title: Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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).
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.forename: Maryam B
authors.initials: MB
authors.lastname: Lustberg
authors.forename: Priya
authors.initials: P
authors.lastname: Balasubramanian
authors.forename: Brandon
authors.initials: B
authors.lastname: Miller
authors.forename: Alejandra
authors.initials: A
authors.lastname: Garcia-Villa
authors.forename: Clayton
authors.initials: C
authors.lastname: Deighan
authors.forename: Yongqi
authors.initials: Y
authors.lastname: Wu
authors.forename: Sarah
authors.initials: S
authors.lastname: Carothers
authors.forename: Michael
authors.initials: M
authors.lastname: Berger
authors.forename: Bhuvaneswari
authors.initials: B
authors.lastname: Ramaswamy
authors.forename: Erin R
authors.initials: ER
authors.lastname: Macrae
authors.forename: Robert
authors.initials: R
authors.lastname: Wesolowski
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Layman
authors.forename: Ewa
authors.initials: E
authors.lastname: Mrozek
authors.forename: Xueliang
authors.initials: X
authors.lastname: Pan
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Summers
authors.forename: Charles L
authors.initials: CL
authors.lastname: Shapiro
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Chalmers
mesh_terms: D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000097:blood / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000097:blood / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000097:blood / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000097:blood* / Q000378:metabolism; D001943:Breast Neoplasms / Q000097:blood* / Q000378:metabolism / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000097:blood / Q000378:metabolism; D045744:Cell Line, Tumor; D000071858:Epithelial Cell Adhesion Molecule; D066246:ErbB Receptors / Q000097:blood / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D053538:Keratin-18 / Q000097:blood / Q000378:metabolism; D053539:Keratin-19 / Q000097:blood / Q000378:metabolism; D053533:Keratin-8 / Q000097:blood / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000097:blood / Q000378:metabolism; D061986:MCF-7 Cells; D018613:Microscopy, Confocal; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D011379:Prognosis; D011446:Prospective Studies; D014746:Vimentin / Q000097:blood / Q000378:metabolism; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D066246:ErbB Receptors; D053538:Keratin-18; D053539:Keratin-19; D053533:Keratin-8; D017493:Leukocyte Common Antigens; D014746:Vimentin; C067980:CD68 antigen, human; D000097382:CD68 Molecule