title: Method for semi-automated microscopy of filtration-enriched circulating tumor cells.
abstract: 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.
  
  
  
  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.
  
  
  
  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 μm(2). 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.
  
  
  
  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.
authors.affiliation: "Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France.|INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France.|Univ Paris Sud, Université Paris-Saclay, F-94270, Le Kremlin-Bicêtre, France.
authors.forename: Emma
authors.initials: E
authors.lastname: Pailler
authors.affiliation: "Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France.|INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France.
authors.forename: Marianne
authors.initials: M
authors.lastname: Oulhen
authors.affiliation: "Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France.|INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France.
authors.forename: Fanny
authors.initials: F
authors.lastname: Billiot
authors.affiliation: Pathology Imaging, Leica Biosystems, F92737, Nanterre, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Galland
authors.affiliation: Department of Biopathology, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Auger
authors.affiliation: "Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France.|INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France.|Univ Paris Sud, Université Paris-Saclay, F-94270, Le Kremlin-Bicêtre, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Faugeroux
authors.affiliation: Imaging and Cytometry Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Laplace-Builhé
authors.affiliation: Department of Medicine, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Besse
authors.affiliation: Department of Medicine, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
authors.forename: Yohann
authors.initials: Y
authors.lastname: Loriot
authors.affiliation: Department of Medicine, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
authors.forename: Maud
authors.initials: M
authors.lastname: Ngo-Camus
authors.affiliation: Pathology Imaging, Leica Biosystems, F92737, Nanterre, France.
authors.forename: Merouan
authors.initials: M
authors.lastname: Hemanda
authors.affiliation: "Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France.|INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France.
authors.forename: Colin R
authors.initials: CR
authors.lastname: Lindsay
authors.affiliation: INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France.|Department of Medicine, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Soria
authors.affiliation: "Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France.|INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France.|Department of Biopathology, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Vielh
authors.affiliation: "Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France. francoise.farace@gustaveroussy.fr.|INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France. francoise.farace@gustaveroussy.fr.|Univ Paris Sud, Université Paris-Saclay, F-94270, Le Kremlin-Bicêtre, France. francoise.farace@gustaveroussy.fr.
authors.forename: Françoise
authors.initials: F
authors.lastname: Farace
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D057205:Automation, Laboratory; D014408:Biomarkers, Tumor / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D002469:Cell Separation; D048430:Cell Shape; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology*; D008856:Microscopy, Fluorescence; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases
keywords: Circulating tumor cells; FA-FISH; Filtration enrichment; Fluorescent staining; Predictive biomarkers