title: In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis.
abstract: BACKGROUND
  Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data.
  
  
  METHODS
  This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45(pos) leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45(neg) cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively.
  
  
  RESULTS
  By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45(neg) subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45(neg) cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment.
  
  
  CONCLUSIONS
  This study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies.
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Bulfoni
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.|Department of Oncology, University Hospital of Udine, P.le S.Maria della Misericordia 15, 33100, Udine, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Gerratana
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Del Ben
authors.affiliation: Institute of Pathology, University Hospital of Udine, P.le S.Maria della Misericordia 15, 33100, Udine, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Marzinotto
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.|Institute of Pathology, University Hospital of Udine, P.le S.Maria della Misericordia 15, 33100, Udine, Italy.
authors.forename: Marisa
authors.initials: M
authors.lastname: Sorrentino
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.|Institute of Pathology, University Hospital of Udine, P.le S.Maria della Misericordia 15, 33100, Udine, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Turetta
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.
authors.forename: Giacinto
authors.initials: G
authors.lastname: Scoles
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Toffoletto
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.
authors.forename: Miriam
authors.initials: M
authors.lastname: Isola
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.
authors.forename: Carlo Alberto
authors.initials: CA
authors.lastname: Beltrami
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.|Institute of Pathology, University Hospital of Udine, P.le S.Maria della Misericordia 15, 33100, Udine, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Di Loreto
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy. antonio.beltrami@uniud.it.
authors.forename: Antonio Paolo
authors.initials: AP
authors.lastname: Beltrami
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy.|Department of Oncology, University Hospital of Udine, P.le S.Maria della Misericordia 15, 33100, Udine, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Puglisi
authors.affiliation: Department of Medical and Biological Sciences, University of Udine, P.le Kolbe 4, 33100, Udine, Italy. daniela.cesselli@uniud.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Cesselli
mesh_terms: D000328:Adult; D014408:Biomarkers, Tumor / Q000097:blood*; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology*; D018572:Disease-Free Survival; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D005260:Female; D006801:Humans; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating; D011379:Prognosis
chemical_list: D014408:Biomarkers, Tumor