Quantification of Cerebrospinal Fluid Tumor DNA in Lung Cancer Patients with Suspected Leptomeningeal Carcinomatosis

Introduction Cerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring neoplastic processes of the central nervous system. We hypothesize that analysis of CSF-tDNA in patients with advanced lung cancer improves the sensitivity of leptomeningeal disease (LMD) diagnosis and enables central nervous system response monitoring. Methods We applied CAPP-Seq using a lung cancer-specific sequencing panel to 81 CSF, blood, and tissue samples from 24 patients with advanced lung cancer who underwent lumbar puncture (LP) for suspected LMD. A subset of the cohort (N = 12) participated in a prospective clinical trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment with. Results CSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearman’s ρ, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). Conclusions Detection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA may improve the sensitivity of LMD diagnosis, enable improved prognostication, and drive therapeutic strategies that account for spatial heterogeneity in resistance mechanisms. ### Competing Interest Statement Disclosure of Potential Conflicts of Interest: A.M.N., A.A.A., and M.D. are co-inventors on patent applications related to cancer biomarkers. A.M.N. reports consultancy with CiberMed. A.A.A. and M.D. are consultants for Genentech and Roche. J.J.C. served as a consultant with Lexent Bio. A.A.A. has served as a consultant for Chugai, Gilead, and Celgene. A.A.A. reports ownership interest in CiberMed and FortySeven. M.D. has served as a consultant for AstraZeneca, Novartis, Bristol Myers Squibb, Gritstone Oncology, and Boehringer Ingelheim. M.D. and A.A.C have received research funding from Varian Medical Systems. M.D. has received research funding from Illumina. S.N. have received research funding from AstraZeneca. M.D. and A.A.A. report ownership interests in CiberMed and Foresight Diagnostics. ### Funding Statement This work was supported by grants from the National Cancer Institute (M.D. and A.A.A., R01CA188298 and R01CA254179), the US National Institutes of Health Director's New Innovator Award Program (M.D.; 1-DP2-CA186569), the Virginia and D.K. Ludwig Fund for Cancer Research (M.D. and A.A.A.), the CRK Faculty Scholar Fund (M.D.), the Doris Duke Charitable Foundation (T.D.A.), the Japan Society for the Promotion of Science KAKENHI (S.N. 21K15546 and 23K06648) and Japanese respiratory society fellowship (S.N.). Visualizations created with BioRender.com. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Stanford University School of Medicine gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript