CSF-Seq enables transcriptome-wide profiling of cerebrospinal fluid and identifies prognostic signature of leptomeningeal disease

Hayden Gephart, M.; Umeh Garcia, M.; Barisano, G.; Nunez Perez, P.; Trinh, T.; Taiwo, R.; Herrick, D.; Roy-O'Reilly, M.; Lee, S.; Spiliotopoulous, E.; Weixel, C.; Burnside, G.; Godfrey, B.; Zhang, Y.; Chernikova, S.; Tosoni, S.; Granucci, M.; Riviere-Cazaux, C.; Coffey, G.; Villanueva, E.; Burns, T.; Nagpal, S.; Ngo, T.

doi:10.64898/2026.05.21.725787

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bioRxiv · 10.64898/2026.05.21.725787

CSF-Seq enables transcriptome-wide profiling of cerebrospinal fluid and identifies prognostic signature of leptomeningeal disease

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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.

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Hayden Gephart, M., Umeh Garcia, M., Barisano, G., Nunez Perez, P., Trinh, T., Taiwo, R., Herrick, D., Roy-O'Reilly, M., Lee, S., Spiliotopoulous, E., Weixel, C., Burnside, G., Godfrey, B., Zhang, Y., Chernikova, S., Tosoni, S., Granucci, M., Riviere-Cazaux, C., Coffey, G., Villanueva, E., Burns, T., Nagpal, S., Ngo, T.. 2026-05-26. CSF-Seq enables transcriptome-wide profiling of cerebrospinal fluid and identifies prognostic signature of leptomeningeal disease. https://doi.org/10.64898/2026.05.21.725787

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