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01.04.2014 | Research article | Ausgabe 2/2014 Open Access

Breast Cancer Research 2/2014

Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients

Breast Cancer Research > Ausgabe 2/2014
Maryam B Lustberg, Priya Balasubramanian, Brandon Miller, Alejandra Garcia-Villa, Clayton Deighan, Yongqi Wu, Sarah Carothers, Michael Berger, Bhuvaneswari Ramaswamy, Erin R Macrae, Robert Wesolowski, Rachel M Layman, Ewa Mrozek, Xueliang Pan, Thomas A Summers, Charles L Shapiro, Jeffrey J Chalmers
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​bcr3622) contains supplementary material, which is available to authorized users.
Maryam B Lustberg, Priya Balasubramanian, Charles L Shapiro and Jeffrey J Chalmers contributed equally to this work.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

MBL, PB, CLS and JJC conceived of and designed the study. All authors collected and assembled the data. XP, MBL, PB, CLS and JJC analyzed and interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.



Circulating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or it can be initially not present. We designed the present prospective trial to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM-dependent enrichment and/or isolation technology.


A total of 32 patients with metastatic breast cancer were enrolled, and blood samples were processed using a previously described negative depletion immunomagnetic methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep sequential labeling was performed to label and fix cell-surface markers followed by permeabilization for cytokeratins (CK) 8, 18 and 19. Multiparametric flow cytometry (FCM) analysis was conducted using a BD LSR II flow cytometer or a BD FACSAria II or FACSAria III cell sorter. Immunocytochemical staining on postenrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM).


CD45-negative/CK-positive (CD45− CK+) populations with EpCAM + and EpCAM − expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM + and EpCAM − populations that were CK + and coexpressing the pan-hematopoietic marker CD45 were also noted. There were more CK + EpCAM − events/ml than CK + EpCAM + events/ml in both the CD45− and CD45+ fractions (both statistically significant at P ≤ 0.0005). The number of CK + CD45− and CK + CD45+ events per milliliter in blood samples (regardless of EpCAM status) was higher in patient samples than in normal control samples (P ≤ 0.0005 and P ≤ 0.026, respectively). Further, a significant fraction of the CK + CD45+ events also expressed CD68, a marker associated with tumor-associated macrophages. Higher levels of CD45-CK + EpCAM − were associated with worse overall survival (P = 0.0292).


Metastatic breast cancer patients have atypical cells that are CK + EpCAM − circulating in their blood. Because a substantial number of these patients do not have EpCAM + CTCs, additional studies are needed to evaluate the role of EpCAM − circulating cells as a prognostic and predictive marker.
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