Stem cell and epithelial-mesenchymal transition markers on circulating tumor cells in patients with metastatic breast cancer.

Abstract

Abstract Abstract #107 Background: Stem cell like tumor cells have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, these cells may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT), which allows them to travel to the site of metastasis formation without getting affected by conventional treatment. Assuming that metastasis requires a dissemination of tumor stem cells or tumor cells showing EMT, it was the purpose of this study to identify these cells among circulating tumor cells (CTC) in blood samples of 28 metastatic breast cancer patients.
 Materials and Methods: 5 ml blood was analyzed for EpCAM, MUC-1 and HER-2 transcripts with the AdnaTest BreastCancer (AdnaGen AG). The recovered c-DNA was additionally multiplex tested for three EMT markers [Twist, Akt2, PI3K (TAP)] and separately for the tumor stem-cell marker 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 resulting PCR fragments were separated and quantified using the Agilent Bioanalyzer 2100.
 Results: Applying an amplicon cut-off value of 0.2 ng/μl for Akt2, 0.15 ng/μl for Twist, 0.25 ng/μl for PI3K and 0.15 ng/μl for ALDH1, 97% of 30 healthy donor samples investigated were negative for TAP and 95% for ALDH1 transcripts. The spiking experiments revealed 80% recovery of the IGROV cells. CTC were detected in 12/28 (43%) breast cancer samples. All samples were further examined for tumor stem cell or TAP markers. In the CTC (+) group 50% were positive for at least one of the TAP markers and 42% for ALDH1. In the CTC (-) group the percentages were 19% and 12%, respectively. The expression of TAP and/or stem cell markers in CTC was compared with the clinical follow up results. In CTC (+) patients diagnosed as non-responders the expression of TAP and ALDH1 was found in 5/12 (40%) samples. In the CTC (+) responder group only one sample expressed TAP and no positive case was found for ALDH1. In the CTC (-) non-responder group 1/14 samples was positive for TAP and ALDH1. 2/14 (14%) of CTC (-) responders were positive for TAP and 1/14 for ALDH1, respectively.
 Conclusion: A major proportion of CTC found in the blood of metastatic breast cancer patients shows EMT and tumor stem cell characteristics supporting the hypothesis that these markers are an indicator for therapy resistant tumor cell populations and, therefore, for an inferior prognosis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 107.