Investigation of origin of circulating free DNA: Is exosomal DNA the carrier

The usefulness of circulating free DNA (cfDNA) for analysis of genetic alterations is largely accepted. We accomplished multicenter prospective study to investigate sequential change of cfDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. The detection frequency was elevated as cancer progressed, and the prognosis of the patients in which cfDNA was detected was poorer than those not detected. These results suggest that appearance of cfDNA was associated with tumor progression. To verify that, animal experiment using immunodeficient mice, NOJ mice, was performed. After transplantation with human lung cancer cell line, H1975 carrying EGFR L858R, and T790M, into dorsal flanks of these mice, systemic metastasis occurred. cfDNA was sequentially analyzed, resulting that the amount of cfDNA was correlated with tumor burden and metastatic status. In spite of these results, origin, kinetics or possible function on tumor progression of cfDNA has not been elucidated. We have reported there were large sized DNA fragments, around 5 Kb, which is longer than 170 bp of cfDNA conventionally detected in peripheral blood of advanced cancer patients. Exosomes, extracellular vesicles detected in peripheral blood has been reported to be involved in tumor progression through vesicle-mediated communication. In general, exosomes deliver protein, lipid and RNA, and few DNA was contained. However, some researchers reported a large proportion of cfDNA was localized in exosomes. To investigate the origin and localization of cfDNA in peripheral blood, we analyzed relationship between cfDNA and exosomal DNA. We isolated both of cfDNA and exosomal DNA simultaneously, from plasma of healthy individuals and advanced non-small cell lung cancer patients, and compared the DNA yield, DNA size distribution and EGFR mutation detection rate. Localization of DNA in exosome is also investigated using fluorescent dye. We used Total Exosome Isolation Kit ® from plasma with/without proteinase K for isolation of exosome from 200 uL plasma, and extracted DNA using Maxwell RSC ® circulating cell free DNA cartridge. The proportion of exosomal DNA /cfDNA (e/c DNA ratio) varied individually, but e/c DNA ratio was lower in plasma from cancer patients than healthy individuals. However, large sized fragments of cfDNA were observed in exosomal DNA in lung cancer patients. These results suggest that exosomal DNA might have some interaction with large sized cfDNA fragments observed in plasma isolated from advanced cancer patients. Citation Format: Chiho Nakashima, Tomonori Abe, Akemi Sato, Tomomi Nakamura, Kazutoshi Komiya, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane. Investigation of origin of circulating free DNA: Is exosomal DNA the carrier [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 3646.