Ghadimi, Keyvan

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 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 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 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.