Abstract 3408: Characteristics of circulating tumor DNA in lung cancer patients

Abstract

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. In metastatic animal model using immunodeficient mice, NOJ mice, the amount or ctDNA was associated with tumor progression such as tumor burden and metastasis. Based on these evidences, we hypothesized that ctDNA has some biological effects on tumor progression. In order to examine that, we analyzed biological and clinical characteristics of ctDNA. ctDNA was extracted from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. Compared to circulating free DNA (cfDNA) isolated from healthy volunteers, size distribution of ctDNA or cfDNA using a capillary electrophoresis system was different, that is one peak around the size of 170 bp in healthy individuals, and two peaks, 170 bp and 5 kb in advanced lung cancer patients. We next 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. The DNA concentration quantified by Quantus®, the fluorescent measurement of dsDNA intercalated dye, 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 were significantly increased in these cases compared to 170 bp fragments. 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. Although the 170 bp short fragments of ctDNA are well known as an apoptotic product, the origin of 5 kb long fragments has not been clarified. We have examined whole ctDNA sequence using next generation sequencing, and compared two fragments. In addition, origins of each fragment have been investigated, and biological effects on tumor progression will be analyzed. Citation Format: Tomonori Abe, Chiho Nakashima, Akemi Sato, 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 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3408.