Abstract 3284: Isolation of circulating tumor cells (CTCs) with mesenchymal and stem cell markers in localized and metastatic breast cancer using a novel negative selection enrichment
Authors: Lustberg Maryam B. , Balasubramanian Priya , Lang Jas C. , Ruppert Amy S. , Carothers Sarah , Berger Michael J. , Mrozek Ewa , Ramaswamy Bhuvaneswari , Layman Rachel C. , Chalmers Jeffrey , Shapiro Charles L. 
Published: February 2011

Abstract
Background: Breast circulating tumor cells (CTCs) are commonly isolated by positive selection enrichment technology, which targets the epithelial cell adhesion activating molecule, EpCAM. However, CTCs with low or no EpCAM expression, such as those from basal and normal-like subtypes, are more likely to be missed by this method. We developed a novel negative enrichment technology to detect CTCs with mesenchymal and stem cell markers in localized and metastatic breast cancer (BC).
Patients and Methods: Twenty nine patients with localized and metastatic BC initiating chemotherapy were enrolled. CTCs were isolated in 10 mL of peripheral blood using negative selection with immunomagnetic tagging and removal of CD45 positive cells at 3 time points: pretreatment, after one cycle of treatment (TP1) and at end of treatment or at disease progression in metastatic patients (TP2). Immunocytochemical staining for nucleus, cytokeratin (8,18,19), vimentin, and CD44 was completed on available samples. Double staining for nucleus and cytokeratin with high nucleus to cytoplasm ratio defined a CTC. Enrollment is ongoing.
Results: Median age was 57 yrs (range 28-78 yrs); stage distributions were I-2 (7%), II-10 (35%), III-3 (10%), and IV-14 (48%); 19 (66%) were estrogen receptor positive (ER+), 4 (14%) estrogen and progesterone receptor negative (ER-PR-HER2 non-overexpressing) and 6 (21%) HER2 overexpressing. Negative enrichment yielded an average log10 depletion of nucleated cells of 2.74 and an overall, average log10 depletion of 5.2 (&gt;100,000 enrichment). No CTCs were identified in 5 healthy volunteers or in buffy coats purchased from the Red Cross. CTCs were identified in all stages at pretreatment but decreased to 0 in three patients by TP1. Baseline median CTC level was 373/mL in localized BC (range 2.2-1975/mL) and 761/mL in metastatic BC (range 9.9-47513/mL). In localized BC, the median percent (%) change in CTCs was +2% at TP1 (n= 13; range −100% to +14945%) and −95% at TP2 (n=5; range −100% to −13%). In metastatic BC, median CTC numbers decreased by 41% at TP1 (n=11; range −100% to +4222%) and increased at TP2 in 2 patients by 231% and 730%. Baseline CTC levels and changes at TP1 were not significantly different between localized and metastatic groups by the Wilcoxen Rank Sum test. All tested CTCs expressed vimentin and CD44. In addition to CTCs, a population of cells without detectable cytokeratin expression but positive for the other markers was identified in some samples. Further characterization of these cells for epithelial mesenchymal transition markers is underway.
Conclusions: CTCs identified with this novel negative selection method have both mesenchymal and stem cell markers in localized and metastatic BC. Higher CTC numbers with epithelial characteristics are detected with this method relative to what is reported with positive selection.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3284.