Low cytokeratin- and low EpCAM-expressing circulating tumor cells in pancreatic cancer.

Abstract

11046 Background: To date, detecting circulating tumor cells (CTCs) in the peripheral blood of pancreatic patients using standard immuno-capture techniques has met with limited success. As pancreatic cancer is prone to metastasize at distant sites, and therefore should have high numbers of CTCs, it is possible that immuno-capture methods are not suitable for this disease. Using a microfiltration approach, we show that CTCs are present in the peripheral blood in over 75% of pancreatic cancer patients, and that two distinct subtypes can be identified. Methods: Pancreatic patient samples were provided by Medical College of Wisconsin, Milwaukee, WI. CellSieve microfilters, with precision 7 micron diameter pores distributed in uniform arrays were employed. 7.5 mL of whole blood was diluted in pre-fixation solution and filtered through CellSieve microfilters. CTCs collected by this size exclusion technique were fixed, permeabilized, and stained with DAPI, an antibody cocktail against cytokeratin 8, 18 and 19 (FITC), EpCAM (PE), and CD45 (Cy5). CTCs, defined as cytokeratin positive and CD45 negative, were found in two distinct subtypes. One subtype had the “classic” characteristics of a CTC, with high EpCAM and cytokeratin expression, identifiable cytokeratin filamentation, and a cancer-like nuclear structure. The second subtype is indicative of a CTC undergoing epithelial-mesenchymal transition (EMT), with low or no EpCAM, weak cytokeratin expression, and a smooth oval nuclear structure. Results: The “classic” CTCs were found in ~20% (n=40) of patient samples. The EMT-like CTCs were found in ~75% of the same patient cohort. Neither cell was present in any healthy subjects (n=30). EMT-like CTCs consistently lacked EpCAM expression and commonly presented as multi-cell clusters, or microemboli, in ~40% of the cases. Conclusions: We show that two distinct CTC subtypes circulate in the blood of most pancreatic patients. The low expression of cytokeratin and EpCAM of the EMT-like subtype implies that immuno-capture based CTC isolation methods have limited utility for pancreatic cancer. Further, this subtype provides a useful strategy for tracking pancreatic CTCs over the course of treatment.