Abstract P3-02-09: Expression of Stem Cell and Epithelial-Mesenchymal Transition Markers in Primary Breast Cancer Patients with Circulating Tumor Cells
Authors: Fehm T , Hoffmann O , Wagner P , Goldnau C , Neubauer H , Kimmig R , Wallwiener D , Kasimir-Bauer S. 
Published: April 2011

Abstract
Background: CTCs are suggested as potential surrogate markers for minimal residual disease, the precursor of metastatic disease. Stem cell like tumor cells have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, to be able to disseminate and metastasize, CTCs must be able to perform epithelial-mesenchymal transition (EMT). In metastatic breast cancer, CTCs were shown to express these characteristics. So far, no data are available for primary breast cancer. Therefore, we studied the expression of (1) the EMT markers Twist1, Akt2, PI3K-alpha and (2) ALDH1 as one of the cancer stem cell markers in CTCs isolated from 347 patients with primary breast cancer. Data were correlated with the presence of disseminated tumor cells (DTCs) in the bone marrow (BM) and clinicopathological data of the patients.
Materials and Methods: 2 x 5 ml blood were analyzed for CTCs with the AdnaTest BreastCancer (AdnaGen AG, Hannover, Germany) for the detection of EpCAM, MUC-1, HER-2, and beta-Actin transcripts. The recovered c-DNA was additionally multiplex tested for Twist1, Akt2, PI3K-alpha and separately for ALDH1. The analytical sensitivity was determined by the detection of a low number of target cells (5 IGROV cells spiked into 5 ml blood of healthy donors) using the AdnaTest BreastCancer procedure. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Healthy donor samples without spiked tumor cells were used to determine the specificity of the test. The expression of CD34 was analyzed in a subset of samples to exclude potential interference of normal hematopoietic stem cells. Furthermore, two BM aspirates from each of these patients were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Results: 97% of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts, respectively. The spiking experiments revealed a recovery rate of 80%. CTCs were detected in 79/347 (23%) patients. All samples were further examined for the expression of ALDH1 and EMT markers. At least one of the EMT markers was expressed in 95/322 patients (30%) and ALDH1 was present in 45/247 patients (18%), respectively. Interestingly, 27% of the ALDH1-positive and 42% of the EMT-positive patients were CTC-negative based on the cut-off level determined for CTC-positivity applying the AdnaTest BreastCancer. Twelve patients initially positive for CTCs and positive for at least one of the EMT markers or ALDH1 or both were tested for CD34 expression. However, none of the samples was positive. DTC in the BM could be analyzed in 343 patients, resulting in a positivity rate of 21% (72/343 patients). The presence of CTCs, EMT and ALDH1 expression was not correlated to any of the prognostic markers including DTC in the BM.
Conclusion: Our data indicate that (1) a subset of primary breast cancer patients shows EMT and stem cell characteristics and (2) the currently used detection methods for CTCs are not efficient to identify a subtype of CTCs which underwent EMT or display a cancer stem cell phenotype. (3) The clinical relevance on prognosis and therapy response has to be further evaluated in a prospective trial.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-09.