PURPOSE OF THE STUDY: The detection of circulating tumor cells (CTCs) in the blood of cancer patients has raised considerable interest over the last decade as a promising biomarker of minimal residual disease in various solid tumors. Although hematogenous spread has not been deemed a major issue for ovarian cancer, there are several studies demonstrating the presence and prognostic impact of CTCs in ovarian cancer patients. Recently, we developed a protocol for the enrichment of CTCs from ovarian cancer blood samples, achieving a high purity of the target cells and enabling high specificity.
EXPERIMENTAL PROCEDURES: Blood samples were enriched using a density gradient centrifugation (OncoQuick, Greiner Bio–One) followed by a micro–fluidic based isolation of the target cells using the Parsortix™ system (Angle plc.). The captured target cells were lysed, RNA was extracted (RNeasy Micro Kit, Qiagen) and transcribed into cDNA (ssVILO, Life Technologies). After a pre–amplification step (PreAmp Master Mix, Life Technologies), the transcript levels of selected genes (EpCAM, PPIC, TUSC3, EMP2, LAMB1, MAL2, FN1) were measured using RT–qPCR. Additionally, density gradient enriched cells were immuno–fluorescently stained targeting CK8/18/19, PPIC, TP53 and CD45.
SUMMARY: By combining a density gradient based pre–enrichment and the micro–fluidic Parsortix™ technology we achieved an up to 106–fold depletion of blood cell contamination. The analysis of CTC–related transcripts in the enriched samples indicated the presence of CTCs in 78% of the ovarian cancer blood samples taken at primary diagnosis (N=15) and in 80% of the samples taken at relapse (N=9). Concordant results of immuno–fluorescent staining and RT–qPCR were obtained in just 30% of ovarian cancer samples. By adding further 22 RT–qPCR markers, 92% of all cancer patients (n=13) and 100% of the ovarian cancer patients (n=7) were classified as being CTC–positive by RT–qPCR.
CONCLUSIONS: In conclusion, we developed a protocol which enabled the detection of CTC–related transcripts in the majority of ovarian cancer blood samples, which is a clear improvement in sensitivity as compared to earlier studies employing density gradient centrifugation as the only enrichment step. Our results are currently validated in the large cohort of platinum resistant ovarian cancer patients provided by a multi–center Gannet53 study.
Citation Format: Eva Obermayr, Elisabeth Maritschnegg, Paul Speiser, Eva Schuster, Barbara Holzer, Nina Pecha, Robert Zeillinger. THE MOLECULAR DETECTION OF CIRCULATING TUMOR CELLS IN THE BLOOD OF OVARIAN CANCER PATIENTS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP04.