Introduction: Circulating tumor cells (CTC) measured with the CellSearch system in patients with metastatic carcinomas are associated with poor survival. The CellSearch system uses immunomagnetic enrichment targeting the EpCAM antigen. A frequently raised question is what the frequency and significance is of CTC that do not express EpCAM within the CTC of the individual patient and between different patients. To investigate this, a device was constructed that collects the blood discarded by the CellSearch system and passes the blood through micro sieves with 5um pores to enrich for the larger CTC.

Methods: A sample collection device was attached to the waste line of the CellTracks Autoprep (AP). The blood discarded after immunomagnetic separation was detected in the waste line and collected into a separate 50ml conical tube for each patient sample. After collection, the blood was filtered through microfabricated silicon nitride filters with pore diameters of 5 micrometer (Aquamarijn, Zutphen, The Netherlands) with a pressure below 40mbar. To evaluate recovery on the filter the COLO320(median size 11μm), SKBR3(16μm) and T24(16μm) cell lines where used. To evaluate recovery from the AP waste the SKBR3(445000 EpCAM antigens) and T24(2167 EpCAM antigens) cell lines where used. 300 cells where spiked in 7.5ml of blood collected in CellSave tubes from healthy volunteers. All samples were processed the day after collection.

Results: The average recovery on filter from whole blood is 29% for COLO320 and ≥80% for SKBR3 and T24. The average recovery of the SKBR3 cell line with the CellSearch system is 91% and 5% are recovered from the AP waste using filtration. In contrast, the average recovery of T24 cell line with the CellSearch system is only 30% but 51% are recovered from the AP waste after filtration. The carryover between spiked and unspiked samples collected from the AP waste was <1.3% of the spiked cells for unspiked samples with a mean of 0.9%.

Conclusions: We present a method and device that enables the identification and characterization of CTC not detected by the CellSearch system which allows a systematic evaluation of the clinical relevance of these CTC.

Citation Format: Guus van Dalum, Aufried T.M. Lenferink, Leon W.M.M. Terstappen. Detection of EpCAM negative circulating tumor cells in CellSearch waste. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1459. doi:10.1158/1538-7445.AM2013-1459