Single-Cell Analyses of Prostate Cancer Liquid Biopsies Acquired by Apheresis

Abstract

Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency. We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity. Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses. Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. .

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).…”

“…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). In addition, the large numbers of CTCs can be used to explore heterogeneity and the subclonal architecture of systemic cancer as demonstrated by Lambros and colleagues in prostate cancer (13). A glimpse on the attainable CTC counts provided our estimation of CTC counts for the whole DLA product.…”

“…Increasing the detection-probability for one CTC to 95% requires a blood volume of 45 ml corresponding to six CS assays. The high CTC-counts in DLA products and the use of a larger DLA product fraction facilitated already the cultivation of organoids from CTCs in prostate cancer (13), which is barely possible with 10 ml PB samples. Likely, processing the whole DLA product will more dramatically increase the detection rates.…”

“…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.…”

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."…”