title: Evaluation of proliferation and apoptosis markers in circulating tumor cells of women with early breast cancer who are candidates for tumor dormancy.
abstract: BACKGROUND
  Clinical dormancy is frequently observed in breast cancer. In the present study, we aimed to characterize circulating tumor cells (CTCs) in dormancy candidates (DC) with early breast cancer in terms of proliferation and apoptosis.
  
  
  METHODS
  Cytospins of peripheral blood mononuclear cells (PBMCs) were obtained from DC (n = 122) who were disease-free for at least 5 years and from metastatic patients (n = 40) who relapsed more than 5 years after surgery. Sequential samples from eight DC (n = 36) who maintained a prolonged disease-free status and from eight DC (n = 27) presenting late relapse during follow-up, were also analyzed. PBMCs were triple stained with a pancytokeratin, antibody along with anti-Ki67 and anti-M30 antibodies as proliferation and apoptosis markers, respectively.
  
  
  RESULTS
  CTCs were identified in 40 (33%) of 122 DC and in 15 (37.5%) of 40 metastatic patients. In total, twenty-five (62.5%) DC had exclusively dormant (Ki67(-)/M30(-)), seven (17.5%) had proliferative Ki67(+)/M30(-), four (10%) had apoptotic Ki67(-)/M30(+) and four (10%) had both phenotypes of proliferative and apoptotic CTCs. In comparison, 53.4% of CTC-positive metastatic patients had exclusively dormant and 46.6% had proliferative CTCs; none had apoptotic CTCs (P = 0.039). Among all CTCs detected in DC patients, 82.4% were dormant, whereas in the nondormant population, 32.5% were proliferative and 67.5% apoptotic. The respective percentages in metastatic patients were 59.1%, 100% and 0% (P <0.0001). Moreover, apoptotic CTCs prevailed among nondormant CTCs detected in sequential samples from DC who remained in a prolonged disease-free status compared to those presenting late relapse during follow-up (70.6% versus 43.5% (P = 0.0002)).
  
  
  CONCLUSIONS
  The apoptotic index of CTCs is increased during clinical dormancy, whereas the proliferation index is increased on relapse. In addition, apoptotic CTCs are more frequently encountered during follow-up in DC patients who remain disease-free compared to those with subsequent late relapse, suggesting that monitoring proliferation and apoptosis in CTCs during clinical dormancy merits further investigation as a tool for predicting late disease recurrence.
authors.affiliation: Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes University Campus, Heraklion, 71003, Crete, Greece. mspiliotaki@med.uoc.gr.
authors.forename: Maria
authors.initials: M
authors.lastname: Spiliotaki
authors.affiliation: Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes University Campus, Heraklion, 71003, Crete, Greece. mavrudis@med.uoc.gr.|Department of Medical Oncology, University General Hospital of Heraklion, Voutes, P.O BOX 1352, Heraklion, 71110, Crete, Greece. mavrudis@med.uoc.gr.
authors.forename: Dimitris
authors.initials: D
authors.lastname: Mavroudis
authors.affiliation: Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes University Campus, Heraklion, 71003, Crete, Greece. KKapranoy@hotmail.gr.
authors.forename: Kyriaki
authors.initials: K
authors.lastname: Kapranou
authors.affiliation: Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes University Campus, Heraklion, 71003, Crete, Greece. harismarko@gmail.com.
authors.forename: Harris
authors.initials: H
authors.lastname: Markomanolaki
authors.affiliation: Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes University Campus, Heraklion, 71003, Crete, Greece. kalergi@med.uoc.gr.
authors.forename: Galatea
authors.initials: G
authors.lastname: Kallergi
authors.affiliation: Department of Medical Oncology, University General Hospital of Heraklion, Voutes, P.O BOX 1352, Heraklion, 71110, Crete, Greece. phillipkoinis@gmail.com.
authors.forename: Filippos
authors.initials: F
authors.lastname: Koinis
authors.affiliation: Department of Medical Oncology, University General Hospital of Heraklion, Voutes, P.O BOX 1352, Heraklion, 71110, Crete, Greece. konkalbakis@yahoo.gr.
authors.forename: Kostas
authors.initials: K
authors.lastname: Kalbakis
authors.affiliation: Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes University Campus, Heraklion, 71003, Crete, Greece. georgsec@med.uoc.gr.|Department of Medical Oncology, University General Hospital of Heraklion, Voutes, P.O BOX 1352, Heraklion, 71110, Crete, Greece. georgsec@med.uoc.gr.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Georgoulias
authors.affiliation: Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes University Campus, Heraklion, 71003, Crete, Greece. agelaki@med.uoc.gr.|Department of Medical Oncology, University General Hospital of Heraklion, Voutes, P.O BOX 1352, Heraklion, 71110, Crete, Greece. agelaki@med.uoc.gr.
authors.forename: Sofia
authors.initials: S
authors.lastname: Agelaki
mesh_terms: D000328:Adult; D000368:Aged; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000378:metabolism* / Q000473:pathology; D018275:Carcinoma, Lobular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018572:Disease-Free Survival; D005260:Female; D006801:Humans; D053538:Keratin-18 / Q000378:metabolism*; D007633:Keratins / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism*; D008875:Middle Aged; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011379:Prognosis
chemical_list: D014408:Biomarkers, Tumor; D053538:Keratin-18; D019394:Ki-67 Antigen; C561463:M30 cytokeratin-18 peptide, human; D010446:Peptide Fragments; D007633:Keratins