Conclusions
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 CTC are not efficient to identify a subtype of CTC which underwent EMT. (3) The clinical relevance on prognosis and therapy response has to be further evaluated in a prospective trial.
Methods
A total of 2 × 5 ml of blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1, HER2 and beta-Actin transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [TWIST1, Akt2, phosphoinositide kinase-3 (PI3Kα)] and separately for the tumor stem cell marker ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Two BM aspirates from all patients were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.
Results
Ninety-seven percent of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts, respectively. CTC were detected in 97/502 (19%) patients. At least one of the EMT markers was expressed in 29% and ALDH1 was present in 14% of the samples, respectively. Interestingly, 5% of the ALDH1-positive and 18% of the EMT-positive patients were CTC-negative based on the cut-off level determined for CTC-positivity applying the AdnaTest BreastCancer. DTC in the BM were detected in 107/502 (21%) patients and no correlation was found between BM status and CTC positivity (P = 0.41). The presence of CTC, EMT and ALDH1 expression was not correlated to any of the prognostic clinical markers. Conclusions: 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 CTC are not efficient to identify a subtype of CTC which underwent EMT. (3) The clinical relevance on prognosis and therapy response has to be further evaluated in a prospective trial.