title: EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low.
abstract: OBJECTIVE
  The potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes.
  
  
  RESULTS
  Cancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups (p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB.
  
  
  METHODS
  Ten cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB.
  
  
  CONCLUSIONS
  EpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.
authors.affiliation: Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.|Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Ring
authors.affiliation: Department of Surgery, Lenox Hospital New York, New York, NY 10065, USA.
authors.forename: Neal
authors.initials: N
authors.lastname: Mineyev
authors.affiliation: Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.|Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Weizhu
authors.initials: W
authors.lastname: Zhu
authors.affiliation: Advanced Cell Diagnostics, Research and Development, Hayward, CA 94545, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Park
authors.affiliation: BD Biosciences, Research and Development, San Jose, CA 95131, USA.
authors.forename: Chip
authors.initials: C
authors.lastname: Lomas
authors.affiliation: Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA.|Division of Hematology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Vasu
authors.initials: V
authors.lastname: Punj
authors.affiliation: Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA.|Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Yu
authors.affiliation: Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.|Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Dany
authors.initials: D
authors.lastname: Barrak
authors.affiliation: Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Victoria
authors.initials: V
authors.lastname: Forte
authors.affiliation: Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.|Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Tania
authors.initials: T
authors.lastname: Porras
authors.affiliation: Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Debu
authors.initials: D
authors.lastname: Tripathy
authors.affiliation: Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.|Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Julie E
authors.initials: JE
authors.lastname: Lang
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D057167:Claudins / Q000235:genetics* / Q000378:metabolism; D000071858:Epithelial Cell Adhesion Molecule; D005240:Feasibility Studies; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018189:Immunomagnetic Separation; D061986:MCF-7 Cells; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D012334:RNA, Neoplasm / Q000235:genetics*; D017423:Sequence Analysis, RNA; D013997:Time Factors
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D057167:Claudins; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D012334:RNA, Neoplasm
keywords: EpCAM; IE/FACS; breast cancer; circulating tumor cells