Senologie - Zeitschrift für Mammadiagnostik und -therapie 2015; 12 - A13
DOI: 10.1055/s-0035-1550454

Detection and molecular characterization of circulating tumor cells in ovarian cancer patients – establishment of a multi-marker gene panel

C Blassl 1, J Kuhlmann 2, S Kasimir-Bauer 3, P Wimberger 2, H Neubauer 4, T Fehm 5
  • 1Heinrich-Heine University, Department of Obstetrics and Gynecology, Duesseldorf, Deutschland
  • 2Medical Faculty and University Hospital Carl Gustav Carus, Department of Obstetrics and Gynecology, Dresden, Deutschland
  • 3University Hospital of Essen, Department of Obstetrics and Gynecology, Essen, Deutschland
  • 4Heinrich-Heine University Duesseldorf, Department of Obstetrics and Gynecology, Duesseldorf, Deutschland
  • 5Heinrich-Heine University Duesseldorf, Department of Obstetrics and Gynecology, Duesseldorf, Deutschland

Circulating tumor cells (CTC) are present in approximately 20% of ovarian cancer patients at primary diagnosis and were shown to correlate with decreased overall survival. However, considering that CTCs with epithelial-mesenchymal-transition (EMT)- or stem-like traits are supposed to be involved in metastatic progression and recurrence, we established a multi-marker panel for molecular characterization of CTCs, detecting EMT- and tumor stem cell associated transcripts.

Dual priming oligonucleotide primers for three independent multiplex RT-PCR panels were designed for detection of transcripts, associated with epithelial-traits (EpCAM, Muc1, CK5 and CK7), EMT-traits (N-Cadherin, Vimentin, Slug, CD117, CD146, CD49f and Snai1) and tumor stem cell traits (CD44, ALDH1A1, Nanog, SOX2, Notch1, Notch4, Oct4 and Lin28). For determining assay sensitivity, defined numbers of OvCar-3 ovarian cancer cells, expressing the majority of above mentioned transcripts, were spiked into the blood of healthy donors. Blood samples were subjected to immunomagnetic tumor cell enrichment, RNA-isolation and reverse transcription using the AdnaTest OvarianCancer. Resulting cDNA was amplified with our epithelial marker panel and PCR-products were assessed by capillary electrophoresis.

Primer specifity and PCR-performance of all three multiplex panels was successfully validated in genomic-DNA and cDNA of OvCar-3 cells. Spiking experiments revealed an assay sensitivity of 5 cells/5 ml blood. Positivity for EpCAM and Muc1 in blood samples, spiked with 5, 10 and 25 OvCar-3 cells, was 100% concordant to EpCAM and Muc1 positivity detected by comparatively performed Adnatest OvarianDetect.

We established and validated a multiplex RT-PCR panel for epithelial genes in tumor cells and focus now on the validation of the EMT and stem cell multiplex RT-PCR panels for transcriptional characterization of CTCs which we will subsequently apply in clinically documented ovarian cancer patients.