Abstract Liquid biopsy using circulating tumor DNA (ctDNA) has been spread world-wide. We established fully automatic sensitive mutation detection system, mutation-biased PCR and quenched probe system (MBP-QP) method, and accomplished multicenter prospective study to investigate the utility of ctDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). The results of the clinical study showed that ctDNA was frequently detected in lung cancer patients with distant metastasis, and detection of ctDNA was associated with poor prognosis. To investigate the significance of these clinical data, we analyzed biological and clinical characteristics of ctDNA. We examined 130 plasma samples from 92 lung cancer patients in addition to 18 benign pulmonary disease patients and 20 healthy individuals at Saga University Hospital.Circulating free DNA (cfDNA) was extracted from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. The DNA concentration was quantified by Quantus®, the fluorescent measurement of dsDNA intercalated dye, and the DNA size distribution was analyzed by Bioanalyzer®, capillary electrophoresis system. We found difference of cfDNA size distribution between lung cancer patients and healthy individuals: former showed two peaks of 5kb and 170bp, and latter showed single peak of 170bp. The DNA concentration was higher in lung cancer patients compared to those in benign pulmonary disease patients and healthy individuals. Among lung cancer patients, DNA concentration was increased in those with advanced stages, especially in presence of metastasis. In addition, 5 kb fragments DNA was significantly increased in these cases compared to 170 bp fragments DNA. To investigate which fragment contained tumor-derived DNA, 170 bp and 5 kb fragments were separately isolated, and EGFR mutation, L858R was examined. L858R was detected in both ctDNA fragments, 170 bp and 5 kb, indicating that both sized DNA fragments contain tumor-derived DNA. In order to evaluate DNA resolution, we measured the concentrations of DNase1 and DNase1L3 in plasma, but no significant difference was observed among three groups. Although the 170 bp fragments DNA are well known as an apoptotic product, the origin of 5 kb long fragments DNA has not been clarified. We hypothesized that long fragment DNA has a mechanism to escape from DNase, extracellular vesicles or DNA associated protein. In order to examine that, We analyze whether long fragment DNA is associated with extracellular vesicles or DNA associated protein. Citation Format: Tomonori Abe, Chiho Nakashima, Akemi Sato, Yohei Harada, Eisaburo Sueoka, Shinya Kimura, Naoko Aragane. Characteristics of circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 420.