BSLM-13 Extraction and Whole Transcriptome Sequencing of Cell Free Rna From Human Cerebrospinal Fluid: A Method to Identify Biomarkers of Brain Health and Disease

BACKGROUND Brain biopsy carries a morbidity risk of ~5%, which has significantly limited samples available for research, hindering our understanding and treatment of brain metastases (BrM) and Leptomeningeal Disease (LMD). However, cerebrospinal fluid (CSF) – which circulates the CNS, significantly interfacing with brain tumor tissues and carries tumor cell-free nucleic acids and LMD cells – provides a critically understudied biospecimen to understand BrM/LMD biology. We previously found brain tumor-specific cell-free DNA (cfDNA) in CSF, even when not detectable in blood; and showed that the mutant fraction of cfDNA mirrored treatment response and disease progression. Expanding to study CSF cell-free mRNA (cfRNA) allowed us to extend from mutation-driven cancers (e.g., BRAF/melanoma, EGFR/lung cancer) to investigate the transcriptional profiles of not only cancer, but also brain-resident cells. METHODS We have banked, and have ready-access to, 300+ CSF samples from patients with brain tumors and LMD, many of whom were treated at Stanford Hospital (2012 – 2023). We then leveraged a combination of total RNA extraction and whole transcriptome RNA sequencing to interrogate gene expression patterns present in CSF cfRNA. A method we term CSF-Seq. RESULTS CSF-Seq has revealed expression profiles in LMD CSF not found in CSF of non-LMD cancers, like gliomas, and non-cancer controls, like hydrocephalous. Importantly, CSF-Seq accurately recapitulates our previous panel-based qPCR method, demonstrating upregulated cancer-associated genes like CEACAM6 and MUC1 in breast and lung LMD, and ubiquitous expression of brain-associated genes, like MBP, across all CSF samples. This demonstrates our ability to retrieve well-established, validated target genes, while positioning us to discover novel gene signatures in LMD. CONCLUSION These results serve as a starting point for identifying key gene expression signatures associated with development of LMD and BrM progression. Further investigation focused on tumor-brain crosstalk, and gene signatures associated with response to treatment, will provide promising targets for therapeutic advancement.