Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism 
by which cells change phenotype during carcinogenesis, as well as tumor 
metastasis. Whether EMT is involved in cancer metastasis has a specific, 
practical impact on the field of circulating tumor cells (CTCs). Since the 
generally accepted definition of a CTC includes the expression of epithelial 
surface markers, such as EpCAM, if a cancer cell loses its epithelial surface 
markers (which is suggested in EMT), it will not be separated and/or identified 
as a CTC. We have developed, and previously reported on the use of, a purely 
negative enrichment technology enriching for CTCs in the blood of squamous cell 
carcinoma of the head and neck (SCCHN). This methodology does not depend on the 
expression of surface epithelial markers. Using this technology, our initial 
data on SCCHN patient blood indicates that the presence of CTCs correlates with 
worse disease-free survival. Since our enrichment is not dependent on epithelial 
markers, we have initiated investigation of the presence of mesenchymal markers 
in these CTC cells to include analysis of: vimentin, epidermal growth factor 
receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have 
demonstrated not only presumed CTCs that express and/or contain: a nucleus, 
cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells 
that contain all of the aforementioned proteins except cytokeratins, suggesting 
that the cells have undergone the EMT process. We suggest that our negative 
depletion enrichment methodology provides a more objective approach in 
identifying and evaluating CTCs, as opposed to positive selection approaches, as 
it is not subjective to a selection bias and can be tailored to accommodate a 
variety of cytoplasmic and surface markers which can be evaluated to identify a 
multitude of phenotypic patterns within CTCs from individual patients, including 
so-called EMT as presented here.