Abstract 1446: Detection of circulating tumor cells for malignant epitherial tumor using a novel cytometry-based system.
Authors: Ito Yukiko , Sawada Takeshi , Watanabe Masaru , Fujimura Yuu , Hashimoto Jun , Kodaira Makoto , Yunokawa Mayu , Yamamoto Harukaze , Yonemori Kan , Shimizu Chikako , Tamura Kenji , Fujiwara Yasuhiro , 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. We have developed a novel flow-cytometry-based CTCs detection and sorting system (On-Chip Sort) independent of EpCAM expression of tumor cells. EpCAM-positive cell line KATO-III and EpCAM-negative cell line A549 and PC-14 were used for the preclinical study. Various numbers of cells were spiked into blood and the sensitivity in detection was evaluated in a blinded manner. The recovery rate ranged from 40.5 - 157.3% with our system and 0 - 89.8% with the CellSearch system. A significantly higher recovery rate was observed with our system (90 - 102%) than with the CellSearch system (0%) when the PC-14 cells were spiked, suggesting a superior sensitivity of our system in capturing EpCAM-negative tumor cells. These data imply an advantageous potential of our detection system.
Here we report the results of a clinical feasibility study on the detection of CTCs in patients with malignant epithelial tumors, including breast cancer, ovarian cancer, cervical cancer and primary unknown cancer, etc.
Sixteen mL 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. Twenty-four patients with epithelial malignancies and two healthy donors were recruited at National Cancer Center Hospital. The results of the CTC enumeration were compared with those by CellSearch®.
In 24 blood samples from cancer patients, CTCs detected by our system ranged from 0 - 829 CTCs (median, 4.5), and 83.3% (20/24) of the samples were above the threshold level (≧ 2 / 4ml). On the other hand, with CellSearch®, only 29% of the samples had a ≧ 2 / 7.5 ml, threshold level.
In 2 blood samples from the healthy donors, 1 CTC was detected by our system and CellSearch® detected none.
These results of a clinical feasibility study showed our system was significantly more sensitive for CTC enumeration in various cancer patients.
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, BT-20 cells spiked into normal blood were isolated and profiled Phosphoinositide-3-kinase catalytic alpha (PIK3CA) mutations status correctly.
In the near future, we plan to apply this approach to clinical samples of breast cancer patients and investigate the correlation of PIK3CA mutations in sorted CTCs, circulating free DNA and primary lesions.
(Data to be updated prior to presentation)
Citation Format: Yukiko Ito, Takeshi Sawada, Masaru Watanabe, Yuu Fujimura, Jun Hashimoto, Makoto Kodaira, Mayu Yunokawa, Harukaze Yamamoto, Kan Yonemori, Chikako Shimizu, Kenji Tamura, Yasuhiro Fujiwara, Yasuhiro Koh, Fumiaki Koizumi. Detection of circulating tumor cells for malignant epitherial tumor using a novel cytometry-based 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 1446. doi:10.1158/1538-7445.AM2013-1446