Burns, Terry

Abstract Cerebrospinal fluid (CSF) is the biofluid most proximal to the central nervous system (CNS), yet its proteomic organization across neurological disease remains unresolved. We assembled a meta-atlas of 35 CSF proteomic datasets (2016–2026) comprising 10,560 quality-controlled samples spanning neurodegenerative, neuroinflammatory, neuro-oncological, infectious and structural-injury contexts. Meta-analysis of cross-cohort detection defined a 747-protein core mass-spectrometry-accessible CSF proteome that generalizes to held-out cohorts. We derived thirteen consensus meta-programs that transfer across independent cohorts and span the major neural, barrier, immune and plasma compartments. Unexpectedly, these programs are dominated by a single axis of variation opposing complement- and plasma-associated inflammation against neuronal-synaptic homeostasis. This injury–homeostasis axis is diagnosis-spanning and tracks clinical severity and disease activity across diverse neurological conditions. We show that a two-protein C5–NRCAM model can approximate the injury–homeostasis axis, bridging deep proteomic profiling toward bedside assessment.

Abstract Introduction Leptomeningeal disease (LMD) is the seeding of tumor cells in the leptomeninges and the cerebrospinal fluid (CSF) and carries a poor prognosis of 2-4 months. CSF sampling is required to study LMD, but genomic analysis may not be able to identify non-genomic and post-translational modifications. Proteomics can address this gap but is limited by reproducibility and disease heterogeneity. Integrating proteomic with genomic data may prioritize driver genes that promote LMD. Methods 23 patients with breast cancer-associated LMD with matched CSF proteomic and genomic data were studied. Variant allele frequencies (VAFs) for genes TP53, NFE2L2, ERBB2 and PIK3CA were derived, and proteomic profiling was performed on matched CSF. Spearman correlations between driver-gene VAF and protein expression were computed, restricting to genes with VAF>0 in ≥ 3 samples, and Benjamini-Hochberg correction was applied across gene-protein pairs. Proteins significantly associated with VAF were analyzed using over representation analysis (ORA) and gene segment enrichment analysis (GSEA) with KEGG, Gene Ontology, and MSigDB. Results TP53 VAF was associated with upregulation of proteins involved in glycolysis and cell proliferation, with enrichment of hallmark glycolysis (FDR=2.6x10-8), mTORC1 signalling (FDR=8.5x10-8), and reactive oxygen species pathways (FDR=2.7x10-3), and downregulation of complement (FDR=6.8 x 10-6) and coagulation cascades (FDR=0.011). NFE2L2 VAF was associated with epithelial-mesenchymal transition by both GSEA (FDR=2.3x10-9) and ORA (FDR=4.6x10-3), TGF-β signalling (FDR=0.015) and glycoprotein metabolism (FDR=1.6x10-7). ERBB2 VAF was associated with an upregulation of cell-cycle regulation (FDR=6.4x10-10), mTORC1 pathway (FDR=3.2x10-4), and a downregulation of proteins involved in cell-cell adhesion (FDR=4.0x10-9), axon guidance (GSEA FDR=0.025), and developmental growth (GOBP FDR=3.4x10-4). Conclusion Distinct CSF proteomic signatures were associated with specific driver mutations, implicating metabolic and oxidative stress pathways (TP53), stromal remodelling (NFE2L2), and disruption of CNS structural pathways (ERBB2). Validation in prospective LMD cohorts is needed to confirm these mutation-specific signatures and inform diagnostic and therapeutic strategies.

Abstract Background Peritumoral brain edema (PTBE) is a frequent complication of CNS tumors and radiation therapy (RT). PTBE is primarily treated with corticosteroids, but prolonged use is associated with severe toxicities including myopathy and immunosuppression. Bevacizumab, an alternative treatment, is not recommended in patients with thromboembolic events and intracerebral hemorrhage. Plasma kallikrein is a key driver of tumor-related edema. By targeting this pathogenic enzyme, the orally delivered inhibitor, RZ402, may provide therapeutic value by controlling edema and decreasing corticosteroid use. Methods In this single-patient investigational new drug protocol, we report results from use of RZ402 for treatment of cerebral edema related to metastatic melanoma and RT. Results A 74-year-old man with BRAF-wildtype melanoma had a right frontal and basal ganglia hemorrhagic metastasis not amenable to resection, treated with stereotactic RT (30 Gy, 5 fractions), followed by ipilimumab and nivolumab. MRI obtained 5 months after RT showed increased edema associated with midline shift. After 12 weeks of treatment with high-dose dexamethasone (16 mg daily), edema remained persistent and patient was unable to tolerate dose reduction below 4 mg daily. Course was complicated by a pulmonary embolus prompting initiation of anticoagulation, leading to deferral of bevacizumab. Patient was treated with RZ402, which was tolerated well with no reported toxicities. Dexamethasone was discontinued over 4 weeks. Follow up imaging 6 weeks after RZ402 initiation and off dexamethasone demonstrated stability without rebound edema. Patient did experience adrenal insufficiency characterized by severe fatigue, managed with low-dose prednisone. RZ402 was discontinued after 8 weeks of treatment with patient remaining clinically stable. Conclusion Despite persistent edema after prolonged use of corticosteroids, the selective plasma kallikrein inhibitor RZ402 was associated with radiographic stabilization and discontinuation of dexamethasone for this patient. These findings support prospective evaluation of RZ402 in treatment of symptomatic cerebral edema related to CNS tumors.

ABSTRACT Leptomeningeal disease (LMD) is a rapidly fatal complication of systemic cancer for which sensitive diagnostic tools and informative biomarkers remain limited. Here, we introduce CSF-Seq, a method for whole-transcriptome sequencing of cell-free RNA (cfRNA) from human cerebrospinal fluid (CSF), designed to enable molecular profiling of LMD and other central nervous system (CNS) conditions. Using a prospectively collected CSF biobank, we analyzed 125 samples spanning multiple pathologies, including breast and lung LMD, glioblastoma, traumatic brain injury, and non-cancer neurological controls. Through optimized RNA extraction, library preparation, and deep sequencing, CSF-Seq generated robust and reproducible transcriptome-wide profiles despite the low abundance and fragmentation of cfRNA in CSF. CSF transcriptomes exhibited disease-specific expression, separating LMD from non-cancer controls and from non-LMD cancers, independent of CSF collection modality. Tumor-associated epithelial transcripts, including CEACAM6 and MUC1, were consistently enriched in LMD samples, whereas immune and CNS-associated transcripts were broadly detected across disease states, consistent with contributions from both tumor and non-tumor sources. Cross-site processing of matched samples demonstrated high concordance, indicating preservation of sample-specific transcriptional signatures across independent workflows. Importantly, we identified a collection method– independent LMD gene expression signature that was significantly associated with overall survival, supporting its potential prognostic relevance. Together, these findings establish CSF-Seq as a technically robust and clinically informative platform for transcriptomic biomarker discovery in CNS metastatic disease, offering a minimally invasive approach for disease characterization, risk stratification, and longitudinal monitoring in patients with LMD.