Efficient leukocyte depletion by a novel microfluidic platform enables the molecular detection and characterization of circulating tumor cells

// Eva Obermayr 1 , Elisabeth Maritschnegg 1 , Christiane Agreiter 1 , Nina Pecha 1, 2 , Paul Speiser 1, 2 , Samir Helmy-Bader 2 , Sabine Danzinger 3 , Michael Krainer 4 , Christian Singer 3 and Robert Zeillinger 1 1 Molecular Oncology Group, Department of Obstetrics and Gynecology, Comprehensive Cancer Center, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria 2 Department of Obstetrics and Gynecology, Clinical Division for General Gynecology and Gynecological Oncology, Comprehensive Cancer Center, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria 3 Department of Obstetrics and Gynecology, Division of Senology, Comprehensive Cancer Center, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria 4 Department of Medicine I, Clinical Division of Oncology, Comprehensive Cancer Center, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria Correspondence to: Eva Obermayr, email: eva.obermayr@meduniwien.ac.at Keywords: circulating tumor cells, microfluidics, gene expression analysis, ovarian cancer Received: December 15, 2016      Accepted: September 30, 2017      Published: November 28, 2017 ABSTRACT RT-qPCR is a highly sensitive approach to detect rare transcripts, as derived from circulating tumor cells (CTCs) in the blood of cancer patients. However, the presence of unwanted leukocytes often leads to false positive results. Here, we evaluated whether the micro-fluidic Parsortix TM technology is appropriate to remove these leukocytes and thereby finally to improve the overall approach. In this study, we established a workflow including the micro-fluidic Parsortix TM technology for the molecular detection of CTC related transcripts. Background levels of EpCAM , PPIC , TUSC3 , and MAL2 were efficiently removed due to an up to 10 6 -fold depletion of leukocytes. The presence of these gene markers was observed in Parsortix TM -enriched blood samples from patients with primary and recurrent gynecological cancer (32% and 14%), as well as in 86% of the metastatic breast cancer samples, at a very high specificity. In the ovarian cancer samples, PPIC was the most prominent gene marker, contributing to all positive cases and at least to 70% of the positive cases after pre-amplification of the respective target genes. Expanding the analytical panel up to 29 gene markers further increased the positivity rate (primary gynecological cancer: 95%, recurrent gynecological cancer: 100%, metastatic breast cancer: 92%). The established workflow strongly improved the overall molecular analysis of the target cells by the efficient removal of contaminating cells, and, thereby offers great promise for the molecular characterization of CTCs.