Abstract 1735: Evaluation of a novel flow cytometry-based system for the detection of circulating tumor cells
Authors: Watanabe Masaru , Uehara Yuri , Yamashita Namiko , Fujimura Yuu , Nishio Kaori , Suzuki Junko , Koizumi Fumiaki , Koh Yasuhiro 
Published: August 2012

Abstract
Background: The presence and number of circulating tumor cells (CTCs) in the blood of solid tumor patients is predictive of clinical outcome. To date the CellSearch system is the only FDA-approved CTCs enumeration system for advanced breast, prostate and colon cancers. However, sensitivity issues due to EpCAM-based enrichment and limited capability for subsequent molecular analysis need to be addressed for the use of CTCs in the clinical setting. We have developed a flow cytometry-based CTCs detection system independent of EpCAM expression which allows enumeration and characterization of CTCs for multiple molecular analyses. Here we report the results of preclinical study with this novel flow cytometry-based CTCs detection system. Materials and Methods: EpCAM-positive gastric cancer cell line KATO-III and lung cancer cell line PC-9 and EpCAM-negative lung cancer cell line A549 were used for this study. Cells were spiked into 1 mL or 4 mL of peripheral blood from healthy donors. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells followed by fixation and labeling with Alexa Fluor 700-CD45, PE-EpCAM and/or FITC-cytokeratin (CK) antibodies. The number of cancer cells in the enriched sample was counted using a novel FISHMAN-R flow cytometry system. CTCs enumeration and a head-to-head comparison with CellSearch system were carried out in a blinded manner at two different sites, Shizuoka cancer center (SCC) and National cancer center Tokyo (NCC) in parallel. Results: Various numbers of KATO-III and PC-9 cells were spiked into 1 mL of blood for a pilot study and sensitivity in detection was evaluated. Recovery rate ranged from 108.8 to 160.0% for KATO-III and from 70.0 to 74.7% for PC-9 cells, respectively. Good correlation was observed between the observed number and the expected number of cancer cells by regression analysis demonstrating a slope of 1.07 and a correlation coefficient (R2) of 0.99 for KATO-III and a slope of 0.75 and R2 of 1.00 for PC-9 cells, respectively. Then we proceeded to the spike-in experiment in 4 mL of blood and this study was performed at two different sites. Recovery rate ranged from 89.0 to 120.0% at SCC and from 88.0 to 130.0% at NCC, respectively, suggesting the robustness of our detection system. In the following head-to-head comparison study with CellSearch system in a blinded manner, the average recovery rate was 90.3% at SCC, 87.6% at NCC and 62.0% by CellSearch system, respectively. EpCAM-negative lung cancer cell line A549 cells were also spiked into 4 mL of peripheral blood and enumerated by our system and CellSearch system. Markedly higher recovery rate was observed with our system (91.9%) than with CellSearch system (28.8%), suggesting superior sensitivity of our system in capturing EpCAM-negative tumor cells. Conclusions: These data imply the potential of our detection system and further evaluation with clinical samples should be conducted.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1735. doi:1538-7445.AM2012-1735