Abstract 2386: Triple fluorescence analysis of estrogen receptor alpha (ER) and ErbB2 status of circulating tumor cells (CTC) in breast cancer: from cell models to metastatic patients
Authors: Bock Carolin , Kuhn Christina , Majunke Leonie , Hofmann Simone , Jäger Bernadette , Theil Conrad , Jeschke Udo , Rack Brigitte , Doisneau-Sixou Sophie F. 
Published: August 2012

Abstract
Purpose: Hormone therapy and anti-ErbB2 (HER-2) therapy are prescribed according to the hormone status (ER/PR protein expression) and ErbB2 expression of the initial tumor. Several trials have shown the prognostic relevance of circulating tumor cells (CTC) in early and metastatic breast cancers. As it appears that the CTC, and consequently the metastatic cells, may have a very different receptor status than the related primary tumors, it is of the highest relevance to analyze ER and ErbB2 expression of the CTC. Experimental procedures: We developed a triple fluorescence technique to visualize simultaneously cytokeratin (8/18, 8/19), ER and ErbB2 on each individual cell. Using this protocol, we analyzed ER and ErbB2 expression in various breast cancer cell lines (MCF-7, T47D, Cama-1, ZR75, SK-Br-3, HCC 3153, MDA-MB-231), and compared ER expression in the different cell lines using also immunostaining. We were then able to test the CTC presence (CK positivity) and ER and ErbB2 expressions on blood samples (cytospins) collected from 15 metastatic breast cancer patients. Results: The 2-step triple fluorescence protocol was optimized to visualize CK in green (DyLight488 conjugate), ER in red (cy3 conjugate) and ErbB2 in blue (AMCA conjugate). Using in parallel this triple fluorescence protocol and the reference immunostaining technique, we could analyze that ER-positive cell lines express very different levels of ER and within one cell line, cells can express low, moderate and/or high levels of ER. The identification of cells that express a very low level of ER is compulsory as the CTC from one unique patient can potentially express differently ER. Consequently, ER-positivity and -negativity have to be clearly defined. CTC analysis from patient blood samples was then performed with an individual assessment, for each single isolated cell, of cytokeratin (epithelial marker that peripheral blood cells should not express) and of ER and ErbB2 expressions. We were able to identify, among the CTC positive patients, changes between primary tumor status and CTC status, e.g. ER-positive primary tumors with both ER-positive and ER-negative CTC. Those ER-negative CTC may direct towards tamoxifen resistance and have to be identified among other ER-positive cells, similarly as any ErbB2 negative CTC. Conclusion: We demonstrated that triple immunofluorescence is very relevant, first to identify CTC, and then to allow an individual assessment of ER and ErbB2 expression, at the protein level. Moreover, the approach we developed allows to characterize a population of CTC with a very low expression of ER. Large multicentric cohorts of breast cancer patients treated with adjuvant therapy should now be analyzed for both ER and ErbB2 status of the CTC, compared to the primary tumor profile. The expected results should lead to optimal individualized treatments.
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 2386. doi:1538-7445.AM2012-2386