Method for semi-automated microscopy of filtration-enriched circulating tumor cells

Pailler et al. BMC Cancer (2016) 16:477 DOI 10.1186/s12885-016-2461-4 TECHNICAL ADVANCE Open Access Method for semi-automated microscopy of filtration-enriched circulating tumor cells Emma Pailler1,2,3†, Marianne Oulhen1,2†, Fanny Billiot1,2, Alexandre Galland4, Nathalie Auger5, Vincent Faugeroux1,2,3, Corinne Laplace-Builhé6, Benjamin Besse7, Yohann Loriot7, Maud Ngo-Camus7, Merouan Hemanda4, Colin R. Lindsay1,2, Jean-Charles Soria2,7, Philippe Vielh1,2,5 and Françoise Farace1,2,3* Abstract Background: Circulating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients. Methods: Spiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner. Results: Two combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45− cells, cytomorphological staining, then scanning and analysis of CD45− cell phenotypical and cytomorphological characteristics. CD45− cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 μm2. The second assay sequentially combined fluorescent staining, automated selection of CD45− cells, FISH scanning on CD45− cells, then analysis of CD45− cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 μm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals. Conclusions: The semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic. Keywords: Circulating tumor cells, Filtration enrichment, Fluorescent staining, FA-FISH, Predictive biomarkers Background Circulating tumor cells (CTCs) captured from blood as a liquid biopsy are currently a very active and promising area in translational cancer research [1]. CTCs are very rare cells – occurring at rates as low as one cell per 106 * Correspondence: † Equal contributors 1 “Circulating Tumor Cells” Translational Platform AMMICA CNRS UMS3655– INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805 Villejuif, France 2 INSERM U981 “Identification of Molecular Predictors and new Targets for Cancer Treatment”, F-94805 Villejuif, France Full list of author information is available at the end of the article or 107 leukocytes – which detach from a primary tumor or metastatic site, circulate in the peripheral blood and may colonize secondary sites to form metastasis [2, 3]. To date, identification and characterization of CTCs has been hampered by their rarity, their phenotypical and genetic heterogeneity and the technical limitation of current assays [4]. As a consequence, CTC detection methods commonly rely on two steps, a first enrichment step based on either CTC phenotypical or physical properties, and a secondary step of detection to increase the sensitivity of the assay. The semi-automated CellSearch © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pailler et al. BMC Cancer (2016) 16:477 platform (Janssen Diagnostics, LLC, Raritan, USA) enriches CTC candidates through ferromagnetic beads coated with epithelial cell adhesion molecule (EpCAM) specific antibody and defines CTCs according to the presence of cytokeratins (CK8, CK18 and CK19) and the absence of the common leukocyte antigen CD45 [5]. In prospective multicenter studies in metastatic breast, prostate (mPCa) and colon cancers [6–8], CTC levels using this platform were shown to have prognostic significance, and extensive analytical validation and clinical qualification led the U.S. Food and Drug Administration (FDA) to approve CellSearch as an aid to prognosis in these tumors. In non-small-cell lung cancer (NSCLC), CTC levels were also reported to have a prognostic value, but the number of CTCs caught by the CellSearch remains low in this tumor type even in patients with metastatic advanced disease [9]. The potential reason for this is that CTCs have lost epithelial features, expressing markers of the epithelial-mesenchymal transition (EMT) which can be missed by the CellSearch [10–12]. In addition to prognostic utility, CTCs are currently extensively studied to detect predictive biomarkers and screen eligible patients for targeted therapies [13–20]. Moreover analyses performed on CTCs could be repeated at different time-points during treatment to monitor response and guide therapeutic decisions. Performing molecular analysis in CTCs analyzed by the CellSearch is relatively challenging in cancers such as NSCLC due to the low numbers of CTCs captured and the processing conditions of the system. The limitations of the CellSearch and other antibodybased capture methods have stimulated the development of other technologies for CTC enrichment and detection [4]. An alternative approach consisting of distinguishing CTCs from normal hematopoietic cells according to size is being actively explored. Several filter devices have been described including ISET (ISET, isolation by size of epithelial tumor cells, Rarecells, Paris, France) [21–23], ScreenCell [24], the Accucyte system [25], CellSieve [26], the Portable Filter-based Microdevice [27], VyCAP [28], the Siemens prototype [29] and the Parsortix system [30]; allowing identification of CTCs according to cytomorphological, phenotypical or molecular characteristics. Ours and oth (...truncated)