Analysis of EpCAM positive cells isolated from sentinel lymph nodes of breast cancer patients identifies subpopulations of cells with distinct transcription profiles

INTRODUCTION: The presence of tumor cells in the axillary lymph nodes is the most important prognostic factor in early stage breast cancer. However, the optimal method for sentinel lymph node (SLN) examination is still sought and currently many different protocols are employed. To examine two approaches for tumor cell detection we performed, in sequence, immunomagnetic enrichment and RT-PCR analysis on SLN samples from early stage breast cancer patients. This allowed us to compare findings based on the expression of cell surface proteins with those based on detection of intracellular transcripts. METHODS: Enrichment of EpCAM and Mucin 1 expressing cells from fresh SLN samples was achieved using magnetic beads coated with the appropriate antibodies. All resulting cell fractions were analyzed by RT-PCR using four chosen breast epithelial markers (hMAM, AGR2, SBEM, TFF1). Gene expression was further analyzed using RT-PCR arrays and markers for epithelial to mesenchymal transition (EMT). RESULTS: Both EpCAM and Mucin 1 enriched for the epithelial-marker expressing cells. However, EpCAM-IMS identified epithelial cells in 71 SLNs, whereas only 35 samples were positive with RT-PCR targeting breast epithelial transcripts. Further analysis of EpCAM positive but RT-PCR negative cell fractions showed that they had increased expression of MMPs, repressors of E-cadherin, SPARC and vimentin, all transcripts associated with the process of epithelial to mesenchymal transition. CONCLUSIONS: The EpCAM IMS-assay detected tumor cells with epithelial and mesenchymal-like characteristics, thus proving to be a more robust marker than pure epithelial derived biomarkers. This finding has clinical implications, as most methods for SLN analysis today rely on the detection of epithelial transcripts or proteins.