Introduction

At present, the CellSearch system is the only validated method for the detection of circulating tumor cells (CTC) that has been cleared by the U.S. Food and Drug Administration. This system, designed for the enumeration of CTC in 7.5 mL of blood, detects CTC based on their expression of EpCAM and cytokeratins and negativity for CD45. However, the number of CTC that are detected in patients with metastatic carcinomas is in most cases too small to reliably determine tumor heterogeneity and to be representative as a ‘liquid biopsy’. Our aim is to identify and isolate a sufficient number of circulating tumor cells in virtually all metastatic cancer patients to enable their characterization and to represent a real-time liquid biopsy. For this purpose we used Diagnostic LeukApheresis (DLA) to increase the blood volume to be analyzed. We developed several techniques to isolate CTC from DLA to enable a multicenter comparison of CTC detection in DLA products.

Methods

DLAs were performed for ∼1 hour to obtain 40 mL of product containing ∼4 x10⁁9 mononuclear cells representing ∼1 liter of blood. Using CellSearch a maximum of 2 mL of DLA could be processed for EpCAM+ CTC (Fisher et al. doi: 10.1073/pnas.1313594110) and EpCAM- CTC (de Wit et al doi: 10.1038/srep12270). Using filtration through microsieves with 5 μm pores a maximum of 1.0 mL of DLA could be processed. To process 18 mL DLA product protocols were developed for leukocyte depletion using RosetteSep™ (StemCell Technologies, USA) and for EpCAM selection using an anti-EpCAM coated column (Leukocare AG). All enriched cell fractions were stained using CD45 PerCP, Cytokeratins PE and the nuclear dye DAPI, followed by fluorescence microscopy scanning and analysis.

Results

Leukocyte depletion using the RosetteSep™ CTC Enrichment cocktail was first optimized using small sample volumes (1 mL) spiked with cells from cancer cell lines. Depletion of leukocytes ranged from 3.1 to 3.9 logs with an average recovery of spiked cancer cells of 50-60%. Isolation of CTC expressing EpCAM was pursued using anti-EpCAM coated columns and optimized for selection and release of EpCAM expressing cells by passage of cells from cancer cell lines through the column resulting in 34-100% recovery. Both procedures were scaled up to enable processing of 18 mL of DLA. Leukocytes were depleted using RosetteSepTM by 3.1 - 3.9 logs whereas with anti-EpCAM columns only 1.7 - 1.8 logs depletion were reached. Using RosetteSepTM 21% and with the anti-EpCAM coated columns 2% of the tumor cells spiked into 18ml DLA were recovered.

Conclusion

Standard operating procedures were developed to isolate CTC in DLA's from breast, prostate cancer and lung cancer patients for evaluation and comparison in the EU sponsored consortiums CTCTrap (www.utwente.nl/tnw/ctctrap/) and CANCER-ID (www.CANCER-ID.eu). Isolation of EpCAM expressing CTC using the anti-EpCAM coated columns will need further optimization before it can proceed to multicenter comparison.

Citation Format: Kiki C. Andree, Anouk Mentink, Martin Scholz, Roland Kirchner, Rui P. Neves, Christiane Driemel, Rita Lampignano, Hans Neubauer, Dieter Niederacher, Tanja Fehm, Wolfram T. Knoefel, Johannes C. Fischer, Nikolas H. Stoecklein, Leon WMM Terstappen. The isolation of CTC from diagnostic leukapheresis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1532.