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For patients with metastatic cancer from the lung, tumor cells release their cell-free DNA (cfDNA) into the CSF. We applied a nanopore single-molecule sequencing approach to analyze the fragmentation, methylation, and hydroxymethylation patterns present in CSF-derived cell-free DNA from patients with metastatic lung cancer to the brain. We compared the cancer cfDNA finding to non-cancer healthy controls and their CSF cell-free DNA. Among cancer patients, we observed enriched mono-nucleosome levels and significantly higher mono-\/trinucleosome ratios in cancer patients. Comparison with plasma-derived cfDNA further confirmed the unique fragmentation features of CSF-derived cfDNA. Distinct methylation and hydroxymethylation patterns were observed between cancer and control CSF samples. We observed significantly lower degree of hydroxymethylation in cancer patients compared with healthy controls and the affected genes had different pathway profiles. Overall, CSF cfDNA in patients with non-small cell lung cancer brain metastases had distinct profiles of DNA fragmentation, methylation, and hydroxymethylation.<\/jats:p>","DOI":"10.1093\/narcan\/zcag021","type":"journal-article","created":{"date-parts":[[2026,8,16]],"date-time":"2026-08-16T11:26:12Z","timestamp":1786879572000},"source":"Crossref","is-referenced-by-count":0,"title":["Multi-omic cell free DNA profiles of cerebral spinal fluid from lung cancer metastasis to the brain"],"prefix":"10.1093","volume":"8","author":[{"given":"Tianqi","family":"Chen","sequence":"first","affiliation":[{"name":"Division of Oncology, Department of Medicine, Stanford University, School of Medicine , Stanford, CA 94305 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangqi","family":"Bai","sequence":"additional","affiliation":[{"name":"Division of Oncology, Department of Medicine, Stanford University, School of Medicine , Stanford, CA 94305 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