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         headline:Expression profiling of circulating tumor cells in metastatic breast cancer
         description: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.
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      name:Division of Breast and Soft Tissue Surgery, Department of Surgery, Norris Comprehensive Cancer Center (NCCC), University of Southern California (USC), Los Angeles, USA
      name:Atara Biotherapeutics, Brisbane, USA
      name:Division of Hematology and Medical Oncology, Department of Medicine, University of California San Francisco (UCSF), San Francisco, USA
      name:Section of Breast Care Surgery, UCSF Department of Surgery, San Francisco, USA
      name:Division of Oncology, USC Department of Medicine, NCCC, Los Angeles, USA
      name:UCSF Department of Laboratory Medicine, San Francisco, USA
      name:Division of Hematology and Medical Oncology, Department of Medicine, University of California San Francisco (UCSF), San Francisco, USA
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