BACKGROUND:Prostate cancer is the most common cancer in men and represents a major public health problem. The current method for the diagnosis or screening of prostate cancer is invasive and costly. There have been renewed and innovative studies searching for urinary biomarkers to aid in the diagnosis of prostate cancer, especially with technologies based on urinary exosomes. However, technologies based on urine exosomes usually need expensive machines such as an ultracentrifuge and they are difficult to standardize, which hinder their application in clinical laboratories. We have optimized and standardized the isolation of urinary exosomes with the precipitation method. We have found that urinary exosomal prostate-specific antigen (PSA) can be quantified by automatic Elecsys total PSA technique. OBJECTIVE:In this study, our objective is to utilize urinary exosomes from prostate cancer for the development of a test to aid in its diagnosis. METHODS:Exosomes from the prostate cancer cell line LNCaP was used to set up the technique. To analyze urine samples from patients, the methods include the collection of first-void urine using the Colli-Pee device, the isolation of urine exosomes using the optimized precipitation method, and the quantification of exosomal PSA by Elecsys total PSA. RESULTS:This will be a 2-year study. We will start including patients and controls in the last quarter of 2024. We expect the results to be published in the second quarter of 2027. CONCLUSIONS:This is the first study to quantify urinary exosomal PSA using the Elecsys total PSA technique for the diagnosis of prostate cancer. This study emphasizes techniques that are suitable for implementation in clinical laboratories, which will facilitate the application of urinary exosomes to simplify and improve the diagnosis and screening of prostate cancer. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID):PRR1-10.2196/63551.

Background Prostate cancer is the most common cancer in men and represents a major public health problem. The current method for the diagnosis or screening of prostate cancer is invasive and costly. There have been renewed and innovative studies searching for urinary biomarkers to aid in the diagnosis of prostate cancer, especially with technologies based on urinary exosomes. However, technologies based on urine exosomes usually need expensive machines such as an ultracentrifuge and they are difficult to standardize, which hinder their application in clinical laboratories. We have optimized and standardized the isolation of urinary exosomes with the precipitation method. We have found that urinary exosomal prostate-specific antigen (PSA) can be quantified by automatic Elecsys total PSA technique. Objective In this study, our objective is to utilize urinary exosomes from prostate cancer for the development of a test to aid in its diagnosis. Methods Exosomes from the prostate cancer cell line LNCaP was used to set up the technique. To analyze urine samples from patients, the methods include the collection of first-void urine using the Colli-Pee device, the isolation of urine exosomes using the optimized precipitation method, and the quantification of exosomal PSA by Elecsys total PSA. Results This will be a 2-year study. We will start including patients and controls in the last quarter of 2024. We expect the results to be published in the second quarter of 2027. Conclusions This is the first study to quantify urinary exosomal PSA using the Elecsys total PSA technique for the diagnosis of prostate cancer. This study emphasizes techniques that are suitable for implementation in clinical laboratories, which will facilitate the application of urinary exosomes to simplify and improve the diagnosis and screening of prostate cancer. International Registered Report Identifier (IRRID) PRR1-10.2196/63551

Currently, the diagnosis of genitourinary cancer depends on medical images and/or biological exams combined with tissue biopsy. This method for diagnosis is expensive, painful, and invasive. Liquid biopsies obtained from bodily fluids like blood and urine are less invasive and more easily repeatable. Exosome is a recent hot topic as a source of liquid biopsy. Liquid biopsies by exosome markers have the potential to change the nature of diagnostic mode of genitourinary cancer. In this chapter, we focus on exosome application in the field of molecular diagnosis for kidney cancer, bladder cancer, and prostate cancer. First, we describe the basic knowledge of exosome. Next, we present current liquid biopsy of exosome technologies. Finally, we demonstrate the potential of liquid biopsy of exosome markers in advancing the diagnosis in genitourinary cancers. We believe that liquid biopsies of exosome markers will facilitate the precision for the management of genitourinary cancer.

Small extracellular vesicles (EVs) are characterized by the membrane expression of CD63, CD81 and CD9 tetraspanins. Their size is inferior to 200 nm. They share the same characteristics as the native cells and are found in human fluids. Specific membrane protein biomarkers expressed on small EV are useful for the diagnosis of tumoural pathologies. Clear cell renal cell carcinoma (CCRCC) is diagnosed by imaging examinations and/or tissue biopsy. Carbonic anhydrase IX (CAIX) is a powerful biomarker of CCRCC. The detection of CAIX on small EV from the urine of patients could constitute a liquid biopsy for CCRCC. We have set up a specific protocol for the preparation, the immunostaining characterization and the transmission electron microscopy observation of small EVs isolated from the urine of CCRCC patients. The background labelling was significantly reduced. We successfully detected biomarkers on urinary small EVs from CCRCC patients. This technique could be extended with antibodies directed against other EV biomarkers for the detection and the monitoring of cancer diseases.

Serum exosomes frequently are used for liquid biopsy. Serum exosomes normally are isolated using ultracentrifugation; however, ultracentrifugation is time-consuming, labor intensive and requires a high-speed centrifuge. Many commercial kits use a precipitation-based method; however, this process can result in substantial contamination. We developed a new method to isolate pure serum exosomes. We isolated serum exosomes using precipitation, extracted them using acetone, then isolated them again by precipitation. We used transmission electron microscopy (TEM) to examine the morphology of serum exosomes. TEM indicated that our isolated exosomes were pure with typical morphology and with a size ranging from 40 to 150 nm. Flow cytometry revealed expression of exosome markers, CD63, CA81 and CD9. Our double precipitation method enables ready extraction of pure exosomes from serum. Our double precipitation method simplifies detection of serum exosomal biomarkers for diagnosis and prognosis of disease.

INTRODUCTION:Ischaemic stroke is the leading cause of adult disability. Thus, a strategy based on an efficient antiplatelet therapy has been developed. The monitoring of antiplatelet therapy is now limited to high risk and poor prognosis patients. Indeed, the biological monitoring of the antiplatelet therapy with available platelet function assays do not provide a global integrative approach. Platelet transmission electron microscopy, recently validated for assessing distinct ultrastructural abnormalities is a reliable morphological platelet structural analysis tool which could be used to collect all the ultrastructural platelet characteristics and assess the degree of activation of platelets.METHODS AND ANALYSIS:Our pilot prospective and descriptive study will include 50 consecutive patients hospitalized for an ischaemic stroke. We expect to identify ultrastructural characteristics that will be correlated with the degree of platelet activation to guide clinicians in decision making regarding the antiplatelet therapy strategy.ETHICS AND DISSEMINATION:The French Ethics Committee (comité de protection des personnes d'Ile-de-France VII) approved the information notice that will be given to participants and the protocol of this trial (protocol No 21-031).The results of the trial will be disseminated through scientific publications.TRIAL REGISTRATION NUMBER:NCT05004233.

Background: We hypothesized that the fine needle aspiration (FNA) supernatant from tumor might contain tumor-derived exosomes. The objective of this pilot study was to test if tumor-derived exosomal RNA could be found in FNA supernatants for molecular diagnosis of cancer.Methods: 10 FNA samples from pancreatic tumor were included. After the routine recuperation of cellular material by centrifugation, the cell-free Cytolyt liquid was collected instead of being discarded. 10 ml Cytolyt was used to isolate the exosomes. Transmission electronic microscopy (TEM) was used to examine the presence of exosomes. The exosomal marker CD63 was analyzed by flow cytometry. The exosomal RNA was extracted. RT-qPCR was performed to detect the GAPDH and the tumor marker of glypican 1 gene expression.Results: TEM confirmed the presence of exosomes from FNA supernatants. Flow cytometry showed a strong positive expression of exosome marker CD63. The concentration of exosomal RNA ranged from 18.81 to 354.75 ng/μl with an average of 81.76 ng/μl. The average exosomal RNA quantity was 1390.01 ng (range from 319.77 to 6030.75 ng) with an average 260/280 ratio of 2.12. GAPDH was detectable in all samples. Exosomal glypican 1 was detected in all samples of pancreatic ductal adenorcarcinomas (3/3) and absent from benign cystic samples (3/3). Furthermore, exosomal glypican 1 was positive in one sample with a non-contributive cytology and in one sample in which no malignant cell was found.Conclusion: This is the first report that the supernatants from FNA biopsy may contain tumor-derived exosomal RNA. These tumor-derived exosomes from FNA may provide a new liquid biopsy for the molecular diagnosis of cancer.

Background 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. Objective 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. Methods 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. Results This study is currently underway with funding support from the CHU Saint-Etienne of France. Conclusions 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. Trial Registration ClinicalTrials.gov NCT04053855; https://clinicaltrials.gov/ct2/show/NCT04053855 International Registered Report Identifier (IRRID) DERR1-10.2196/24423

Objective: The objective of this study was to assess the possibility of using urinary exosomal CA9 mRNA as a novel liquid biopsy for the molecular diagnosis of bladder cancer. Patients and Methods: A total of 168 bladder cancer patients and 90 control subjects were included in the study. An isolation kit was used to isolate urinary exosomes. Transmission electron microscopy (TEM) was used to examine the presence of exosomes. Flow cytometry was used to examine the exosomal marker CD63. The expression level of exosomal CA9 mRNA was detected by RT-qPCR. The diagnostic performance of urinary urinary exosomal CA9 mRNA was evaluated. Results: TEM confirmed the enriched exosomes from urinary bladder patients. Flow cytometry indicated a strong positive expression of exosome marker CD63. Successful extraction of RNA was performed from exosome samples. The level of urinary exosomal CA9 mRNA was significantly higher in bladder cancer group than in control group (p<0.001). The area under the ROC curve was 0.837 (95% CI: 0.743-0.859) with a sensitivity of 85.18% and a specificity of 83.15% for the diagnosis of bladder cancer. Conclusion: We found that the urinary exosomes were abundant in the urine of bladder cancer patients. CA9 mRNA could be detectable in urinary exosomes. The urinary exosomal CA9 mRNA may present a new liquid biopsy for the diagnosis of bladder cancer.

BACKGROUND AND PURPOSE:Biological response to clopidogrel prescribed after a non-cardioembolic ischemic stroke or transient ischemic attack (TIA) has been little studied. The aim of our study (AAPIX) was to assess this response and investigate the agreement between different biological assays in revealing poor responders. METHODS:Patients hospitalized following a non-cardioembolic ischemic stroke or transient ischemic attack (TIA) and prescribed clopidogrel were consecutively included from September 2013 to November 2015 in the Stroke Center of Saint-Etienne Hospital. Blood was drawn after 5 to 8 days of standard-dose clopidogrel. Light transmission aggregometry (LTA) and flow cytometric assays, using vasodilator-stimulated phosphoprotein [VASP] and CD62P, were accomplished for all patients. Transmission electron microscopy (TEM) was performed for a poor clopidogrel-responder and for a patient with discordant platelet assay results (platelet reactivity index (PRI) >50% and maximum platelet aggregation <70%), after activation with adenosine diphosphate (ADP) 10 µM. RESULTS:72 patients were included. According to LTA, VASP assay and CD62P test results, 65%, 71% and 0% of patients, respectively, had a low response to clopidogrel, indicating poor agreement between these assays. Images of ADP-activated platelet samples from a patient manifesting a low response to clopidogrel and from a patient with discordant platelet assay results showed an ultrastructural pattern typical of activation and a state of slight activation, respectively. CONCLUSIONS:Platelet function results obtained using different assays for patients having experienced a non-cardioembolic ischemic stroke or TIA were discordant. Transmission electron microscopy could be useful in certain clinical contexts when platelet function assay results disagree.

Background and purpose: Low biological response to Clopidogrel prescribed after non cardioembolic ischemic stroke or transient ischemic attack (TIA) is a major clinical problem and could explain the recurrence of vascular events. Platelet alpha 2-adrenoreceptors are involved in the high residual platelet reactivity in stable coronary artery disease patients on dual antiplatelet therapy. In the present study we investigated the impact of platelet alpha 2-adrenoreceptors on ADP-induced platelet aggregation and on ADP-induced platelet membrane CD62P (P-selectin) expression, a marker of platelet activation on blood samples from patients hospitalized at the acute phase of a non cardioembolic ischemic stroke or TIA. Methods: 72 consecutive patients were prospectively recruited over the course of two years in a monocentric study. Patients received a daily 75 mg-dose of Clopidogrel. ADP-induced platelet aggregation was measured alone, with low dose epinephrine or with atipamezole, a selective alpha blocker of alpha 2-adrenoreceptors, by Light Transmittance Aggregometry (LTA). Platelet membrane expression of P-selectin was measured by flow cytometry with either ADP alone or combined with epinephrine. Results: Epinephrine at low dose stimulated ADP-induced platelet membrane expression of CD62P whereas Atipamezole significantly inhibited 10 mu M ADP-induced platelet aggregation. Conclusions: Our study showed the role of platelet alpha 2-adrenoreceptors in biological low response to Clopidogrel for patients hospitalized for a non-cardioembolic ischemic stroke or TIA. Atipamezole could improve the status of biological response to Clopidogrel.

A recently released kit (PerFix EXPOSE) was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12). The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24 h post clopidogrel dose from 19 patients hospitalized for a non-cardio-embolic ischemic stroke and treated with clopidogrel monotherapy for at least five days in an observational study. The platelet PerFix method was based on adaptation of the volume of the sample, the centrifugation speed and the incubation temperature. Poor agreement between prevention by adenosine diphosphate (ADP) of PGE(1)-induced cAMP-mediated VASP phosphorylation and ADP induced aggregation assessed by Light Transmittance Aggregometry was found. We found a significant correlation between the PLT VASP/P2Y12 kit and the PerFix EXPOSE kit. The PerFix EXPOSE kit may also be helpful to monitor adverse effects of second-generation tyrosine kinase inhibitors on platelets.

The serology of patients with Rheumatoid arthritis (RA) is characterized by persistently raised levels of autoantibodies: Rheumatoid Factors (RhF) against Fc of IgG, and to citrullinated (Cit) protein/peptide sequences: ACPA, recognizing multiple Cit-sequences. B cell depletion therapy based on rituximab delivers good clinical responses in RA patients, particularly in the seropositive group, with responses sometimes lasting beyond the phase of B cell reconstitution. In general, ACPA levels fall following rituximab, but fluctuations with respect to predicting relapse have proved disappointing. In order to identify possible immunodominant specificities within either IgG- or IgA-ACPA we used a Multiplex bead-based array consisting of 30 Cit-peptides/proteins and 22 corresponding native sequences. The kinetics of the serum ACPA response to individual specificities was measured at key points (Baseline, B cell depletion phase, Relapse) within an initial cycle of rituximab therapy in 16 consecutive patients with severe, active RA. All had achieved significant decreases in Disease Activity Scores-28 and maintained B cell depletion in the peripheral blood (<5 CD19+cells/μl) for at least 3 months. At Baseline, mean fluorescence intensity shown by individual IgG- and IgA-ACPA were strongly correlated (R2 = 0.75; p < 0.0001) but IgA-ACPA were approximately 10-fold lower. Data were Z-normalised in order to compare serial results and antibody classes. At Baseline, a total of 68 IgG- and 51 IgA-ACPA had Z-scores ≥ 1 (above population mean) were identified, with at least one Cit-antigen identified in each serum. ACPA to individual specificities subsequently fluctuated with 3 different patterns. Most 51/68 (75%) IgG- and 48/51 IgA-ACPA (94%) fell between Baseline and Depletion, of which 57% IgG- and 65% IgA-ACPA rebounded pre-Relapse. Interestingly, 17/68 IgG-ACPA (25%) and some IgA-ACPA (3/51; 6%) transiently increased from Baseline, subsequently falling pre-Relapse. Individual responses to particular Cit-epitopes were not linked to particular patterns of fluctuation, but IgG- and IgA-ACPA to individual Cit-antigens often followed similar courses. Some new IgG- and IgA-ACPA, generally to different Cit-antigens however, arose at Relapse in 4 patients. The complexities of the ACPA response after rituximab may therefore reflect its ability to deplete or modify the function of parent B cell clones, which varies between patients. Although relapse following rituximab invariably follows naïve B cell exit from the bone marrow, these studies show that interactions between both ‘new’ and residual autoreactive memory B cells may be key to resumption of symptoms. The lack of identification of any immunodominant specificity suggests that the process of citrullination, rather than any particular Cit-antigen drives the autoimmune response in RA patients.

Drug–drug interactions may contribute to the variability of the response of clopidogrel. Several hypotheses have been proposed concerning the potential modification of clopidogrel pharmacokinetics and pharmacodynamics by fluoxetine. This open‐label crossover study assessed the effect of fluoxetine on the pharmacological activity of clopidogrel in healthy volunteers. Eight healthy male volunteers received a single 600‐mg loading dose of clopidogrel followed by 20 mg of fluoxetine on 4 days and then 20 mg of fluoxetine plus 600 mg of clopidogrel on the fifth day. Eleven blood samples were withdrawn after clopidogrel administration to determine plasma concentrations of clopidogrel active metabolite ( CAM ) and platelet function. Platelet aggregation was measured by light transmittance aggregometry ( LTA ) and platelet reactivity index by flow cytometric vasodilator‐stimulated phosphoprotein ( VASP ) analysis. The areas under the curve and maximum plasma concentrations of CAM were, respectively, 20.6 and 25.3% lower after co‐administration of fluoxetine compared with administration of clopidogrel alone. The percentage maximum platelet aggregation values in the presence of 5 μ m and 10 μ m adenosine diphosphate, measured by LTA , were, respectively, 13.9 and 22.4% lower after fluoxetine co‐administration. The platelet reactivity index measured by the flow cytometric VASP method was 36.8% lower when clopidogrel was administered in conjunction with fluoxetine.

Summary The existence of poor biological response to clopidogrel has been shown in some patients. Despite the increasing number of studies, this phenomenon remains difficult to quantify. We performed a systematic review to estimate the prevalence of poor biological response to clopidogrel and investigate the factors known to modulate this. An exhaustive search was performed. Altogether 171 publications were identified, providing data for a total of 45,664 subjects. The estimated prevalence of poor biological response to clopidogrel ranged from 15.9% to 49.5% according to the platelet function assay employed. The assays most frequently used were light transmittance aggregometry (LTA), the vasodilator-stimulated phosphoprotein (VASP) assay and the Verify -now® assay. For all these assays, higher cut-off values were associated with a lower prevalence of poor biological response to clopidogrel. However, when choosing a fixed cut-off point for each assay, the prevalence of poor biological response to clopidogrel was highly variable suggesting that other factors could modulate poor biological response to clopidogrel. Finally, none of the studied factors could apparently explain the variability of poor biological response to clopidogrel. This meta-analysis shows that the prevalence of poor biological response depends on the assay employed, the cut-off value and on various unidentified additional factors.

Background: Previous studies have shown an important risk of cardiovascular events in patients with clopidogrel biological non-response, and data have shown considerable, unexplored heterogeneity. Objectives: To evaluate the magnitude of cardiovascular risk associated with clopidogrel non-response and to explore heterogeneity. Methods: This was a systematic review and meta-analysis of prospective studies of patients treated with clopidogrel for symptomatic atherothrombosis, evaluated by light transmission aggregometry with ADP and monitored prospectively for clinical ischemic events. Results: Fifteen studies were included, totaling 3960 patients, of whom 25% were considered to be clopidogrel non-responders. The global relative risk (RR) for recurrent ischemic events in clopidogrel non-responders was 3.5 [95% confidence interval (CI) 2.4-5.2, P < 0.0001]. The results of the different studies were heterogeneous (Cochran P = 0.01 and I2 = 52%). The most recent studies yielded lower RRs [global RR = 2.9 (95% CI 2.3-3.8) after 2007, and global RR = 6.6 (95% CI 3.7-11.9) before 2007, P = 0.01]. Heterogeneity was present in the group of studies in which more than 10% of patients took glycoprotein (GP)IIb-IIIa inhibitors [Cochran P = 0.003 and I2 = 70%; RR = 3.8 (95% CI 2.9-5.1)] and was absent in the other studies [Cochran P = 0.88 and I2 = 0; RR = 2.5 (95% CI 1.7-3.6)]. The RR was significantly higher in studies using higher ADP maximal aggregation cut-offs (> 65%) for clopidogrel non-response than in studies using lower cut-offs [RR = 5.8 (95% CI 3.2-10.3) and RR = 2.9 (95% CI 2.2-3.7), respectively, P = 0.03]. Conclusions: The risk of ischemic events associated with clopidogrel non-response is now more precisely defined. The risk is heterogeneous across studies, possibly because of an interaction with GPIIb-IIIa inhibitors and the use of different cut-offs to identify non-responders.

A sensible ultra-performance LC-MS method was developed and validated for the quantification of clopidogrel active metabolite in human plasma, with clopidogrel D4 as internal standard. Plasma pretreatment involved a one-step protein precipitation with acetonitrile. The separation was performed by reverse-phase chromatography on a C8 column. The method was linear over the concentration range of 1-150 ng/mL. The intra- and inter-day precision values were below 17% and accuracy was from 1.7 to 7.5% at all quality control levels. The lower LOQ was 0.8 ng/mL. Sample analysis time was reduced to 5 min including sample preparation (limited to protein precipitation). The present method was successfully applied to a clopidogrel active metabolite pharmacokinetic study following oral administration to healthy volunteers.

patients with heterozygous defects of the receptor. Our results might also be relevant to the use of the VASP phosphorylation assay for monitoring individual response to clopidogrel, which has been recommended for identifying patients who are poor responders to the drug [2]. The VASP phosphorylation assay has been used for this purpose in many studies [10,11], but the cut-off value for the identification of patients who are poor responders or resistant to the drug has not been clearly identified yet. Fromour observation that the assay is insensitive to mild defects of P2Y12, it can be concluded that clopidogreltreated patients who display a normal PRI with the VASP phosphorylation assay should also not be considered resistant to clopidogrel, because they may have a high percentage of their platelet receptors inhibited by the drug.