Abstract 4149: Genetic analysis of circulating tumor cells using 3D immunostain and FISH
Authors: Mishima Yuji , Matsusaka Satoshi , Takagi Koichi , Mikuniya Mariko , Terui Yasuhito , Hatake Kiyohiko 
Published: August 2014

Abstract
Background: Circulating tumor cells (CTCs) offer a non-invasive approach to characterize metastatic tumor cells. However, the low recovery rates of CTCs by methods performed in most clinical laboratory have limited their usefulness. Recent studies have suggested that discordance may exist between Her-2 status of a patient's CTC and that of primary breast tumor. To investigate Her-2 status in detail, we developed a new technique of the FISH analysis of CTCs. Most CTCs enrichment techniques are anti-EpCAM antibody based. However, some tumor cells express low or no EpCAM. So, we adopted the strategy independent of EpCAM antigenecity in order to reliably analyze genes even with very few CTCs. Material and methods: The method for Her-2 analysis that we established was based on three-dimensional multi-color imaging. Briefly, CTCs of tumor patients were concentrated by negative selection from peripheral blood by using antibodies against WBC, RBC, and platelets. The concentrated CTCs were labeled by fluorescent monoclonal antibodies against pan-cytokeratin and CD45. The specimens were then hybridized with FISH probes and were mounted in DAPI with antifade reagent. The preparations were screened for a series of Z-axis optical sections with a confocal microscope and 3D images were generated using Fluoview software. To inspect the availability of our method, we analyzed the peripheral blood or effusion specimens of patients with gastrointestinal tumors. This study was approved by the Institutional Review Board of Japanese Foundation for Cancer Research. Results: CTCs (Cytokeratin+/CD45-/DAPI+) were easily discriminated from remaining hematopoietic cells (CD45+). And these immunocytochemical staining had no crossover on FISH signal by the advantage of confocal imaging. In addition, three-dimensional imaging reconstruction enabled counting of DNA probe spots that differed in the Z plane but were at the same XY position in the cell. Recovery rates of tumor cells spiked into normal blood averaged 79%. By the analysis of the several cases of peripheral blood and the colon fiber biopsy derived from the same patients, it was suggested that the expression of EpCAM in CTCs was distinctly lower than it in the primary tumor site. Conclusions: Because our method does not depend on expression of EpCAM, even the CTCs that express little or no EpCAM can be identified. In addition, the three-dimensional imaging with high spacial/wavelength resolution made it possible to analyze Her-2 gene status of CTCs easily and accurately. Using this methods, we are currently working on the investigation of Her-2 state in CTCs derived from patients with gastrointestinal tumor and its relation of Her-2 status of primary tumor and metastases.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4149. doi:10.1158/1538-7445.AM2011-4149