This is a case-control, single-center study. The study team is composed of urologists, oncologists, and biologists from Centre Hospitalier Universitaire (CHU) Saint-Etienne, which is a public and university medical center for patient care and medical research. CHU Saint-Etienne supports medical research projects and provides complete administrative guidance and methodological aid to implement medical research. Before the project began, many meetings were held to discuss the methodological issues as well as administrative issues such as informed consent. Research assistants will be engaged in document collection and sample collection. The study scheme is presented in Figure 1.

The primary objective of this study is to quantify urinary exosomal PSA from prostate cancer with clinical laboratory–based techniques.

The secondary objective of this study is to compare the use of transmission electron microscopy (TEM), flow cytometry (FCM), and reverse transcription–quantitative polymerase chain reaction (RT-qPCR) for the detection of tumor urinary exosomes from patients with prostate cancer.

The primary end point is to quantify urinary exosomal PSA.

The secondary end point is to compare the percentage of detection according to the different techniques (TEM, FCM, and RT-qPCR).

Patients who meet the following criteria are eligible for inclusion into the group of patients with prostate cancer: (1) aged >50 years; (2) a positive biopsy for prostate cancer; (3) without urinary tract infection; (4) benefiting from social security; and (5) having accepted and signed the consent form.

Patients who meet the following criteria are eligible for inclusion into the group of patients with benign prostatic hyperplasia (BPH): (1) aged >50 years; (2) a histologically confirmed diagnosis of BHP; (3) without urinary tract infection; (4) benefiting from social security; and (5) having accepted and signed the consent form.

Patients who meet the following criteria are eligible for inclusion into the control group: (1) aged >50 years; (2) normal PSA levels; (3) no clinical evidence of BPH or prostate cancer; (4) prostate volume less than 30 cm³; (4) without urinary tract infection; (5) benefiting from social security; and (6) having accepted and signed the consent form.

Patients meeting any of the following criteria are excluded from the study: (1) aged <50 years; (2) taking treatment or hormones known to modify the level of serum PSA in the 3 to 6 months preceding inclusion; (3) a urinary tract infection or history of prostate cancer; (4) history of invasive therapeutic procedures for BPH or for symptoms of urinary tract disorders within 6 months prior to inclusion; (5) a history of concurrent bladder or renal tumors in the 6 months preceding inclusion; and (6) no signature of the consent form.

After checking the inclusion and exclusion criteria, the study information leaflet will be given to patients. Patients will have a 1-hour reflection period. A 20-mL first-void urine sample will be collected using the Colli-Pee device in the department before oncological treatment. Urine collection corresponds to the end of a patient’s participation in this study.

Urine samples are kept in refrigerator and processed within 24 hours after collection. Urine is centrifuged at 3000 g for 15 minutes at 4 °C to remove the exfoliated urinary cells. The supernatant is aliquoted and used for the isolation of urinary exosomes.

To isolate urinary exosomes, 5 mL of the urine supernatant will be used. Urinary exosomes are isolated using the polyethylene-glycol (PEG)–based precipitation method. We have optimized the PEG-based technique for the isolation of urinary exosomes. After the adjustment of urine pH, a solution of PEG is mixed with urine. The mixture is incubated at 4 °C for 2 hours. The mixture is centrifuged at 4500 g at 4 °C to collect urinary exosomes. The exosome pellet is suspended in 200 µL of phosphate buffered saline (PBS) solution for the quantification of PSA.

In both exosomes isolated from the prostate cancer cell line LNCaP and those isolated from urine samples, the concentration of total PSA is determined in the automatic analyzer Cobas 8000 (Roche Diagnostics). In the Cobas 8000, total PSA is measured in a c602 module using Elecsys total PSA reagent kits based on electrochemiluminiscent immunoassays in a “sandwich” configuration. The concentration of total PSA is expressed in µg/mL.

We will characterize urinary exosomes using TEM, FCM, and nanoparticle tracking analysis. TEM is performed on the exosome pellets, which are suspended in PBS. A 10-µL urinary exosome suspension is placed on 200 mesh copper grids (AGS138; Oxford Instruments) and allowed to adhere for 20 minutes. The excess sample is wicked off using a piece of filter paper. The grids are washed 3 times with distilled water. The grids are incubated with a drop of staining solution Uranyless (Delta Microscopy) for 20 seconds and blotted with a filter paper. The urinary exosomes are observed at an accelerating voltage value of 100 kV using a Hitachi electron microscope (H800; Hitachi).

For FCM analysis, the technique uses Aldehyde/sulphate latex beads (A37304; ThermoFisher). First, 1 µL of Aldehyde/sulphate latex beads is added to 100 µL of exosome suspension and mixed well. Second, 900 µL of PBS is then added, and the mixture is incubated for 2 hours at room temperature on a rotary wheel. Third, 100 µL of 100 mM glycine is then added, and the mixture is incubated at room temperature for 30 minutes. The mixture is then pelleted by centrifugation at 2650 g for 5 minutes, and the supernatant is removed. The pellet is washed by the addition of 1mL PBS+0.5% fetal bovine serum solution. The mixture is centrifuged at 2650 g for 5 minutes. Finally, the pellet is resuspended in PBS, and the antibody of antihuman CD63-FITC (IM1165U; Beckman Coulter), CD81-FITC (B25329; Beckman Coulter), or CD9-FITC (IM1755U, Beckman Coulter) or the antibody of isotype control (A07795; Beckman Coulter) is added. The incubation with antibody is performed for 30 minutes in the dark. After twice washing with PBS, the staining is observed by using conventional FCM.

Nanoparticle tracking analysis for urinary exosomes is performed using NanoSight LM10 fitted with a 488-nm laser and 500-nm long-pass filter (Malvern Instruments). The urinary exosome sample is diluted in PBS to a final volume of 1 mL. The diluted sample is then injected into the laser chamber. Particle counts per mL and size distribution of particles in solution are collected and calculated.

To calculate the necessary number of patients with prostate cancer needed for inclusion, our hypotheses were as follows: 75% detection is considered insufficient (P0=0.75) and 90% is sufficient (P1=0.90), unilateral α=5%, and power=95%. Based on these assumptions, using the 1-step Fleming method, 65 participants are required. To verify the specificity, we will also include 65 patients with BPH and 65 controls.

For quantitative analysis, the number of observations available, mean, SD, median, minimum, and maximum will be calculated and compared. For qualitative analysis, absolute and relative frequencies (expressed in percentages) will be calculated and compared. Sensitivity and specificity will be calculated. The area under the receiver operating characteristic curve will be determined.

The sponsor (via the research technicians or the investigators) aims to respond to any request for access to data within a maximum of 1 month. Furthermore, only personnel authorized by the sponsor (investigators, research engineers, and research technicians) and representatives of the health authorities will have access to this information.

Study data will be collected directly in observation notebooks. These data will be validated by the investigator, who will sign (electronically) the observation notebooks.

The information collected as part of this study includes (1) demographic data (age, gender, weight, and height); (2) medical history (stage and grade of cancer); and (3) results of different analysis techniques.

Missing data will need to be justified. Any correction made in the case report form must be traceable.

This study has received ethics approval by the ethics committee of CHU Saint-Etienne (IRBN262024/CHUSTE). No compensation will be provided to the participants.