Abstract 2760: Proof of principle studies for detection of circulating renal cancer cells from blood samples using diverse technologies
Authors: Maertens Yvonne , Humberg Verena , Steinestel Julie , Boegemann Martin , Schrader Andres J. , Bernemann Christof 
Published: July 2017

Abstract
Background: During the past years, much progress has been made in detection and analysis of circulating tumor cells (CTCs) in several tumor entities, including prostate cancer, breast cancer or lung cancer. However, little is known about circulating tumor cells in patients suffering from clear cell renal cell carcinomas. The majority of technologies detecting CTCs is based on expression of epithelial markers on the surface of these cells, e.g. EpCAM. Additionally, biophysical approaches have been invented to detect CTCs based on size, invasive capacity or density. In order to be able to detect CTCs in patient samples, in vitro establishment of the most accurate isolation procedure followed by precise detection techniques has to be performed. Aim of our studies was to build a stable in vitro fundament of isolation and subsequent detection of CTCs in ccRCC patients. Methods: We made use of 4 different technologies, all of which have been approved for detection of CTCs in distinctive tumor entities. We used EpCAM based positive enrichment of CTCs, Ficoll densitiy centrifugation followed by CD45-positive cell depletion, rosette formation followed by CD45 positive cell depletion as well as size and deformability based enrichment technologies by using the Parsortix system. Furthermore, by using 4 phenotypically distinct ccRCC cell lines, we tried to detect markers unique for tumor cells in demarcation to blood cells. Results: By performing spiking experiments of renal cancer cells, we found the highest recovery rates by using the size based Parsortix system. Interestingly, the most established technique of EpCAM based isolation failed in three out of four cell lines to recover more than 40%. Expression of well-established markers for ccRCC, like carboanhydrase (CA)-9, could be detected in renal cancer cell lines. However, expression was also found in blood samples of healthy donors. Another marker used for immunohistochemical diagnosis of ccRCC, PAX8, showed weak to absent expression in established renal cancer cell lines. The highest specificity to detect renal cancer cells in blood samples was found when analyzing KRT8 or KRT 19 expression. Conclusion: Our results demonstrate that firstly, using the EpCAM based CTC enrichment, which is the basis of the CellSearch system, which up to now is the only methodology approved by the FDA, the majority of renal cancer cells will presumably not be detected in blood samples. This seems largely due to low or absent expression of EpCAM on renal cancer cells. The usage of the size based Parsortix system showed the highest recovery rates and should therefore be analyzed in more detail on samples of ccRCC patients. Secondly, an exclusive marker for defining a renal CTC is still missing. Some well-established ccRCC markers, like CA-9, failed to specifically detect renal cancer cells in blood samples, as they were either present also in healthy blood samples or absent in renal cancer cell lines.
Citation Format: Yvonne Maertens, Verena Humberg, Julie Steinestel, Martin Boegemann, Andres J. Schrader, Christof Bernemann. Proof of principle studies for detection of circulating renal cancer cells from blood samples using diverse technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2017-2760