The focus of the study was to implement a new workflow for circulating tumor cells (CTCs) characterization that would allow the analysis of CTCs on a cytomorphological and molecular level in patients with diagnosed gynecological cancer. Our findings may be useful in future cancer patient management. The study introduces a size-based enrichment (MetaCell(®)) method for the separation of viable CTCs, followed by CTCs culturing in vitro and gene expression characterization. It is based on the observation of CTCs and DTCs (Disseminated Tumor Cells) in several case studies of ovarian, endometrial and cervical cancer by means of cytomorphology and gene expression profiling. The viability of the enriched CTCs was estimated using vital and lethal fluorescence nuclear staining. This type of staining may be predictive for the success rate of subsequent CTC growth in vitro. To identify CTCs in the enriched CTC fraction, cytomorphological evaluations based on vital fluorescence staining were followed by gene expression analysis of tumor-associated (TA) genes. Cytokeratin expression (KRT7, KRT19) was analyzed in combination with MUC1, MUC16, CD24, CD44 and ALDH1. Gene expression analysis has shown that short-term in vitro culture enhanced the differentiation process of the captured CTCs growing on a membrane. On the other hand, redundant white blood cells captured on the membrane were eliminated during a short-term culture. The most frequently elevated genes in ovarian cancer (serous type) are EPCAM, KRT19 and MUC1. It has been demonstrated that CTC presence revealed by cytomorphological evaluation may be usefully complemented by TA-gene expression analysis, to increase the sensitivity of the analysis.

3rd Department of Surgery 1st Faculty of Medicine Charles University Prague and University Hospital Motol Prague Czech Republic

Department of Biochemistry Institute of Laboratory Diagnostics University Hospital Kralovske Vinohrady Srobarova 50 Prague Czech Republic

Department of Histology and Embryology Wroclaw Medical University Chalubinskiego 6a 50 368 Wroclaw Poland

Department of Laboratory Genetics Institute of Laboratory Diagnostics University Hospital Kralovske Vinohrady Srobarova 50 Prague Czech Republic

Division of Surgical Oncology and Department of Oncology Wroclaw Medical University Plac Hirszfelda 12 53 413 Wrocław Poland

Lower Silesian Oncology Centre pl Hirszfelda 12 53 413 Wroclaw Poland