Clonotypic Cell-Free Dna(Cfdna) In The Cerebrospinal Fluid (Csf) Of Patients With Aggressive Lymphomas

Current methods for detection of CNS involvement in lymphoma (CSF cytology, flow cytometry) have very limited sensitivity, particularly in cases of parenchymal brain involvement. Early detection is however critical to institute CNS-directed therapy that can avert dismal outcomes of overt CNS recurrence. Clonotype-specific cfDNA can be detected in the plasma of patients with lymphoma using next-generation sequencing (NGS), and residual cfDNA-based minimal residual disease (MRD) assay can predict impending recurrence (Roschewski et al., Lancet Oncol. 2015). cfDNA has not been systematically evaluated in the CSF, yet it may hold promise as a sensitive and specific method to detect CNS invasion. To evaluate the ability of an NGS-MRD assay in the CSF to detect CNS invasion, we examined CSF and plasma samples from patients with aggressive lymphomas who either had overt CNS disease, or who were without evident CNS invasion, but at high clinical risk. Genomic DNA from primary tumors was analyzed for tumor-specific clonotype using NGS of rearranged IGK, IGH (VJ or DJ), or IGL loci (Adaptive Biotechnologies; Carlson CS et al., Nat Commun. 2013). Tumor-specific clonotypes from each case were selected for subsequent tracking by NGS-MRD in CSF and plasma samples. Clonotype copy numbers are expressed per mL for acellular CSF, and clonotype frequency per all B-cells. NGS identified median 3 (range, 2-7) dominant immunoglobulin sequences in each primary lymphoma (N=16), with median dominant clonotype frequency 50.3% (range, 26.8-9.28%). In the CSF, the NGS-MRD assay detected the dominant clonotype in 9 out of 16 samples, including all (N=4) with overt CNS invasion (sensitivity=100%), of which 2 had parenchymal disease only with negative standard CSF cytology, flow cytometry, or IGH PCR. Median detectable cfDNA clonotype in the CSF was 1,077 copies per mL (range, 2-5,620), with median clonotype frequency of 28.4% (range, 0.1-98.5%). cfDNA copy counts were significantly higher in cases with positive CSF cytology than those with parenchymal or clinically occult disease (P=.0016). We observed no significant correlation between the red cell count in the CSF and the clonotype cfDNA concentration (P=0.73) or frequency (P=0.62) suggesting that the CSF cfDNA was not due to contamination by blood. There was also no evident correlation between cfDNA in plasma and CSF. Our results suggest that NGS-MRD assay for cfDNA in the CSF can identify intraparenchymal or leptomeningeal CNS invasion with high sensitivity, including cases not identifiable by traditional methods. Prognostic significance of detecting lymphoma-specific cfDNA in the CSF of some high-risk patients without overt CNS disease will be further explored in a larger sample. Pre-treatment NGS-MRD assay could be prospectively tested to predict the risk of CNS recurrence, with a future potential to enable more accurate selection of patients for CNS prophylaxis therapy. Citation Format: Adam J. Olszewski, Anna Chorzalska, Diana O. Treaba, John L. Reagan, Andrew Hsu, Pamela C. Egan, Habibe Kurt, James Butera, William Rafelson, Thomas Ollila, Rabin Niroula, Jordan Robison, Chelsea D. Mullins, Patrycja M. Dubielecka. Clonotypic cell-free DNA(cfDNA) in the cerebrospinal fluid (CSF) of patients with aggressive lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 733.