title: Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients.
abstract: BACKGROUND
  A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction.
  
  
  METHODS
  The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed.
  
  
  RESULTS
  CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84).
  
  
  CONCLUSIONS
  Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.
authors: Department of Hematology and Medical Oncology, Charité, Berlin, Germany.
authors: Zhian
authors: Z
authors: Liu
authors: Alberto
authors: A
authors: Fusi
authors: Eva
authors: E
authors: Klopocki
authors: Alexander
authors: A
authors: Schmittel
authors: Ingeborg
authors: I
authors: Tinhofer
authors: Anika
authors: A
authors: Nonnenmacher
authors: Ulrich
authors: U
authors: Keilholz
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