Comprehensive profiling and clinical utility of CSF-derived cell-free DNA/RNA for evaluation of solid and hematologic malignancies affecting the central nervous system.

3066 Background: Diagnosis and risk stratification of brain lesions is challenging when tissue biopsy is contraindicated due to anatomic or clinical factors. Cerebrospinal fluid (CSF)–based liquid biopsy using cell-free DNA (cfDNA) and RNA (cfRNA) offers a minimally invasive approach to detect neoplastic disease and inform management. We evaluated the clinical utility of a commercially available cfDNA/cfRNA next-generation sequencing assay applied to CSF specimens from patients with CNS lesions of uncertain etiology. Methods: Between 2023 and 2025, cfDNA, cfRNA, and CSF cell pellet RNA profiling using next generation sequencing (NGS) was performed on CSF samples from 1,559 patients, including 1,209 evaluated for suspected primary or secondary solid CNS malignancies and 350 evaluated for CNS involvement by hematologic malignancies. The assay interrogated single-nucleotide variants, insertions/deletions, copy number alterations, gene fusions, lymphocyte clonality, and tumor mutational burden. The depth of sequencing was between 25,000x and 30,000x for cfDNA. For quality control purposes, more than 80 million reads were required for accepting RNA results, while the required percentage of spliced RNA reads was above 20%. Clinical utility and actionability was further assessed in a single-institution cohort of 90 patients by correlating cfDNA/cfRNA results with cytology, flow cytometry, tissue biopsy, and clinical decision-making. Results: Among patients evaluated for solid malignancies in the entire cohort, the most common alterations involved TP53, KMT2C, PIK3CA, EGFR, and DNMT3A, while hematologic cases frequently harbored PIM1, TP53, MYD88, CD79B, TET2, and DNMT3A alterations. In the institutional cohort (median age 57 years), neoplastic cfDNA was detected in 40/90 samples, with recurrent alterations mirroring those of the larger panel. cfDNA profiling demonstrated 100% sensitivity for neoplastic DNA detection compared with current standard-of-care cytology and flow cytometry and identified neoplastic DNA in 37% of cytology-negative and 42% of flow-negative cases. Concordance with contemporaneous tissue biopsy yielded a positive predictive value of 89%, with a sensitivity of 68%, reflecting reduced detection in intraparenchymal lesions with limited CSF exposure. cfDNA results directly influenced treatment decisions in patients evaluated for new intracranial malignancy and for CNS metastasis or recurrence. Conclusions: CSF cfDNA/cfRNA profiling significantly enhanced diagnostic yield in CNS lesions of uncertain etiology, particularly when conventional cytology is negative, or biopsy is infeasible, while also providing actionable molecular information to guide therapy.