Introduction: The presence of circulating tumor cells (CTC) enumerated by the CellSearch system in blood of patients with cancers of epithelial origin is strongly associated with a poor prognosis of these patients. CTC are enriched by targeting the EpCAM antigen, raising the question which EpCAM subtypes are present as well in patients. In the EU-FP7 CTCTrap program, we investigated the presence of EpCAM- CTC in blood samples after depletion of EpCAM+ CTC by CellSearch. Studies were performed and validated at the participating laboratories by distribution and analysis of blood samples spiked with cancer cells and by testing blood from 73 metastatic prostate and 22 metastatic breast cancer patients. Methods: Blood samples were processed according to the standard operating procedures and tools developed in the program (https://www.utwente.nl/tnw/mcbp/protocolsandtools/). First, CellSearch was performed for EpCAM+ CTC, followed by filtration and fluorescent labeling of the blood discarded by CellSearch for EpCAM- CTC. To validate the procedures across the 6 participating clinical sites, 3x 3 tubes with aliquots of healthy donor blood, spiked with either PC3, MDA-MB-231 or no cells, were prepared at one site and shipped to all other sites for simultaneous processing. Metastatic prostate and breast cancer patients were processed with the same procedures. Results: 27% of PC3 cells were recovered by CellSearch and 21% by filtration, leaving 52% unaccounted for. For MDA, 26% were recovered by CellSearch and 18% by filtration, leaving 56% accounted for. Differences in recovery between sites were not significant. In patients both EpCAM+ and EpCAM- CTC were detected (see table). Conclusion: In a multicenter study EpCAM+ and EpCAM- CTC were present in blood of metastatic prostate and breast cancer patients. Spiking experiments showed that the developed methods can be further improved to increase the CTC yield. Molecular characterization of the CTC subtypes and relation with clinical outcome is ongoing.

% metastatic cancer patients with EpCAM+ and EpCAM- CTC 
  % prostate cancer patients (n=73) % breast cancer patients (n=22) 
  CellSearch CTC EpCAM+  CellSearch CTC EpCAM+  
 # CTC 1-4 >4 Total 1-4 >4 Total 
Microsieve CTC EpCAM- 11 19 38 18 32 
 1-4 16 29 14 32 
 >4 19 33 14 14 36 
 Total 25 21 54  41 27 32  
% metastatic cancer patients with EpCAM+ and EpCAM- CTC 
  % prostate cancer patients (n=73) % breast cancer patients (n=22) 
  CellSearch CTC EpCAM+  CellSearch CTC EpCAM+  
 # CTC 1-4 >4 Total 1-4 >4 Total 
Microsieve CTC EpCAM- 11 19 38 18 32 
 1-4 16 29 14 32 
 >4 19 33 14 14 36 
 Total 25 21 54  41 27 32  

Citation Format: Sanne de Wit, Kiki C. Andree, Joost F. Swennenhuis, Elisabetta Rossi, Mariangela Manicone, Riccardo Vidotto, Rita Zamarchi, Marianna Alunni-Fabbroni, Elisabeth K. Trapp, Marie Tzschaschel, Brigitte Rack, Rita Lampignano, Hans Neubauer, Tanja Fehm, Marianne Oulhen, Emeline Colomba, Françoise Farace, Mateus Crespo, Penelope Flohr, Gemma Fowler, Mariane Sousa Fontes, Johann S. de Bono, Leon WMM Terstappen. EpCAM- and EpCAM+ circulating tumor cells in metastatic prostate and breast cancer patients: a multicenter study [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 3787. doi:10.1158/1538-7445.AM2017-3787