Serum Cell-Free DNA Concentration Is Predictive of Survival Outcomes and Its Genetic Mutation Analysis May Be Viable in Obtaining Pathological Information

Introduction: Cell-free circulating DNA (cfDNA) and its genetic mutations have been studied as a non-invasive tool to obtain clinical information in malignant lymphoma. We analysed serum cfDNA concentrations in lymphoma patients and investigated its gene mutation profile in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Methods: Serum samples from lymphoma patients diagnosed at our institution between 2016-2017 were obtained at the time of initial diagnosis. Serum samples of 10 healthy individuals were used as control. cfDNA was extracted using Maxwell RSC cfDNA Plasma Kit (Promega), and concentration was measured by Quantus Fluorometer (Promega). The serum concentration level in each subtype of lymphoma was compared to control using Mann-Whitney U test. Furthermore, progression free survival (PFS) was compared between those with and without elevated cfDNA concentrations using Kaplan-Meier method. We also assessed factors associated with elevated cfDNA. Using cfDNA, multiplex PCR was performed, then a sequence library constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using the Ion AmpliSeq DesignerTM . Genomes were sequenced for the selected 121 target genes using the Ion ProtonTM System. Frequencies of each mutation were compared amongst DLBCL, FL, and DLBCL + FL using Kruskal-Wallis test. Results: We analysed 114 patients with newly diagnosed lymphoma (DLBCL n=63, DLBCL combined with FL n=8, DLBCL combined with extranodal marginal zone B cell lymphoma (MALT) n=1, FL n=20, FL/MALT n=1, Burkitt lymphoma (BL) n=1, MALT n=8, Mantle cell lymphoma (MCL) n=3, Low-grade B cell lymphoma n=2, Hodgkin lymphoma (HL) n=4, Peripheral T cell lymphoma (PTCL) n=3), as well as control (n=10). The median cfDNA concentration in all lymphoma patients was 55.8ng/ml (range 6.2-1220.0ng/ml), which was significantly higher than that for control (median 7.5ng/ml, range 4.4-14.1ng/ml; p