Abstract P2-01-08: Enumeration of heterogeneous circulating tumor cells (CTCs) using size-based method in early, and metastatic, breast cancer patients
Authors: Tozuka K , Nagai SE , Kubo K , Komatsu K , Takai K , Inoue K , Matsumoto H , Hayashi Y , Tsuboi M , Yamada Y , Wang X , Suganuma M 
Published: February 2018

Abstract
Background
The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy, and also a prognostic marker for patients with metastatic breast cancer. One of the main methods to detect CTCs is CellSearch system, which uses immune-magnetic separation followed by immunocytochemistry. A microdevice (CTChip from ClearCell system) can capture and enumerate CTCs based on distinctive physiological differences (size and deformability) between cancer cells and blood cells. CTChip thus obtains a larger CTC yield than affinity-based separation, which enriches a particular subgroup of cells expressing EpCAM. In this study, we enumerate CTCs in peripheral blood from early and metastatic breast cancer patients using a size-based method.
Patients and methods
We examined blood samples from a total of 18 early and metastatic breast cancer patients, after obtaining written informed consent. Blood samples were taken in sodium EDTA tubes after discarding the first 1ml of blood from the syringe. Two ml blood samples were applied to CTChip (ClearCell system), and CTCs were eventually trapped in the microwells of the CTChip. Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4',6-diamidino-2-phenylindole (DAPI) for nuclei: CK8/18-positive, DAPI-positive and CD45-negative cells more than 10 μm in diameter were defined as CTCs. Eight patients were examined using both the CTChip and CellSearch system to compare the yield of CTCs.
Results
Of 18 patients, 6 were de novo stage IV, 6 were recurrent and 6 were early stage breast cancer patients. Of primary tumors, 8 were HER2- and ER and/or PR +, 6 were HER2-and ER- and PR-, 3 were HER2+ and ER and/or PR +, and one was HER2+ and ER- and PR-. Using CTChip, detected CTCs ranged from 3 - 107 cells/2 ml in all cases: 3 - 83 for early stage, 19 - 156 for stage IV and 21 - 146 for recurrent. The number of CTCs found in recurrent patients tended to be higher than in early stage patients. Size-based method using CTChip clearly showed high sensitivity compared with the CellSearch system, which detected CTCs in only 2 cases out of 8. In analysis by immunochemistry, we found CK-negative, CD45-negative and DAPI positive cells with larger diameter (&gt;16 μm) than CK-positive CTCs in most patients, and the numbers were higher in stage IV (8.5 cells of median value) and recurrent (13 cells) patients than in early stage patients (1.5 cells). Our study suggested that CK-negative large cells might be CTCs with epithelial–mesenchymal transition (EMT).
Conclusion
This size-based technology enables us to capture CTCs regardless of EpCAM expression. Enumerated CTCs varied in size and positivity of CK8/18, suggesting the heterogeneity of CTCs. Further research, especially focusing on EMT will be crucial to understand the key mechanism of metastasis and drug resistance.
Citation Format: Tozuka K, Nagai SE, Kubo K, Komatsu K, Takai K, Inoue K, Matsumoto H, Hayashi Y, Tsuboi M, Yamada Y, Wang X, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) using size-based method in early, and metastatic, breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-08.