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Univariate analyses revealed that the World Health Organization (WHO) grade (p = 0.0034), radiographic enhancement (p = 0.0006), and Mib1 index (p = 0.01) were significant predictors of precise CSF-based molecular diagnosis. We precisely diagnosed WHO grade III or IV diffuse gliomas using lumbar CSF obtained from 6 (87%) of 7 patients with tumors harboring any mutation. We established a novel, non-invasive molecular diagnostic method using a chip-based digital PCR system targeting ctDNA derived from CSF with high sensitivity and specifi