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J. O'Flaherty

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-056 - Isolation & enumeration of Circulating Tumor Cells in Non-small Cell Lung Cancer, using Screencell & VitaAssay techniques. (ID 3318)

      09:30 - 16:30  |  Author(s): J. O'Flaherty

      • Abstract

      Circulating Tumour Cells (CTCs) have been the subject of much interest as a potential biomarker however methods for isolating CTCs are still in their infancy. A promising method of CTC detection is ScreenCell. This technique uses polycarbonate filtration membranes containing multiple tiny pores. When blood is made to flow across the membrane, tumour cells are captured due to their greater size. Another such method is the use of the modified invasion assay, VitaAssay. This technique uses CAM (Collagen Adhesion Matrix) coated plates to capture CTCs with an invasive phenotype.

      Peripheral blood samples were obtained from patients with advanced NSCLC using both Screencell & VitaAssay. In addition healthy blood samples spiked with NSCLC cells were also analysed. ScreenCell: Peripheral blood is diluted with specified buffer and drawn across the Screencell filter using a vacuum tube. The filters with captured fixed cells are then stained with H&E and/or immunocytochemistry. VitaAssay: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density centrifugation. PBMCs were seeded onto VitaAssay plates and cultured for 12-18 hrs. The supernatant is removed and the remaining captured cells are enriched for CTCs due to their invasive phenotype. Captured cells are fixed and stained using immunocytochemistry.

      Using the ScreenCell technique CTCs were identified by size & morphology using H&E staining. CTCs were detected in 70% of patient samples with. (n=10) Numbers of CTCs detected ranged from 6-82 per ml of blood. In addition, clumps of tumour cells or Circulating Tumour Microemboli (CTM) were detected in 50% of patient samples. (An example of CTM is illustrated in Fig. 1) Cells captured from NSCLC patients using VitaAssay were stained for EpCAM/pan-Cytokeratin and CD45. EpCAM/Pan-CK positive, CD45 negative cells were classed as CTCs. In healthy blood samples spiked with A549 & H2228 cells, approximately 20% (range 9%-26.4%) of spiked cells were recovered using VitaAssay. In NSCLC patients an average of 30.67 CTCs per ml of blood were identified. (range 14-52, n = 6) (An example of CTCs detected by immunocytochemistry is illustrated in Figs. 2 & 3) Figure 1

      ScreenCell & VitaAssay techniques both appear to be viable methods of isolating & enumerating CTCs, in both model cell-spiking experiments and in NSCLC patient samples, as determined by morphology and antigen expression detected with immunocytochemistry. Of particular interest many of the CTCs isolated using Screencell, were detected as clusters or microemboli. Additional samples are being taken to compare CTC & CTM numbers with clinical outcomes.