Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap)

Abstract

Frequently, the number of circulating tumor cells (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) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting

“…Importantly, none of the patients experienced adverse effects (mild, moderate, or severe). It is of note that since our initial description, DLAs have been carried out in different European hospitals without adverse effects (12,13). The reported mild adverse events are mainly due to (17) and usually occur after longer periods of leukapheresis (e.g., >2-3 h).…”

“…It was calculated that the probability to detect at least one of 1,000 CTCs in 5 l circulating blood (≤1.5 CTC/7.5 ml) with CS is just 34% due to Poisson statistics, assay efficacy of 80 (AE15)%, and an inter-reader variability of 0.8 (16). But promising depletion strategies are underway enabling to analyze at least a large part of the product (12). Therefore, analyzing 5% of DLA products, which was equivalent to a median PB volume of around 60 ml, boosted already the detection probability in the M0 patients that often have less than 1,000 CTC circulating in their total blood volume (11).…”

“…For treatment decisions it will be of utmost importance to harvest more CTCs in more patients in order to close the current gap between patients with cancer cells in circulation and the 30-50% CTC-test negative patients, when only small volumes of peripheral blood are investigated. Very recently, the method was validated by a European multicenter study (CTCTrap) in 32 metastatic prostate and breast cancer patients, demonstrating that the workflow yields indeed significantly higher CTC numbers and that the procedure could be safely performed in different clinical environments (12,13). The underlying principle is that epithelial derived CTCs have a similar density compared to peripheral mononuclear cells (MNCs) (3) and can be harvested by the continuous flow centrifugation of a standard leukapheresis procedure.…”

“…Nevertheless, the utility of CTCs for liquid biopsy in BC is currently challenged and limited by their low frequency (3,11,12). For treatment decisions it will be of utmost importance to harvest more CTCs in more patients in order to close the current gap between patients with cancer cells in circulation and the 30-50% CTC-test negative patients, when only small volumes of peripheral blood are investigated.…”

CTC Technologies and Tools

“…Extrapolation of the frequency distribution of CTC in patients with metastatic prostate cancer, breast, and colorectal cancer showed that an increase of blood volume from 7.5 ml to 600 ml >90% of patients with metastatic cancers will have CTC detected (33). However, at present the available technologies are not yet capable of isolating CTC from the complete DLA volume (34)(35)(36). This procedure baptized Diagnostic Leukapheresis indeed allows for the isolation of a substantial larger number of CTC and enables a true "liquid biopsy."…”