Abstract 5250: Analysis of circulating plasma DNA on metastatic animal model using human lung cancer cell lines
Authors: Aragane Naoko , Sato Akemi , Kobayashi Naomi , Sueoka Eisaburo , Okada Seiji , Kimura Shinya 
Published: August 2015

Abstract
Circulating plasma DNA (cpDNA) has been widely applied to detecting molecular alterations of cancers. However, the mechanisms regarding how tumor derived DNA is released into peripheral blood have not been clarified. In this study, we established animal model with highly metastasis, and analysis relationship between the amount of cpDNA derived from tumor and tumor progression. We recently established a system for monitoring EGFR mutation, T790M using mutation-biased PCR and quenched probe (MBP-QP) method. T790M is a gatekeeper mutation of EGFR, which appears in half of lung cancer patients treated with EGFR tyrosine kinase inhibitors (EGFR-TKI). Because the system is highly sensitive, it was able to successfully detect T790M using circulating plasma DNA (cpDNA). Using this system, we performed a prospective, multicenter, observational study, and showed usefulness of this method for monitoring acquired resistance to EGFR-TKI. In this study, we applied the technique to monitor metastasis in an animal model. To establish an animal model reflecting human metastatic lung cancers, we used NOD/SCID/Jak3null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and dendritic cell function, and complement activation, as well as T and B cell deficiencies. After screening twenty human lung cancer cell lines through expression patterns of E-cadherin and vimentin according to epithelial mesenchymal transition features, an H1975 cell line carrying EGFR mutations, L858R and T790M, was selected for investigation. After inoculation of the cells into the dorsal flanks, metastatic status was monitored. The frequency of metastasis to axillary and abdominal lymph nodes was 79% (11/14), and that to lung and liver occurred in 64% (9/14). Peritoneal dissemination with ascites was also observed in 29% (4/14). In total, one or more of these types of metastasis was observed in 93% (13/14) of the mice. We confirmed the existence of H1975 cells in metastatic lesions by detection of T790M and L858R using MBP-QP system. Next, we investigated the relationship between tumor progression and amount of EGFR mutations with cpDNA analyzed from the area under the mutation peak. The frequency of metastasis increased with time concomitant with increasing detection of EGFR mutations using plasma DNA. Tumor volume of the primary lesion was significantly correlated with area under the mutation peak, whereas that of metastatic lesions for T790M was not. Regardless of tumor volume of metastatic lesions, area under the mutation peak was relatively high. This animal model should be useful to investigate further the mechanisms of circulating tumor derived DNA in peripheral blood and could be applicable to a preclinical trial for a novel anti-cancer agent targeted at metastasis.
Citation Format: Naoko Aragane, Akemi Sato, Naomi Kobayashi, Eisaburo Sueoka, Seiji Okada, Shinya Kimura. Analysis of circulating plasma DNA on metastatic animal model using human lung cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5250. doi:10.1158/1538-7445.AM2015-5250