Abstract 2395: HER-2 FISH analysis and H &amp; E staining of circulating tumor cells pre-isolated using high porosity precision microfilters
Authors: Daniel Adams, Peixuan Zhu, Olga Makarova, Shuhong Li, Platte Amstutz, Cha-Mei Tang
Journal: Cancer Research (2012)

Abstract Background: We have previously presented a novel precision microfilter, CellSieveTM, which achieves rapid (&amp;lt;2 min) and highly efficient (&amp;gt;98%) isolation of circulating tumor cells (CTCs) from peripheral blood identified by fluorescent immunostaining. We continue with the concept of using CTCs as a minimally invasive “liquid biopsy” from a patient's peripheral blood for cancer detection and subtyping. We show CTCs can be analyzed on a microfilter using both molecular determinants and histopathology, namely fluorescent in-situ hybridization (FISH) for the HER-2 mutation and Hematoxylin &amp; Eosin Staining (H&amp;E) for the histological morphology. Cancer subtyping using the molecular characterization of CTCs is a method to provide prognostic determination of breast cancer patients. Further, histopathological diagnosis is still the gold standard for detecting cellular abnormalities in determining further testing. We show CTCs can be rapidly and efficiently collected from blood specimens on CellSieveTM microfilters and analyzed morphologically, immunologically, and molecularly for cancer subtyping and clinical determination. Materials and Methods: CellSieve™ microfilters are produced with 8 µm diameter pores on a clear polymer with approximately 140,000 pores contained within a 9 mm diameter area. Breast cancer cell lines were used to evaluate the assay performance. Enumerated cells were spiked into 7.5 mL whole human blood and mixed with 7.5 mL of a fixation solution. Filtration was performed in a filter holder with the sample drawn by negative pressure at a flow rate of ∼10 mL/min. The cells captured on the filter were fixed and permeabilized within the filter holder. Cells were then stained and identified by fluorescent microscopy by the presence of DAPI, cytokeratin (FITC), and EpCAM (PE), while showing no CD45 marker (Cy5). Filters were then washed and probed directly with a HER-2 DNA probe according to a modified FISH procedure. After microscopic analysis and mutation determination, the filters were flushed with a modified hematoxylin solution followed by an Eosin Y solution. Filters were then remounted and analyzed by light microscopy. Results: We demonstrated that CTC isolation, immunostaining, molecular analysis, and finally morphological determination can be performed rapidly and efficiently on a single sample. Using the antibody combination described above, we counted the CTCs. We then determined HER-2 mutation status of the CTCs using FISH, and finally verified the morphology of the cell type. Conclusions: Microfiltration of CTCs on a CellSieveTM microfilter can be used in a variety of characterization techniques, including immunofluorescence, FISH and histopathological staining. This technique, rapid and efficient capture of CTCs from patient blood, followed by analysis of tumor cells, can be used to determine therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2395. doi:1538-7445.AM2012-2395