title: Expression profiling of circulating tumor cells in metastatic breast cancer.
abstract: Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer. However, since they are extremely rare, little is known about the molecular nature of these cells. We report a novel strategy for the isolation and expression profiling of pure populations of CTCs derived from peripheral blood. We developed a method to isolate CTCs based on immunomagnetic capture followed by fluorescence-activated cell sorting (IE/FACS). After assay validation using the BT474 cell line spiked into blood samples in vitro, RNA from CTCs isolated from the blood of five metastatic breast cancer (MBC) patients was linearly amplified and subjected to gene expression profiling via cDNA microarrays. We isolated a range of 9-993 captured CTCs from five MBC patients' blood and profiled their RNA in comparison to a diverse panel of primary breast tumors (n = 55). Unsupervised hierarchical clustering revealed that CTC profiles clustered with more aggressive subtypes of primary breast tumors and were readily distinguishable from peripheral blood (PB) and normal epithelium. Differential expression analysis revealed CTCs to have downregulated apoptosis, and they were distinguishable from PB by the relative absence of immune-related signals. As expected, CTCs from MBC had significantly higher risk of recurrence scores than primary tumors (p = 0.0073). This study demonstrates that it is feasible to isolate CTCs from PB with high purity through IE/FACS and profile them via gene expression analysis. Our approach may inform the discovery of therapeutic predictors and be useful for real-time identification of emerging resistance mechanisms in MBC patients.
authors.affiliation: Division of Breast and Soft Tissue Surgery, Department of Surgery, Norris Comprehensive Cancer Center (NCCC), University of Southern California (USC), 1510 San Pablo Street Suite 412, Los Angeles, CA, 90033-0800, USA, julie.lang@med.usc.edu.
authors.forename: Julie E
authors.initials: JE
authors.lastname: Lang
authors.forename: Janet H
authors.initials: JH
authors.lastname: Scott
authors.forename: Denise M
authors.initials: DM
authors.lastname: Wolf
authors.forename: Petr
authors.initials: P
authors.lastname: Novak
authors.forename: Vasu
authors.initials: V
authors.lastname: Punj
authors.forename: Mark Jesus M
authors.initials: MJ
authors.lastname: Magbanua
authors.forename: Weizhu
authors.initials: W
authors.lastname: Zhu
authors.forename: Neal
authors.initials: N
authors.lastname: Mineyev
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Haqq
authors.forename: Julia R
authors.initials: JR
authors.lastname: Crothers
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Esserman
authors.forename: Debasish
authors.initials: D
authors.lastname: Tripathy
authors.forename: Laura
authors.initials: L
authors.lastname: van 't Veer
authors.forename: John W
authors.initials: JW
authors.lastname: Park
mesh_terms: D000951:Antigens, Neoplasm / Q000096:biosynthesis; D014408:Biomarkers, Tumor / Q000097:blood*; D053898:Biosynthetic Pathways / Q000235:genetics; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000096:biosynthesis; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D046228:Microarray Analysis; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000097:blood; D009364:Neoplasm Recurrence, Local / Q000235:genetics* / Q000473:pathology; D009360:Neoplastic Cells, Circulating; D011379:Prognosis
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D009363:Neoplasm Proteins