Introduction Frequently the number of CTC isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a ‘liquid biopsy’. In the EU FP7 program CTCTrap we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA; doi: 10.1073/pnas.1313594110) approach to screen liters of blood and thereby substantially increasing the number of CTC available for further characterization. Here we present the results obtained from 32 metastatic cancer patients subjected to DLA in the participating institutions.

Methods Before the DLA procedure, whole blood was drawn in a CellSave blood collection tube and a 7.5 ml aliquot was processed with the ‘gold standard’ reference CellSearch® (Janssen Diagnostics, USA). DLAs from metastatic cancer patients were performed for ≈90 minutes to obtain 40 mL of product containing ≈4x109 mononuclear cells (MNC) representing ≈1 liter of blood. The obtained DLA samples were then divided, fixed with CellSave preservative, prepared and processed with each of the analysis techniques as described in the Standard Operating Procedures developed for DLA in the CTCTrap consortium (

Results DLAs were obtained from 20 metastatic prostate cancer patients and 12 metastatic breast cancer patients at four different European academic medical institutions. Using a SOP for the DLA procedure, similar DLA products (MNC concentration: 64x106/mL, SD = 38x106) could be generated without any noticeable side effects. CTC in 7.5 mL of blood ranged from 0 to 324 (mean = 61, median = 18). DLA processed with CellSearch represented 7 to 212 mL of blood (mean = 100, median = 97), CTC ranged from 0 to 2913 (mean = 330, median = 105). Resulting in a significant increase in CTC yield (p = 0.004) ranging from 0x to 40x (mean = 13, median = 9) when comparing 1mL of whole blood to 1mL of DLA. Filtration of 50x106 WBC of DLA, through 5um microsieves yielded only 0 to 12 CTC (mean = 2, median = 0, n = 16). Leukocyte depletion of 18 mL of DLA followed by filtration yielded 0 to 178 CTC (mean = 37, median = 4, n = 22) not yielding a relative increase versus CellSearch DLA. Leukocyte depletion followed by CellSearch yielded 271 to 1620 CTC (mean = 792, median = 484, n = 3) also not yielding a relative increase versus CellSearch DLA. In 7 patients 0 CTC were detected in 7.5mL of blood, in 4 out of these 7 patients CTC were detected in DLA.

Conclusion The yield of CTC can be significantly increased by the use of DLA in patients with CTC detected in 7.5 mL of blood. Technology to select CTC from DLAs will need to be further improved before one can make optimal use of the large processed blood volumes.

Citation Format: Kiki C. Andree, Anouk Mentink, Joost F. Swennenhuis, Leon W. Terstappen, Nikolas H. Stoecklein, Rui P. Neves, Rita Lampignano, Hans Neubauer, Tanja Fehm, Johannes C. Fischer, Elisabetta Rossi, Mariangela Manicone, Umberto Basso, Piero Marson, Rita Zamarchi, Yohann Loriot, Valérie Lapierre, Vincent Faugeroux, Marianne Oulhen, Francoise Farace, Gemma Fowler, Mariane Sousa Fontes, Berni Ebbs, Maryou Lambros, Mateus Crespo, Penelope Flohr, Johann S. de Bono. Diagnostic leukapheresis results in a significant increase in CTC yield in metastatic breast and prostate cancer [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 1723. doi:10.1158/1538-7445.AM2017-1723