title: Isolation and Characterization of Circulating Tumor Cells in Squamous Cell Carcinoma of the Lung Using a Non-EpCAM-Based Capture Method.
abstract: BACKGROUND
  The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile.
  
  
  METHODS
  Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested.
  
  
  RESULTS
  Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found.
  
  
  CONCLUSIONS
  Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.
authors.affiliation: Division of Oncology, University Hospital of Parma, Parma, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bozzetti
authors.affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Quaini
authors.affiliation: Division of Oncology, University Hospital of Parma, Parma, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Squadrilli
authors.affiliation: Division of Oncology, University Hospital of Parma, Parma, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Tiseo
authors.affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Frati
authors.affiliation: Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma, Italy.
authors.forename: Costanza
authors.initials: C
authors.lastname: Lagrasta
authors.affiliation: Centre for Molecular and Translational Oncology (COMT), Department of Biomedical, Biotechnological and Translational Sciences, Unit of Pathological Anatomy, University Hospital of Parma, Parma, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Azzoni
authors.affiliation: Centre for Molecular and Translational Oncology (COMT), Department of Biomedical, Biotechnological and Translational Sciences, Unit of Pathological Anatomy, University Hospital of Parma, Parma, Italy.
authors.forename: Lorena
authors.initials: L
authors.lastname: Bottarelli
authors.affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
authors.forename: Maricla
authors.initials: M
authors.lastname: Galetti
authors.affiliation: Lung Cancer Unit, IRCCS AOU S. Martino-IST National Institute for Cancer Research, Genoa, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Alama
authors.affiliation: Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma, Italy.
authors.forename: Silvana
authors.initials: S
authors.lastname: Belletti
authors.affiliation: Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Gatti
authors.affiliation: Division of Thoracic Oncology, European Institute of Oncology, Milan, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Passaro
authors.affiliation: Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Gradilone
authors.affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cavazzoni
authors.affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Alfieri
authors.affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Petronini
authors.affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
authors.forename: Mara
authors.initials: M
authors.lastname: Bonelli
authors.affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Falco
authors.affiliation: Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Carubbi
authors.affiliation: Department of Neuroscience, University of Parma, Parma, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pedrazzi
authors.affiliation: Division of Oncology, University Hospital of Parma, Parma, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Nizzoli
authors.affiliation: Division of Oncology, University Hospital of Parma, Parma, Italy.
authors.forename: Nadia
authors.initials: N
authors.lastname: Naldi
authors.affiliation: Division of Medical Oncology, S. Maria Nuova Hospital/IRCCS, Reggio Emilia, Italy.
authors.forename: Carmine
authors.initials: C
authors.lastname: Pinto
authors.affiliation: Division of Oncology, S.Orsola-Malpighi Hospital, Bologna, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ardizzoni
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000951:Antigens, Neoplasm / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000473:pathology*; D016022:Case-Control Studies; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D018628:Gene Dosage; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000235:genetics; D014019:Tissue Donors
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D051496:Receptor, Fibroblast Growth Factor, Type 1