Simple SummaryThere is a growing trend towards exploring the use of non-invasive liquid biopsy (LB) for prostate cancer (PCa) detection. The primary objective of this study is to identify the rate of patients who accepted and were able to collect seminal fluid. The secondary objective was to evaluate the efficiency of our protocol in collecting prostate-derived cells. Our study shows that only one third (139/356) of patients affected by locally advanced PCa, who accepted to collect their seminal fluid, were able to donate. The main favorable predictors for semen collection were young age and lower prostate volume. Putative cancer cells (PSMA + EpCAMhigh) were 2% of the isolated cells in urine and semen. The fraction of EpCAMhighcells over prostate-enriched cells (PSMA+) significantly correlated with patient age in both semen and urine, but not with other clinical parameters.AbstractLiquid biopsy (LB) for prostate cancer (PCa) detection could represent an alternative to biopsy. Seminal fluid (SF) is a source of PCa-specific biomarkers, as 40% of ejaculate derives from the prostate. We tested the feasibility of an SF-based LB by evaluating the yield of semen self-sampling in a cohort of >750 patients with clinically localized PCa. The overall SF collection yield was 18.2% (39% when considering only compliant patients), with about a half of the patients (53.15%) not consenting to SF donation. Independent favorable predictors for SF collection were younger age and lower prostate volume. We implemented a protocol to enrich prostate-derived cells by multi-color flow cytometry and applied it on SF and urine samples from 100 patients. The number of prostate-enriched cells (SYTO-16+ PSMA+ CD45−) was variable, with higher numbers of cells isolated from SF than urine (pvalue < 0.001). Putative cancer cells (EpCAMhigh) were 2% of isolated cells in both specimens. The fraction of EpCAMhighcells over prostate-enriched cells (PSMA+) significantly correlated with patient age in both semen and urine, but not with other clinical parameters, such as Gleason Score, ISUP, or TNM stage. Hence, enumeration of prostate-derived cells is not sufficient to guide PCa diagnosis; additional molecular analyses to detect patient-specific cancer lesions will be needed.