title: Soluble EpCAM levels in ascites correlate with positive cytology and neutralize catumaxomab activity in vitro.
abstract: BACKGROUND
  EpCAM is highly expressed on membrane of epithelial tumor cells and has been detected as soluble/secreted (sEpCAM) in serum of cancer patients. In this study we established an ELISA for in vitro diagnostics to measure sEpCAM concentrations in ascites. Moreover, we evaluated the influence of sEpCAM levels on catumaxomab (antibody)--dependent cellular cytotoxicity (ADCC).
  
  
  METHODS
  Ascites specimens from cancer patients with positive (C+, n = 49) and negative (C-, n = 22) cytology and ascites of patients with liver cirrhosis (LC, n = 31) were collected. All cell-free plasma samples were analyzed for sEpCAM levels with a sandwich ELISA system established and validated by a human recombinant EpCAM standard for measurements in ascites as biological matrix. In addition, we evaluated effects of different sEpCAM concentrations on catumaxomab-dependent cell-mediated cytotoxicity (ADCC) with human peripheral blood mononuclear cells (PBMNCs) and human tumor cells.
  
  
  RESULTS
  Our ELISA showed a high specificity for secreted EpCAM as determined by control HEK293FT cell lines stably expressing intracellular (EpICD), extracellular (EpEX) and the full-length protein (EpCAM) as fusion proteins. The lower limit of quantification was 200 pg/mL and the linear quantification range up to 5,000 pg/mL in ascites as biological matrix. Significant levels of sEpCAM were found in 39% of C+, 14% of C- and 13% of LC ascites samples. Higher concentrations of sEpCAM were detectable in C+ (mean: 1,015 pg/mL) than in C- (mean: 449 pg/mL; p = 0.04) or LC (mean: 326 pg/mL; p = 0.01). Soluble EpCAM concentration of 1 ng/mL significantly inhibited ADCC of PBMNCs on EpCAM overexpressing target cells.
  
  
  CONCLUSIONS
  Elevated concentrations of sEpCAM can be found in a subgroup of C+ and also in a small group of C- patients. We consider that sEpCAM levels in different tumor entities and individual patients should be evaluated prior to applying anti-EpCAM antibody-based cancer therapies, since sEpCAM neutralizes catumaxomab activity, making therapy less efficient.
authors.affiliation: Experimental Oncogenomics, Tyrolean Cancer Research Institute, Innsbruck, Austria. andreas.seeber@uki.at.|Oncotyrol - Center for Personalized Cancer Medicine, Innsbruck, Austria. andreas.seeber@uki.at.|Department of Hematology and Oncology, Medical University of Innsbruck, Innsbruck, Austria. andreas.seeber@uki.at.
authors.forename: Andreas
authors.initials: A
authors.lastname: Seeber
authors.affiliation: Experimental Oncogenomics, Tyrolean Cancer Research Institute, Innsbruck, Austria. agnieszka.martowicz@ki.se.|Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden. agnieszka.martowicz@ki.se.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Martowicz
authors.affiliation: Experimental Oncogenomics, Tyrolean Cancer Research Institute, Innsbruck, Austria. gilbert.spizzo@i-med.ac.at.|Oncotyrol - Center for Personalized Cancer Medicine, Innsbruck, Austria. gilbert.spizzo@i-med.ac.at.|Hemato-Oncological Day Hospital, Hospital of Merano, Merano, Italy. gilbert.spizzo@i-med.ac.at.
authors.forename: Gilbert
authors.initials: G
authors.lastname: Spizzo
authors.affiliation: Department of Internal Medicine, Hospital of Merano, Merano, Italy. thomas.buratti@asbmeran-o.it.
authors.forename: Thomas
authors.initials: T
authors.lastname: Buratti
authors.affiliation: Pathology Laboratory, Hospital of Zams, Zams, Austria. peter.obrist@tyrolpath.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Obrist
authors.affiliation: Experimental Oncogenomics, Tyrolean Cancer Research Institute, Innsbruck, Austria. dominic.fong@i-med.ac.at.|Hemato-Oncological Day Hospital, Hospital of Merano, Merano, Italy. dominic.fong@i-med.ac.at.
authors.forename: Dominic
authors.initials: D
authors.lastname: Fong
authors.affiliation: Department of Hematology and Oncology, Medical University of Innsbruck, Innsbruck, Austria. guenther.gastl@i-med.ac.at.
authors.forename: Guenther
authors.initials: G
authors.lastname: Gastl
authors.affiliation: Experimental Oncogenomics, Tyrolean Cancer Research Institute, Innsbruck, Austria. gerold.untergasser@i-med.ac.at.|Oncotyrol - Center for Personalized Cancer Medicine, Innsbruck, Austria. gerold.untergasser@i-med.ac.at.|Department of Hematology and Oncology, Medical University of Innsbruck, Innsbruck, Austria. gerold.untergasser@i-med.ac.at.
authors.forename: Gerold
authors.initials: G
authors.lastname: Untergasser
mesh_terms: D018033:Antibodies, Bispecific / Q000494:pharmacology*; D000951:Antigens, Neoplasm / Q000378:metabolism*; D001201:Ascites / Q000378:metabolism* / Q000473:pathology*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003602:Cytotoxicity, Immunologic; D000071858:Epithelial Cell Adhesion Molecule; D057809:HEK293 Cells; D006801:Humans; D008103:Liver Cirrhosis / Q000473:pathology; D009369:Neoplasms / Q000473:pathology; D012189:Retrospective Studies
chemical_list: D018033:Antibodies, Bispecific; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; C522419:catumaxomab