Abstract P4-01-16: Detection of EMT, anoikis and stem cell markers in metastatic breast cancer patients under different lines of treatment
Authors: Trapp Elisabeth K , Rack Brigitte , Majunke Leonie , Koch Julian , Hofmann Simone , Friedl Thomas WP , Neugebauer Julia , Jückstock Julia , Jäger Bernadette , Huober Jens , Janni Wolfgang , Alunni-Fabbroni Marianna 
Published: May 2015

Abstract
Background: Metastasis are thought to be induced by occult spreading of tumor cells already during the early phases of the disease. Circulating Tumor Cells (CTCs) are regarded as precursors of distant metastasis, while detaching from the primary tumor and originating micrometastases in distant organs.
Recent evidences pointed to CTC heterogeneity, showing that CTCs can present different phenotypes. Goal of this study was to identify in metastatic breast cancer (mBC) patients CTCs with EMT features and to further characterize them with respect to cellular heterogeneity.
Methods: This prospective ongoing study included mBC patients (n=12) with a median age of 58,5 years (range: 35-78 years), enrolled in a time frame of 7 months while undergoing therapy. The majority of patients were estrogen and/or progesterone receptor positive (11/12) and HER2 non-amplified (10/12). Patients had metastatic lesions in viscera as well as bone (6/12), only bone (2/12), only viscera (2/12), only viscera in combination with locoregional recurrence (1/12), or visceral and bone metastases in combination with locoregional recurrence (1/12). Current therapy was endocrine therapy (4/12), chemotherapy (3/12), chemotherapy and HER2-targeted therapy (2/12), HER2-targeted monotherapy (1/12), antiangiogenic therapy (1/12), or surgical therapy only (1/12). Blood samples, withdrawn at any time point during treatment, were depleted of EpCAM+ cells and CD45+ white blood cells (EpCAM/CD45 depleted fraction) (Mego et al., Int J Cancer 2012;130(4):808-816) and expression of epithelial markers (EpCAM, E-Cadherin, Cytokeratin 8,18,19), mesenchymal markers (n-Cadherin, Vimentin), EMT-inducing factors (Twist1, Snail1, SLUG, Zeb1 and FoxC2), anoikis markers (TrkB1, Bcl2) and stem cell markers (CD24, CD44, CD133) were analyzed by qRT-PCR. CTC counting with the CellSearchTM system (Veridex, Raritan NJ) was run in parallel. Healthy donors (n=10) were included in the study as negative controls.
Results: The data collected so far showed that 50% of the patients were positive for CTCs in the EpCAM+ fraction as detected with the CellSearchTM system, while 33% were still CTC positive in the EpCAM/CD45 depleted fraction. 50% of the patients, found CTC negative with CellSearchTM, were nevertheless positive for the epithelial and EMT markers in the EpCAM/CD45 depleted fraction (EpCAM 25%, E-Cadherin 25%, CK8 16,6%, CK18 16,6%, CK19 16,6%, SLUG 8,3%, Zeb1 25%, Twist1 8,3%, Vimentin 58,3%). N-cadherin, FoxC2, Snail1, TrkB1, CD24, CD44 and CD133 were never detectable.
Conclusions: These preliminary results suggest that mBC patients undergoing different lines of therapy present heterogeneous CTCs. 50% of the patients with undetectable EpCAM+ CTCs, were found CTC positive in the EpCAM/CD45 depleted fraction. mBC patients might be insensitive to treatment due to a selection of resistant CTCs subpopulations with different phenotypes. Additional patients with the same clinical characteristics will be analyzed in the next 6 coming months and updated results will be included.
Citation Format: Elisabeth K Trapp, Brigitte Rack, Leonie Majunke, Julian Koch, Simone Hofmann, Thomas WP Friedl, Julia Neugebauer, Julia Jückstock, Bernadette Jäger, Jens Huober, Wolfgang Janni, Marianna Alunni-Fabbroni. Detection of EMT, anoikis and stem cell markers in metastatic breast cancer patients under different lines of treatment [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-16.