A CSF ctDNA-based molecular panel for minimally invasive diagnosis of primary CNS lymphoma

Background: Primary central nervous system lymphoma (PCNSL) progresses rapidly and requires prompt diagnosis, yet tissue biopsy is invasive and may be infeasible in high-risk patients.Reliable minimally invasive diagnostic strategies are unmet.Methods: In this multicenter diagnostic study, 174 patients with radiologically suspected PCNSL, including 103 with pathologically confirmed PCNSL, were prospectively enrolled across two Chinese centers.Available tumor tissue (TIS), cerebrospinal fluid (CSF) and plasma specimens were sequenced.For comparison, TIS sequencing data from 54 patients with grade 4 gliomas were retrieved from the CGGA database.Publicly available TIS and CSF sequencing data from 29 patients with PCNSL and 28 with grade 4 gliomas at a US center were included for validation.A coarse-to-fine machine-learning framework was applied to develop TIS-based molecular panels.Mutational concordance between matched TIS and liquid biopsy samples was assessed, and panel performance was further validated using CSF circulating tumor DNA (ctDNA).Results: We developed and validated three TIS-based diagnostic panels capable of distinguishing PCNSL from grade 4 glioma.Mutational concordance analysis indicated CSF as the most reliable liquid biopsy source for molecular diagnosis.The random forest-optimized eight-gene panel (N8-RF-PCNSL) enabled CSF ctDNA-based diagnosis of PCNSL (sensitivity ≥ 79%, specificity: 100%, AUC ≥ 0.90) and identified a subset of MYD88 wt PCNSL with 100% specificity across the two Chinese validation cohorts.However, diagnostic performance was limited in the US validation set.Combining N8-RF-PCNSL with CSF interleukin-10 testing further increased sensitivity to 86%-93% while maintaining 100% specificity.This synergistic approach also provided supportive molecular evidence for PCNSL in over 60% of biopsy-infeasible patients, who subsequently demonstrated favorable radiographic responses.Conclusion: We developed and validated a CSF ctDNA-based eight-gene molecular panel (N8-RF-PCNSL) providing minimally invasive molecular support for PCNSL diagnosis.This work supports a practical CSF-first diagnostic strategy to facilitate timely and accurate decision-making in neuro-oncology.