Abstract 5105: Molecular characterization of captured circulating tumor cells with a novel cell sorting system.
Authors: Watanabe Masaru , Sawada Takeshi , Serizawa Masakuni , Ito Yukiko , Fujimura Yuu , Koizumi Fumiaki , Koh Yasuhiro 
Published: August 2014

Abstract
Background: The number of circulating tumor cells (CTCs) has the prognostic significance in solid tumor patients. Because of recent progress in targeted therapies, there is an increasing need to obtain tumor tissues for companion diagnostics. In some tumor types such as lung cancer, it is relatively difficult to obtain tumor tissues due to the tumor location and capturing CTCs could be an alternative approach. The CellSearch system, the only FDA-approved CTCs enumeration system has an advantage in detection of CTCs with high reproducibility, but it is not optimized for capturing tumor cells for subsequent molecular analyses. Thus we have developed a novel benchtop cell sorting system equipped with a disposable microfluidic chip (On-Chip Sort, On-Chip Biotechnologies, Tokyo, JAPAN), which may allow better recovery of CTCs than conventional cell sorting systems for further characterization.
Materials and Methods: The On-Chip Sort is a disposable microfluidic-chip-based cell sorter. The sorting is performed by Push-Pull pulse flow in a micro channel, and this novel cell sorting technique is free of cell damage caused by high pressure, ultrasonic and electrical charge. To evaluate the performance of the On-Chip Sort, various numbers of NCI-H1975 human non-small cell lung cancer cells harboring EGFR mutations (L858R and T790M) were spiked into 5 x 105 K562 human leukemia cells serving as surrogate cells for white blood cells. Spiked samples were fixed, stained with cytokeratin-FITC and CD45-Alexa700, and purified using the “On-Chip Sort” cell sorter. Captured tumor cells (defined as CK+/CD45− cells) were subjected to molecular analyses by pyrosequencing for EGFR mutation after whole genome amplification.
Results: EGFR gene mutations (L858R and T790M) were successfully detected from formalin-fixed and immunofluorescently stained single H1975 cell, suggesting captured tumor cells after sorting process can be used for mutational analyses. Results of in vitro dilution model revealed that the detection limit for pyrosequencing of 10% to 20% EGFR mutant allele following whole genome amplification, suggesting mutations can be detected with a certain amount of normal blood cell carryover after sorting process. In spike-in experiment using H1975 cells (10 to 102 H1975 cells spiked into 5 x 105 K562 cells), we observed recovery and purity rates of 30.4% to 45.0% and 8.6% to 45.0%, respectively, which was sufficient for mutational analysis by pyrosequencing. The detection limit for EGFR gene mutations (L858R and T790M) was observed when twenty-five H1975 cells were spiked into 5 x 105 K562 cells, which was thought to be feasible in the clinical setting.
Conclusions: The novel cell sorting system allowed efficient and convenient isolation of circulating tumor cells. These data demonstrated the potential of our capture system and evaluation using clinical samples should be conducted.
Citation Format: Masaru Watanabe, Takeshi Sawada, Masakuni Serizawa, Yukiko Ito, Yuu Fujimura, Fumiaki Koizumi, Yasuhiro Koh. Molecular characterization of captured circulating tumor cells with a novel cell sorting system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5105. doi:10.1158/1538-7445.AM2013-5105