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Approximately 70%-80% of kidney cancers are clear cell renal cell carcinomas (CCRCCs). Patient management is based on imaging (abdominal ultrasound and computerized tomography), surgical excision of the tumor, and pathological analysis. A tissue biopsy is therefore necessary to confirm the diagnosis and avoid unnecessary nephrectomy. For metastatic cancers, a tissue biopsy is essential for establishing the targeted therapy. This biopsy of tumor material is invasive and painful. Other techniques such as liquid biopsy would help reduce the need for tissue biopsy. The development of a simple biological test for diagnosis is essential. CA9 is a powerful marker for the diagnosis of CCRCC. Exosomes have become a major source of liquid biopsy because they carry tumor proteins, RNA, and lipids. Urine is the most convenient biological liquid for exosome sampling.
The aim of this study (PEP-C study) is mainly to determine whether it is possible to detect urinary exosomal CA9 for the molecular diagnosis of CCRCC.
This study will include 60 patients with CCRCC and 40 noncancer patients. Exosomes will be isolated from urine samples and exosomal CA9 will be detected by transmission electron microscopy, flow cytometry, and reverse transcription-quantitative polymerase chain reaction.
This study is currently underway with funding support from the CHU Saint-Etienne of France.
We expect to demonstrate that urinary tumor exosomes could be a novel liquid biopsy to diagnose CCRCC and to guide clinicians in treatment decision-making.
ClinicalTrials.gov NCT04053855; https://clinicaltrials.gov/ct2/show/NCT04053855
DERR1-10.2196/24423
Renal cell carcinomas are serious and common cancers [
The notion of liquid biopsy has been mentioned in recent publications [
Exosomes are small nanoparticles that are secreted into the extracellular medium by different cell types [
This is a descriptive study (PEP-C study). The investigators will provide eligible patients with an informative notice on the PEP-C study. Upon a patient providing written consent, 100 ml of urine will be collected in a sterile tube for each exosomal analysis. The study design is outlined in
Outline of study. RT-qPCR: reverse transcription-quantitative polymerase chain reaction
There is no available publication in the literature to calculate the number of subjects required for this type of study. Thirty patients receive surgery for removal of CCRCC in our department per year. Therefore, we propose to study 60 patients over 2 years. In addition, 20 control patients without cancer in our department are expected per year. Therefore, we propose studying 40 controls over 2 years. Eligibility criteria for cancer patients and controls are listed in
Adult patient >18 years old
All patients with a renal mass and scheduled for surgery (partial or total nephrectomy)
Patients having accepted and signed the consent form
Patients benefitting from social security
Adult patient >18 years old
Patients hospitalized in the urology department without a known cancer
Patient having accepted and signed the consent form
Patients benefitting from social security
Insufficient volume of urine sample
Patients with a urinary catheter
Patients under court-ordered guardianship or curatorship
Since CD63, CD9, and CD81 are the common markers for exosomes, we will test these three markers for detecting urinary exosomes. CA9 and vascular endothelial growth factor receptor 2 (VEGFR2) are two markers for CCRCC, which are used as markers for tumor exosomes.
The primary objective of this study is to detect urinary exosomes from CCRCC using an exosome marker (CD63) and CCRCC marker (CA9).
The secondary objectives are to assess (1) agreement between identification of CD63+/CA9+ tumor exosomes and patient status (clear cell kidney cancer), (2) agreement between the identification of CD9+/CD63+/CD81+/CA9+ tumor exosomes and patient status (clear cell kidney cancer), and (3) agreement between the identification of CD63+/VEGFR2+ tumor exosomes and patient status (clear cell kidney cancer).
The primary endpoint of the study is to characterize patients for whom CD63+ and CA9+ exosomes are detected. The secondary endpoints are as follows: (1) percentage of CD63+/CA9+ exosomes in patients with CCRCC compared with that of controls, (2) percentage of CD9+/CD63+/CD81+/CA9+ exosomes in patients with CCRCC compared with that of controls, and (3) percentage of CD63+/VGEFR2+ exosomes in patients with CCRCC compared with that of controls.
Exosomes will be isolated using a well-established commercial kit and will be characterized using exosome markers. The tumor markers will be analyzed by transmission electronic microscopy, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and flow cytometry. Transmission electronic microscopy is typically used to characterize exosomal markers. RT-qPCR is a well-established tool for detecting gene expression and is frequently used in exosome analysis. However, conventional flow cytometry may be inconvenient for exosome analysis given the small size of exosomes. Therefore, we will use aldehyde/sulfate latex beads to capture the exosome. This technique was recently proposed to advance conventional flow cytometry as a suitable tool for analyzing exosomal markers [
Data collected during the study will be recorded on a case report form for patients and control subjects. On the initial visit, age, sex, and medical history will be noted. For patients, date of disease diagnosis, surgical procedures, and pathological results will be noted.
A selected group of 60 patients with renal cancer and 40 control subjects will be included in the study (
The diagnostic performance of tumor exosomes will be evaluated by receiver operating characteristic curves. The area under the curve will be determined with its 95% CI. Analyses will be performed using SAS 9.4.
The ethics committee examined the information notice that will be given to participants of this project. This consultation helped to improve the patient information notice on the design and aim of the study so that the participants will clearly understand and decide whether to participate in this study, in agreement with the principle of informed consent.
This study is currently underway with funding support from the CHU Saint-Etienne of France. Recruitment of all patients will be completed in 2 years. The results of the study will then be communicated via presentations and publications.
Research on liquid biopsy has become a hot topic. Liquid biopsy allows for the detection of tumor markers in bodily fluids for the management of cancer patients. Compared with tissue biopsy, liquid biopsy is noninvasive. Liquid biopsy usually utilizes circulating tumor cells, cell-free DNA, and exosomes. Compared with circulating tumor cells and cell-free DNA, the use of exosomes is relatively more recent, but has shown rapid development. Exosomes carry tumor markers such as nucleic acids, proteins, and lipids, including mRNAs, microRNAs, and signaling molecules that reflect the physiological and pathological condition of the cells of origin [
Our previous study demonstrated that CA9 is a powerful marker for the diagnosis of CCRCC [
Several techniques for the isolation of urinary exosomes have been proposed, which can be classified according to the biochemical principle of purification: ultracentrifugation, filtration, immunocapture, or precipitation [
Transmission electron microscopy observation of urinary exosomes.
In conclusion, this will be the first study to evaluate the technical feasibility of detecting urinary exosomal CA9 in CCRCC patients. The results of this study might highlight the strong potential of liquid biopsy through urinary exosomes in the diagnosis of CCRCC. Therefore, if this pilot study is successful, a multicenter study will be envisaged.
External peer report A from CHU St-Etienne.
External peer report B from CHU St-Etienne.
External peer report C from CHU St-Etienne.
clear cell renal cell carcinoma
reverse transcription-quantitative polymerase chain reaction
vascular endothelial growth factor receptor 2
This research is supported by CHU Saint-Etienne, France.
None declared.