Abstract 1445: Clinical feasibility study of a novel cytometry-based-system for the detection of circulating tumor cells of patients with lung cancer.
Authors: Sawada Takeshi , Ito Yukiko , Watanabe Masaru , Fujimura Yuu , Nakamichi Shinji , Kanda Shintaro , Horinouchi Hidehito , Fujiwara Yutaka , Nokihara Hiroshi , Yamamoto Noboru , Tamura Tomohide , Koh Yasuhiro , Koizumi Fumiaki 
Published: August 2014

Abstract
Enumeration and characterization of circulating tumor cells (CTCs) are, as "liquid biopsy," expected to provide useful clinical information on prognosis, cancer staging, drug choice, and therapeutic efficacy. We have developed a novel flow-cytometry-based CTCs detection and sorting system (On-Chip Sort) independent of EpCAM expression of tumor cells. In a preclinical study, various kinds of cell lines were spiked into human blood and the sensitivity in detection was evaluated. Enumeration of the spiked tumor cells was linear over a range of 10 to 1000 cells, with a recovery rate of ≧ 90%. A significantly higher recovery rate was observed with our system (90 - 102%) than with CellSearch® (0%) when EpCAM-negative PC-14 cells were spiked, suggesting a superior sensitivity of our system in capturing EpCAM-negative tumor cells. Here we report the results of a clinical feasibility study on the detection of CTCs in patients with advanced lung cancer.
16mL of peripheral blood was drawn from each case. In our assay, 4mL of samples were hemolyzed and eliminated CD45+ leucocytes by magnetic beads coated with anti-CD45 antibody. After negative CTC enrichment, samples were fixed and labeled with FITC-cytokeratin, PE-EpCAM and Alexa700-CD45 antibodies, and analyzed by using our flow-cytometry-based CTC detection system. 22 advanced lung cancer patients and 3 healthy donors were recruited at National Cancer Center Hospital. The results of the CTC enumeration were compared with those by CellSearch®.
In 22 blood samples from patients, CTCs detected by our system ranged from 0 - 18 CTCs (median, 6.5), and 77.3% (17/22) of the samples were above the threshold level (≧ 4 / 4mL). On the other hand, with CellSearch®, only 27% of the samples had a ≧ 2 / 7.5 mL threshold level. In 2 blood samples from the healthy donors, our system detected 0 and 3 CTCs, and CellSearch® detected 0 and 1 CTC. In the sample from a healthy donor spiked with 18 EpCAM-negative PC-14 cells, our system was able to detect 10 cells; however, CellSearch® could not recover even one cell.
These results of a clinical feasibility study showed our system to be significantly more sensitive for CTC enumeration in advanced lung cancer.
Presently, we are developing a novel approach for molecular analysis of CTCs via sequential immunomagnetic enrichment and FACS sorting to isolate CTCs, followed by real-time PCR analysis using Scorpion Amplified Refractory Mutation System technology. For methods validation, PC-9 and 11_18 cells spiked into normal blood were isolated, and profiled the epidermal growth factor receptor (EGFR) mutations status correctly. In the near future, we plan to apply this approach to clinical samples of lung cancer patients and investigate the correlation of EGFR mutations in sorted CTCs, circulating free DNA and primary lesions.
(Data to be updated prior to presentation)
Citation Format: Takeshi Sawada, Yukiko Ito, Masaru Watanabe, Yuu Fujimura, Shinji Nakamichi, Shintaro Kanda, Hidehito Horinouchi, Yutaka Fujiwara, Hiroshi Nokihara, Noboru Yamamoto, Tomohide Tamura, Yasuhiro Koh, Fumiaki Koizumi. Clinical feasibility study of a novel cytometry-based-system for the detection of circulating tumor cells of patients with lung cancer. [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 1445. doi:10.1158/1538-7445.AM2013-1445