranial glioma CSF samples (n=14). Using aptamer-based proteomics, we identified significant differences in the glioma CSF proteome between lumbar, subarachnoid, and ventricular CSF. Indeed, intracranial CSF in paired lumbar/intracranial samples had nearly 1600 unique aptamers, regardless of which intracranial location was compared to the paired lumbar CSF, including in enrichment style analyses that evaluated the overlap between protein states. Importantly, resection had a significant, evolving longitudinal impact on the CSF proteome, with subsets of proteins associated with early versus delayed post-resection changes, many of which were enriched for inflammatory-related pathways. Interestingly, despite increased contamination from surgical debris, enrichment for plasma-associated proteins decreased with resection (FDR: 0.000), suggesting that a portion of the baseline glioma panel could be derived from plasma-originating analytes due to blood-brain barrier disruption. Despite these challenges, our data still revealed preliminary candidate monitoring biomarkers, such as brevican, that increased with progression (example patient: fold-change of 3.58x and 2.08x through lomustine and bevacizumab, respectively), but not pseudoprogression (Fold change less than 1.5). Serial intracranial CSF samples suggested the early potential for disease monitoring and evaluation of pharmacodynamic impact of targeted therapies, including bevacizumab and pembrolizumab. In conclusion, our available data to date suggest that the intracranial glioma CSF proteome provides an abundant and evolving source of candidate biomarkers to longitudinally discern the impact of resection and systemic treatments.

Abstract Accurate response assessment in patients with high-grade gliomas (HGGs) is complicated by post-treatment radiographic changes that are equivocal for treatment effect versus progression. Plasma-based liquid biopsy assays for circulating tumor DNA (ctDNA) have had limited sensitivity to date, due in part to the presence of the blood-brain barrier that limits the diffusion of tumor-derived analytes out of the central nervous system. To address this challenge, we developed a ctDNA plasma-based assay to detect individualized tumor-derived amplified junctions in patients with HGGs. These junctions occur due to large-scale chromosomal rearrangements, such as those associated with epidermal growth factor receptor (EGFR) or cyclin-dependent kinase 4 (CDK4) amplification. To identify these amplified junctions, whole genome sequencing was performed on resected tissue. Primers were designed to flank junction breakpoints and were then utilized on each patient’s plasma samples to detect the amplified junction via qPCR. Tumor-specific amplified junctions were robustly detected in plasma from 14/14 (100%) patients with IDH-mutant or wild-type grade 4 astrocytomas whose tumor genomes demonstrated amplification-related junctions, located at chromosomes 7, 11, or 12. In comparison, ctDNA could not be detected in three control cases that did not have an amplification or did not have high copy number junctions. Importantly, serial plasma samples obtained prior to and at disease recurrence in 4 patients demonstrated an increasing abundance of amplified junctions that accurately correlated with disease progression in 4/4 patients (100%). In two of these patients, increasing abundance of amplified junctions preceded radiographic changes compatible with disease progression by approximately 2 or 4 months, respectively. In conclusion, tumor-specific amplified junctions could reliably be detected in plasma from patients with IDH-mutant or wild-type grade 4 astrocytomas and correlated with changes in disease burden. Further work is currently ongoing to evaluate these promising results in a larger cohort of patients with serial plasma samples.

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Despite an established standard of care including surgical resection, radiation therapy, and chemotherapy, GBM unfortunately is associated with a dismal prognosis. Therefore, researchers are extensively evaluating avenues to expand GBM therapy and improve outcomes in patients with GBM. In this review, we provide a broad overview of novel GBM therapies that have recently completed or are actively undergoing study in clinical trials. These therapies expand across medical, surgical, and radiation clinical trials. We additionally review methods for improving clinical trial design in GBM.