Abstract 2387: Analysis of the expression of stemness and epithelial-to-mesenchymal transition markers in circulating tumor cells of patients with breast cancer
Authors: Papadaki Maria A. , Kallergi Galaktia , Agelaki Sofia , Polioudaki Hara , Mavroudis Dimitrios , Georgoulias Vassilis , Theodoropoulos Panayiotis A. 
Published: August 2012

Abstract
Introduction: Circulating tumor cells (CTCs) have been identified in peripheral blood (PB) of patients with breast cancer and their presence has been associated with poor disease outcome. Cancer is considered to be driven by cancer stem cells (CSCs) whose connection to metastatic spread remains incompletely understood. In addition, Epithelial-to-Mesenchymal transition (EMT) has been proposed as an essential process in the metastatic cascade. A link between stem cell-like properties and the ability of cancer cells to undergo EMT has recently been suggested. In breast cancer, ALDH1 and Twist have been proposed among others as stemness and EMT markers, respectively. The expression of these markers in CTCs could be an important determinant of increased metastatic risk and resistance to conventional therapies. In the present study we aimed to investigate the co-expression of ALDH1 and Twist in CTCs from patients with metastatic breast cancer, at the single cell level. Patients and Methods: PBMCs’ cytospins were prepared from PB obtained from 150 patients with metastatic breast cancer, prior to the initiation of first-line therapy. Triple immunofluorescence experiments were performed in patients’ samples using the cytokeratin anti-mouse antibody (A45-B/B3) conjugated with Alexa 488 by Zenon technology along with ALDH1 anti-rabbit and Twist anti-mouse antibodies. A total of 500.000 PBMCs per patient were analyzed by the use of ARIOL system. Results: CTCs were identified in 70 out of 150 (47%) patients. A total of 428 CTCs were detected with a median of 2 CTCs per patient (range: 1-27). All patients had detectable ALDH1 expressing CTCs, whereas high ALDH1 expression levels were confirmed in 63% of the total number of CTCs identified. In addition, Twist expression in CTCs was evident in 99% of patients and in 93% of the total CTCs detected. Concerning the co-expression of these molecules in CTCs, the most abundant phenotype was ALDH1high Twist +, identified in 62% of CTCs, with a median expression of 58% per patient. Furthermore, no CTCs expressing the phenotype ALDH1- Twist + were detected in any patient. The correlation between Twist expression and high ALDH1 expression levels in CTCs was statistically significant (p=0.005). Conclusions: The above results demonstrate that stemness and EMT markers are co-expressed in the majority of CTCs from patients with metastatic breast cancer. This observation supports the hypothesis that there is a link between stem-cell like features and EMT in cancer cells. In addition, the co-expression of stemness and EMT markers in CTCs could be related to their metastatic potential and might help to further refine prognosis of patients with breast cancer.
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 2387. doi:1538-7445.AM2012-2387