An effective urobilin clearance strategy based on paramagnetic beads facilitates microscale proteomic analysis of urine.

Urine provides an ideal source for disease biomarker discovery. High-adhesion contaminants such as urobilin, which are difficult to remove from urine, can severely interfere with urinary proteomic analysis. Here, we aimed to establish a strategy based on single-pot, solid-phase-enhanced sample preparation (SP3) technology to prepare samples for urinary proteomics analysis that almost completely eliminates the impact of urobilin. A systematic evaluation of the effects of two urinary protein precipitation methods, two types of protein lysis buffers, and different ratios of magnetic digestion beads on the identification and quantification of the microscale urinary proteome was conducted. Our results indicate that methanol-chloroform precipitation, coupled with efficient lysis facilitated by urea, and subsequent enzymatic digestion using a mix of hydrophilic and hydrophobic magnetic beads offers the best performance. Further applying this strategy to the urine of patients with benign prostatic hyperplasia, prostate cancer and healthy individuals, combined with a narrow window of data-independent acquisition, FGFR4, MYLK, ORM2, GOLM1, SPP1, CD55, CSF1, DLD and TIMP3 were identified as potential biomarkers to discriminate benign prostatic hyperplasia and prostate cancer patients.

Extracellular RNAs from Whole Urine to Distinguish Prostate Cancer from Benign Prostatic Hyperplasia.

RNAs, especially non-coding RNAs (ncRNAs), are crucial players in regulating cellular mechanisms due to their ability to interact with and regulate other molecules. Altered expression patterns of ncRNAs have been observed in prostate cancer (PCa), contributing to the disease's initiation, progression, and treatment response. This study aimed to evaluate the ability of a specific set of RNAs, including long ncRNAs (lncRNAs), microRNAs (miRNAs), and mRNAs, to discriminate between PCa and the non-neoplastic condition benign prostatic hyperplasia (BPH). After selecting by literature mining the most relevant RNAs differentially expressed in biofluids from PCa patients, we evaluated their discriminatory power in samples of unfiltered urine from 50 PCa and 50 BPH patients using both real-time PCR and droplet digital PCR (ddPCR). Additionally, we also optimized a protocol for urine sample manipulation and RNA extraction. This two-way validation study allowed us to establish that miRNAs (i.e., miR-27b-3p, miR-574-3p, miR-30a-5p, and miR-125b-5p) are more efficient biomarkers for PCa compared to long RNAs (mRNAs and lncRNAs) (e.g., PCA3, PCAT18, and KLK3), as their dysregulation was consistently reported in the whole urine of patients with PCa compared to those with BPH in a statistically significant manner regardless of the quantification methodology performed. Moreover, a significant increase in diagnostic performance was observed when molecular signatures composed of different miRNAs were considered. Hence, the abovementioned circulating ncRNAs represent excellent potential non-invasive biomarkers in urine capable of effectively distinguishing individuals with PCa from those with BPH, potentially reducing cancer overdiagnosis.

High level of phosphatidylcholines/lysophosphatidylcholine ratio in urine is associated with prostate cancer.

The altered levels of phospholipids (PLs) and lysophospholipids (LPLs) in prostate cancer (CaP) and benign tissues in our previous findings prompted us to explore PLs and LPLs as potential biomarkers for CaP. Urinary lipidomics has attracted increasing attention in clinical diagnostics and prognostics for CaP. In this study, 31 prostate tissues obtained from radical prostatectomy were assessed using high-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS). Urine samples were collected after digital rectal examination (DRE), and urinary lipids were extracted using the acidified Bligh-Dyer method. The discovery set comprised 75 patients with CaP and 44 with benign prostatic hyperplasia (BPH) at Kyoto University Hospital; the validation set comprised 74 patients with CaP and 59 with BPH at Osaka University Hospital. Urinary lipidomic screening was performed using MALDI time-of-flight MS (MALDI-TOF/MS). The levels of urinary lysophosphatidylcholine (LPC) and phosphatidylcholines (PCs) were compared between the CaP and BPH groups. The (PC [34:2] + PC [34:1])/LPC (16:0) ratio was significantly higher (P < .001) in CaP tissues than in benign epithelial tissues. The urinary PCs/LPC ratio was significantly higher (P < .001) in the CaP group than in the BPH group in the discovery and validation sets.

Identification of the metabolic signatures of prostate cancer by mass spectrometry-based plasma and urine metabolomics analysis.

OBJECTIVE: Prostate cancer (PCa) is one of the most commonly diagnosed cancers among men which is associated with profound metabolic changes. Systematic analysis of the metabolic alterations and identification of new biomarkers may benefit PCa diagnosis and a deep understanding of the pathological mechanism. The purpose of this study was to determine the metabolic features of PCa. METHODS: Plasma and urine metabolites from 89 prostate cancer (PCa) patients, 84 benign prostatic hyperplasia (BPH) patients, and 70 healthy males were analyzed using LC-MS/MS and GC-MS. The Orthogonalised Partial Least Squares Discriminant Analysis (OPLS-DA) was used to find the significantly changed metabolites. The clinical value of the candidate markers was examined by receiver operating characteristic curve analysis and compared with prostate-specific antigen (PSA). RESULTS: Multivariate statistical analyses found a series of altered metabolites, which related to the urea cycle, tricarboxylic acid cycle (TCA), fatty acid metabolism, and the glycine cleavage system. Plasma Glu/Gln showed the highest predictive value (AUC = 0.984) when differentiating PCa patients from healthy controls, with a higher sensitivity than PSA (96.6% vs. 94.4%). Both Glu/Gln and PSA displayed a low specificity when differentiating PCa patients from BPH patients (<53.2%), while the combination of Glu/Gln and PSA can further increase the diagnostic specificity to 66.9%. CONCLUSIONS: The present study showed the metabolic features of PCa, provided strong evidence that the amide nitrogen and the energy metabolic pathways could be a valuable source of markers for PCa. Several candidate markers identified in this study were clinically valuable for further assessment.

National trends in the use of PSA, urinalysis, and digital rectal exam for evaluation of lower urinary tract symptoms in men.

PURPOSE: The purpose of this study was to describe the practices of primary care physicians (PCPs) and urologists in their implementation of the 2010 American Urological Association (AUA) recommendations for the management of benign prostatic hyperplasia (BPH) in a nationally representative sample. METHODS: Data collected from 2008 to 2015 in the National Ambulatory Medical Care Survey (NAMCS) were used. Men aged 45 and older who presented with either a new complaint or exacerbation of lower urinary tract symptoms (LUTS) were included. Primary outcomes were the prevalence and determinants of prostate-specific antigen (PSA) testing, urinalysis (UA), and digital rectal exam (DRE), as all three were included in the AUA guidelines during the time period studied. In logistic regression analyses weighted to reflect national estimates, potential determinants of adherence for each testing modality were examined. RESULTS: Between 2008 and 2015, 878 visits met inclusion criteria, corresponding to 14,399,121 ambulatory visits for new or exacerbated LUTS. Weighted prevalence estimates were 24% for PSA testing (95% CI: 19-29%), 61% for urinalysis (95% CI: 56-66%), and 18% for DRE (95% CI: 15-23%). Age ≥ 75 years was associated with lower prevalence of testing for all three tests, and region was associated with different testing estimates for PSA and UA. Patients referred to urologists were more likely to receive a DRE, although overall rates of DRE decreased per additional year of data. CONCLUSIONS: Adherence to AUA guidelines for evaluation of LUTS in ambulatory visits was low in a nationally representative sample of Americans, particularly for PSA testing and DRE, suggesting substantial discordance between guidelines at the time and practice patterns. Practice patterns also differed by age and region. These discrepancies encourage increased education of providers in the implementation of the guidelines, particularly since they have been updated recently.

Screening, identification of prostate cancer urinary biomarkers and verification of important spots.

Prostate-specific antigen (PSA) has been widely used as the unique serum biomarker for the diagnosis of prostate cancer (PCa). When PSA is moderately increased (e.g., 4-10 ng/ml), it is difficult to differentiate benign prostatic hyperplasia (BPH) from cancer. The diagnostic test (i.e., prostate biopsy) is invasive, adding pain and economic burden to the patient. Urine samples are more convenient, non-invasive and readily available than blood. We sought to determine whether ferritin might be the potential urinary biomarker in prostate cancer diagnosis. Using two-dimensional electrophoresis (2DE) followed by mass spectrometry (MS), differentially expressed urinary proteins among patients with PCa, BPH and normal controls were obtained. The ferritin heavy chain (FTH) gene, ferritin light chain (FTL) gene and protein expression of BPH-1 cells and PC-3 cells were analyzed by real-time quantitative PCR and Western blotting, respectively. Stable FTH or FTL silenced cell lines were generated by small hairpin(sh) RNA lentiviral transfection. The function of the cell lines was evaluated by the colony formation assay, transwell assay, and flow cytometry. Compared with BPH and normal controls, 15 overexpressed proteins, including FTH and FTL, were identified in the urine of the PCa group. FTH and FTL were also highly expressed in PC-3 cell lines compared with BPH-1 cells. FTH-silenced cells showed reduced cell proliferation, migration and increased cell apoptosis. FTL-silenced cells showed increased proliferation and migration abilities. There are differences in urinary proteins among patients with PCa, BPH and normal controls. FTH and FTL play different roles in PCa cells and are potential biomarkers for PCa.

Molecular correlates in urine for the obesity and prostatic inflammation of BPH/LUTS patients.

PURPOSE: Benign prostatic hyperplasia (BPH) is strongly associated with obesity and prostatic tissue inflammation, but the molecular underpinning of this relationship is not known. Here, we examined the association between urine levels of chemokines/adipokines with histological markers of prostate inflammation, obesity, and lower urinary tract symptoms LUTS in BPH patients. METHODS: Frozen urine specimens from 207 BPH/LUTS patients enrolled in Nashville Men's Health Study were sent for blinded analysis of 11 analytes, namely sIL-1RA, CXC chemokines (CXCL-1, CXCL-8, CXCL-10), CC chemokines (CCL2, CCL3, CCL5), PDGF-BB, interleukins IL-6, IL-17, and sCD40L using Luminex™ xMAP® technology. After adjusting for age and medication use, the urine levels of analytes were correlated with the scales of obesity, prostate inflammation grade, extent, and markers of lymphocytic infiltration (CD3 and CD20) using linear regression. RESULTS: sIL-1RA levels were significantly raised with higher BMI, waist circumference and waist-hip ratio in BPH patients after correction for multiple testing (P = 0.02). Men with greater overall extent of inflammatory infiltrates and maximal CD3 infiltration were marginally associated with CXCL-10 (P = 0.054) and CCL5 (P = 0.054), respectively. CCL3 in 15 patients with moderate to severe grade inflammation was marginally associated with maximal CD20 infiltration (P = 0.09), whereas CCL3 was undetectable in men with mild prostate tissue inflammation. There was marginal association of sCD40L with AUA-SI scores (P = 0.07). CONCLUSIONS: Strong association of sIL-1RA in urine with greater body size supports it as a major molecular correlate of obesity in the urine of BPH patients. Increased urine levels of CXCL-10, CCL5, and CCL3 were marginally associated with the scores for prostate tissue inflammation and lymphocytic infiltration. Overall, elevated urinary chemokines support that BPH is a metabolic disorder and suggest a molecular link between BPH/LUTS and prostatic inflammation.

Two long non-coding RNAs, Prcat17.3 and Prcat38, could efficiently discriminate benign prostate hyperplasia from prostate cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) have recently appeared as new players in cancer biology. Recently, a number of new prostate cancer-associated lncRNAs has been listed via RNA-seq approach by Mitranscriptome project. By analyzing this data we chose four lncRNAs (Prcat17.3, Prcat38, Prcat47, and Cat2184.4) and evaluated their expressions and their abilities to discriminate prostate tumors from benign prostate hyperplasia (BPH). METHODS: Fresh Prostate tissue samples (30 BPH, and 30 tumor samples) and urine samples (19 BPH, and 19 tumor samples) were collected and their total RNA extracted for cDNA syntheses. The expression of candidate lncRNAs was assessed by the real-time PCR technique. RESULTS: Our data revealed that the expression levels of PRCAT17.3 (P < 0.0001) and PRCAT38 (P < 0.0002) were significantly upregulated in human prostate cancer tissues, compared to BPH ones. Moreover, the altered expression was much higher for PRCAT17.3 (∼2000 folds) than PRCAT38 (∼50 folds). In contrast, the expression of Cat2184.4 showed a significant down-regulation in tumor samples (P < 0.0001), compared to BPH ones. While the expression level of PRCAT47 was increased in cancer samples, the changes were not statistically significant. In discriminating prostate tumors from BPH samples, Prcat17.3 (AUC-ROC, 0.927) demonstrated a better diagnostic efficacy than Prcat38 (AUC-ROC, 0.778). Moreover, real-time RT-PCR analyses on urine samples of prostate cancer patients revealed that prcat17.3 level is significantly elevated, (P < 0.0197; AUC-ROC value of 0.72), compared to that of BPH patients. CONCLUSION: We introduce here two novel lncRNAs with a potential application in diagnosis of prostate cancer.

Efficacy and safety of a hexanic extract of Serenoa repens (Permixon® ) for the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia (LUTS/BPH): systematic review and meta-analysis of randomised controlled trials and observational studies.

OBJECTIVES: To comprehensively evaluate the efficacy and safety of the hexanic extract of Serenoa repens (HESr, Permixon® ; Pierre Fabre Médicament, Castres, France), at a dose of 320 mg daily, as monotherapy for the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia (LUTS/BPH). MATERIALS AND METHODS: We conducted a systematic review and meta-analysis of randomised controlled trials (RCTs) and prospective observational studies in patients with LUTS/BPH identified through searches in Medline, Web of Knowledge (Institute for Scientific Information), Scopus, the Cochrane Library, and bibliographic references up to March 2017. Articles studying S. repens extracts other than Permixon were excluded. Data were collected on International Prostate Symptom Score (IPSS), maximum urinary flow rate (Qmax ), nocturia, quality of life, prostate volume, sexual function, and adverse drug reactions (ADRs). Data obtained from RCTs and observational studies were analysed jointly and separately using a random effects model. A sub-group analysis was performed of studies that included patients on longer-term treatment (≥1 year). RESULTS: Data from 27 studies (15 RCTs and 12 observational studies) were included for meta-analysis (total N = 5 800). Compared with placebo, the HESr was associated with 0.64 (95% confidence interval [CI] -0.98 to -0.31) fewer voids/night (P < 0.001) and an additional mean increase in Qmax of 2.75 mL/s (95% CI 0.57 to 4.93; P = 0.01). When compared with α-blockers, the HESr showed similar improvements on IPSS (weighted mean difference [WMD] 0.57, 95% CI -0.27 to 1.42; P = 0.18) and a comparable increase in Qmax to tamsulosin (WMD -0.02, 95% CI -0.71 to 0.66; P = 0.95). Efficacy assessed using the IPSS was similar after 6 months of treatment between the HESr and 5α-reductase inhibitors (5ARIs). Analysis of all available published data for the HESr showed a mean improvement in IPSS from baseline of -5.73 points (95% CI -6.91 to -4.54; P < 0.001). HESr did not negatively affect sexual function and no clinically relevant effect was observed on prostate-specific antigen. Prostate volume decreased slightly. Similar efficacy results were seen in patients treated for ≥1 year (n = 447). The HESr had a favourable safety profile, with gastrointestinal disorders being the most frequent ADR (mean incidence of 3.8%). CONCLUSION: The present meta-analysis, which includes all available RCTs and observational studies, shows that the HESr (Permixon) reduced nocturia and improved Qmax compared with placebo and had a similar efficacy to tamsulosin and short-term 5-ARI in relieving LUTS. HESr (Permixon) appears to be an efficacious and well-tolerated therapeutic option for the long-term medical treatment of LUTS/BPH.

Association of ferritin with prostate cancer.

PURPOSE: Prostate specific antigen (PSA) has been widely used as the unique serum biomarker for the diagnosis and/ or pre-diagnosis of prostate cancer (PCa). However, the diagnostic value of PSA is a subject of ongoing debate owing to its lack of specificity, especially when the PSA level is moderately increased (e.g., 4-10 ng/ml). Thus, we suggest the need for identification of a new biomarker to discriminate PCa cases from benign prostatic hyperplasia (BPH) and normal controls (N). METHODS: Urine or tissue samples of PCa patients, BPH patients, and normal controls were systematically collected. The expression of ferritin light chain (FTL) and ferritin heavy chain (FTH) was verified by immunohistochemistry in the tissue. The concentration of urinary ferritin was measured by Access Immunoassay. The level of creatinine in the urine was detected on a HITACHI 7080 system to calculate the ferritin-creatinine ratio (FCR). The data were statistically analyzed using the rank sum test. RESULTS: Immunohistochemical characterization of tissues in patients with PCa and BPH was conducted. We found representative immunohistochemical expression of FTL and FTH, with strong staining intensity in PCa and weak staining intensity in BPH. Furthermore, there were differences in urinary FCR among the three groups, with significant differences in the PCa group (134.46±47.01) compared to both the BPH (24.18±3.17, p = 0.009) and control (6.42±0.82, p = 0.003) groups. In contrast, there was no significant difference between the BPH and N groups (p = 0.649). CONCLUSIONS: Ferritin is a potential urinary biomarker to discriminate between PCa and BPH patients.

[The use of ProstaDoz in patients with benign prostatic hyperplasia and coexistent high-grade prostatic intraepithelial neoplasia].

INTRODUCTION: In recent years there has been an increasing interest in the drug treatment of benign prostatic hyperplasia (BPH) to improve the patients quality of life without surgical treatment. In this connection, phytotherapeutic drugs and biologically active dietary supplements (BADS), consisting of a combination of microelements, vitamins and plant extracts, have been increasingly used in addition to synthetic preparations. OBJECTIVE: To investigate the clinical effectiveness of dietary supplement ProstaDoz in the treatment of BPH with coexistent high-grade prostatic intraepithelial neoplasia (PIN). MATERIALS AND METHODS: The study group comprised 30 men with BPH and high-grade PIN who were diagnosed with primary biopsy and received ProstaDoz for three months. The control group included 18 patients with the same diagnosis who were treated with Tamsulosin. RESULTS: After a 3-month intake of the ProstaDoz, a statistically significant reduction was observed in symptom scores for BPH and the quality of life index by 18% (p<0.001) and 16.7% (p<0.001), respectively. The residual urine volume and total serum PSA decreased by 28.3% (p<0.001) and 46.8% (p<0.001), respectively. Unlike the control group, the rate of prostate cancer detection was 6.25 times lower in patients taking ProstaDoz, and 16% of them had lower grade PIN compared with baseline or no PIN at all. CONCLUSION: The findings suggest that using ProstaDoz in patients with BPH and high-grade PIN contributes to improving the quality of life (QoL) of patients and the regression of dysplasia.

Increased Urinary Adenosine Triphosphate in Patients With Bladder Outlet Obstruction Due to Benign Prostate Hyperplasia.

BACKGROUND: Diagnosis of bladder outflow obstruction (BOO) in patients with lower urinary tract (LUT) symptoms is challenging without using invasive urodynamic tests. Recently, we showed in vitro that urothelial strips from patients with benign prostatic hyperplasia (BPH) release more ATP than controls. Here, we tested whether urinary ATP can be used as a wall tension transducer non-invasive biomarker to detect BOO in patients with BPH. METHODS: 79 male patients with BOO and 22 asymptomatic controls were recruited prospectively. Patients were asked to complete the International Prostate Symptom Score (IPSS) questionnaire and to void at normal desire into a urinary flowmeter; the postvoid residual volume was determined by suprapubic ultrasonography. Urine samples from all individuals were examined for ATP, creatinine, and lactate dehydrogenase. RESULTS: BOO patients had significantly higher (P < 0.001) urinary ATP normalized by the voided volume (456 ± 36 nmol) than age-matched controls (209 ± 35 nmol). Urinary ATP amounts increased with the voided volume, but the slope of this rise was higher in BOO patients than in controls. A negative correlation was detected between urinary ATP and flow rate parameters, namely maximal flow rate (r = -0.310, P = 0.005), Siroky flow-volume normalization (r = -0.324, P = 0.004), and volume-normalized flow rate index (r = -0.320, P = 0.012). We found no correlation with LUT symptoms IPSS score. Areas under the receiver operator characteristics (ROC) curves were 0.91 (95%CI 0.86-0.96, P < 0.001) for ATP alone and 0.88 (95%CI 0.81-0.94, P < 0,001) when adjusted to urinary creatinine. CONCLUSIONS: Patients with BOO release higher amounts of ATP into the urine than the control group. The high area under the ROC curve suggests that urinary ATP can be a high-sensitive non-invasive biomarker of BOO, which may have a discriminative value of detrusor competence when comparing BPH patients with low urinary flow rates. Prostate 76:1353-1363, 2016. © 2016 Wiley Periodicals, Inc.

The utility of urine-circulating miRNAs for detection of prostate cancer.

BACKGROUND: In this paper, the utility of urine-circulating microRNAs (miRNAs) as the potential biomarker of prostate cancer (PCa), the second most prevalent male cancer worldwide, was evaluated. METHODS: Cancerous (N=56) and non-cancerous (N=16) prostate tissues were analysed on TaqMan Low Density Array, with the initial screening of 754 miRNAs in a subset of the samples. The abundance of selected miRNAs was analysed in urine specimens from two independent cohorts of patients with PCa (N=215 overall), benign prostatic hyperplasia (BPH; N=23), and asymptomatic controls (ASC; N=62) by means of quantitative reverse transcription PCR. RESULTS: Over 100 miRNAs were found deregulated in PCa as compared with non-cancerous prostate tissue. After thorough validation, four miRNAs were selected for the analysis in urine specimens. The abundance of miR-148a and miR-375 in urine was identified as specific biomarkers of PCa in both cohorts. Combined analysis of urine-circulating miR-148a and miR-375 was highly sensitive and specific for PCa in both cohorts (AUC=0.79 and 0.84) and strongly improved the diagnostic power of the PSA test (AUC=0.85, cohort PCa1), including the grey diagnostic zone (AUC=0.90). CONCLUSIONS: Quantitative measurement of urine-circulating miR-148a and miR-375 can serve as the non-invasive tool for sensitive and specific detection of PCa.

Molecular Diagnostic of Prostate Cancer From Body Fluids Using Methylation-Specific PCR (MS-PCR) Method.

BACKGROUND: Worldwide prostate cancer (PCa) represents the 2nd leading cause of cancer related deaths among men. Currently, the screening for early detection of PCa is based on determination of serum prostate-specific antigen (PSA) levels. But this biomarker presents some disadvantages related to its specificity and sensitivity. In our study, we want to determine if methylation levels of the glutathione S-transferase P1 (GSTP1) gene could be used as a new biomarker for the early detection of PCa and to distinguish between malignant and benign pros-tatic lesions. METHODS: To determine the methylation levels of the GSTP1 gene, 31 men with histopathological diagnosis of prostate adenocarcinoma and 34 men with the histopathological diagnosis of benign prostatic hyperplasia (BPH) as controls were included in the study group. The genomic DNA was extracted from urine samples. We analyzed the methylation levels of the GSTP1 gene by methylation-specific polymerase chain reaction (MS-PCR) method. RESULTS: In prostate cancer patients 27 of 31 (87%) presented hypermethylated levels of the GSTP1 gene, whereas 4 of 34 (11.8%) BPH patients had hypermethylated levels of the GSTP1 gene. Further, in the case of these four patients a second biopsy was done, which confirmed the diagnosis of prostate adenocarcinoma. Using the receiver operating curve (ROC), we obtained a specificity of 87% and a sensitivity of 98% for the GSTP1 gene. CONCLUSIONS: We can conclude that GSTP1 represents a new molecular biomarker which can aid in early detection of PCa and be used to discriminate between benign and malignant prostatic lesions from body fluids by noninvasive methods.

WT1 immunoenzyme staining using SurePath(™) processed urine cytology helps to detect kidney disease.

OBJECTIVES: Damage and detachment of podocytes and loss into the urine have been implicated in the progression of kidney diseases. The purpose of this study was to investigate the potential role of urine cytology based on SurePath(™) combined with immunoenzyme staining using Wilms' tumour 1 (WT1) antibody as a podocyte marker in the discrimination of normality and non-renal urinary tract disease from kidney disease. METHODS: Sixty-six patients with kidney disease, 45 patients with lower urinary tract disease and 30 healthy volunteers were examined. Urine cytology slides were prepared using the SurePath method and immunoenzyme stained with WT1 antibody, and the number of WT1-positive cells was counted. RESULTS: In kidney disease, WT1-positive cells were found in 33 (50%) of 66 samples. No WT1-positive cells were found in 45 patients with lower urinary tract disease or in 30 healthy volunteers. The positive rates for WT1 varied with disease type, but not significantly: immunoglobulin A (IgA) nephropathy, (14/23); membranous glomerulonephritis, (4/10); Henoch-Schönlein purpura nephritis, (3/5); diabetic glomerulopathy, (5/5); minor glomerular abnormality/minimal change nephrotic syndrome (0/4). CONCLUSIONS: The results suggest that WT1 immunoenzyme staining of urine cytology can be used to detect some types of kidney disease.

Detection of miRNAs in urine of prostate cancer patients.

BACKGROUND AND AIM: Prostate cancer (PCa) is the second most prevalent oncologic disease among men worldwide. Expression of various transcripts, including miRNAs, is markedly deregulated in cancerous prostate tissue. This study aimed at identifying a PCa-specific expression profile of miRNAs for subsequent use in noninvasive diagnostics. MATERIALS AND METHODS: MiRNA expression was profiled in 13 PCa tissues using human miRNA microarrays. Highly expressed miRNAs were selected for the analysis in urine of patients with PCa (N=143) and benign prostate hyperplasia (BPH; N=23) by means of real time PCR, while miRNAs showing the expression differences in relation to clinical variables were further analyzed in 52 PCa and 12 noncancerous prostate tissues (NPT) on TaqMan Low Density Arrays (TLDA). RESULTS: Analysis of miRNA expression in prostate tissue linked miR-95 to aggressive form of PCa. This miRNA was up-regulated in high grade (P=0.041), the TMPRSS2-ERG fusion-positive tumors (P=0.026), and in patients with subsequently developed biochemical recurrence (BCR; P=0.054) after radical prostatectomy. MiRNAs highly expressed in PCa tissues were also detectable in urine from PCa patients. Moreover, the urinary levels of miR-21 had significant discriminatory power (P=0.010) to separate PCa patients from BPH, while the combined analysis of urinary miR-19a and miR-19b was prognostic for BCR. In PCa, the diagnostic potential of urinary miRNA panel (miR-21, miR-19a, and miR-19b) was higher than that of the PSA test (AUC=0.738 vs. AUC=0.514). CONCLUSIONS: Measurement of urinary levels of PCa-specific miRNAs could assist in more specific detection of PCa and prediction of BCR.

[A Study of the Efficacy of the Additional Administration of Tadalafil in Japanese Men with α1-Blocker-Resistant LUTS/BPH].

Alpha 1-blockers are widely used at present for lower urinary tract symptoms (LUTS)/benign prostatic hyperplasia (BPH). However, some patients experience little improvement of symptoms, and it is difficult to provide additional treatment. We have additionally administered tadalafil to patients with inadequate symptom improvement, despite treatment with alpha-1 blockers. The subjects were 57 patients with a diagnosis of LUTS/BPH who showed a poor response to treatment with alpha-1 blockers for 1 month or more (international prostate symptom score [IPSS] ≥8 and/or quality of life [QOL] index ≥3). Tadalafil 5 mg was administered on consecutive days to patients orally receiving alpha-1 blockers. We determined IPSS, the QOL index, overactive bladder symptom scores (OABSS), maximum urine flow, residual urine volume, and the sexual health inventory for men (SHIM) before, and 4, 8, and 12 weeks after administration, and then evaluated improvement effects. IPSS, the QOL index, OABSS, and SHIM showed significant improvement (P ＜0.05) at 4 weeks after the start of treatment and onward. IPSS and the QOL index showed greater improvement effects at 8 and 12 weeks. Residual urinary volume was significantly improved only at 8 weeks. However, the maximum urine flow showed no improvement at any time point. Our results demonstrated the additional administration of tadalafil to patients with LUTS showing poor responses to alpha-1 blockers to improve LUTS/BPH symptoms as well as sexual function.

Urinary prostate protein glycosylation profiling as a diagnostic biomarker for prostate cancer.

BACKGROUND: Serum prostate-specific antigen (sPSA) measurement is widely used as opportunistic screening tool for prostate cancer (PCa). sPSA suffers from considerable sensitivity and specificity problems, particularly in the diagnostic gray zone (sPSA 4-10 µg/L). Furthermore, sPSA is not able to discriminate between poorly-, moderately-, and well-differentiated PCa. We investigated prostatic protein glycosylation profiles as a potential PCa biomarker. METHODS: Differences in total urine N-glycosylation profile of prostatic proteins were determined between healthy volunteers (n = 54), patients with benign prostate hyperplasia (BPH; n = 93) and newly diagnosed PCa patients (n = 74). Variations in N-glycosylation profile and prostate volume were combined into one urinary glycoprofile marker (UGM). Additionally, differences in N-glycosylation were identified between Gleason <7, =7, and >7. RESULTS: The UGM was able to discriminate BPH from PCa, overall and in the diagnostic gray zone (P < 0.001). The UGM showed comparable diagnostic accuracy to sPSA, but gave an additive diagnostic value to sPSA (P < 0.001). In the diagnostic gray zone the UGM performed significantly better than sPSA (P < 0.001). A significant difference was found in core-fucosylation of biantennary structures and overall core-fucosylation of multiantennary structures between Gleason < 7 and Gleason > 7 (P = 0.010 and P = 0.020, respectively) and between Gleason = 7 and Gleason > 7 (P = 0.011 and P = 0.025, respectively). CONCLUSIONS: The UGM shows high potential as PCa biomarker, particularly in the diagnostic gray zone. Further research is needed to validate these findings.

Effects of hexanic extract of Serenoa repens (Permixon® 160 mg) on inflammation biomarkers in the treatment of lower urinary tract symptoms related to benign prostatic hyperplasia.

BACKGROUND: Chronic prostatic inflammation (CPI) could be a cause of symptomatic or complicated benign prostatic hyperplasia (BPH). In previous in vitro and in vivo studies, Hexanic Extract of Serenoa repens (HESr) namely Permixon(®) has demonstrated potent anti-inflammatory properties. With the aim to provide new insight onto HESr anti-inflammatory properties in human we explore its effect on CPI biomarkers in men with lower urinary tract symptoms (LUTS) related to BPH using a non-invasive method and investigate links between biomarkers and clinical symptoms. METHODS: An international, randomized, double-blind, parallel-group, tamsulosin-controlled study was carried out in 206 men with BPH-related LUTS. Patients received oral daily HESr 320mg or tamsulosin 0.4 mg during 3 months. The first urine stream after digital rectal examination (DRE) was collected at Day 1 and Day 90 and mRNA was extracted from prostatic epithelial cells desquaming in the lumen of the glands and seminal plasma fluid after DRE. mRNA quantification of the 29 most significant published inflammation markers in BPH and protein detection in urine was performed. RESULTS: At D90, a decrease in mean gene expression was observed for 65.4% of the markers detected in the HESr group versus 46.2% in the tamsulosin group. In the 15 most frequently expressed genes, this difference was higher (80% vs. 33% respectively). Three proteins (MCP-1/CCL2, IP-10/CXCL10, and MIF) were detected. At D90, a decrease in the number of patients who expressed MCP-1/CCL2 and IP-10/CXCL10 was observed only in the HESr group. Moreover, MIF expression was significantly reduced by HESr compared with tamsulosin (P = 0.007). Finally, in contrast to tamsulosin, the subgroup of patients treated by HESr and who over expressed MIF at baseline, had a higher response to the International Prostate Symptom Score (I-PSS) than those who did not over express this protein (mean I-PSS change: -6.4 vs. -4.5 respectively). As the study is exploratory, results should be confirmed in a powered clinical study. CONCLUSIONS: These results showed for the first time at clinical level the anti-inflammatory properties of HESr, already indicated in BPH-related LUTS. Thus, HESr could be of interest to prevent unfavourable evolution in patients with CPI.

Comparison of mRNA, Protein, and Urinary Nucleic Acid Levels of S100A8 and S100A9 between Prostate Cancer and BPH.

BACKGROUND: Infections and inflammation in the prostate play a critical role in carcinogenesis, and S100A8 and S100A9 are key mediators in acute and chronic inflammation. Therefore, we investigated the differences of S100A8/A9 expression between prostate cancer (CaP) and benign prostatic hyperplasia (BPH) tissues, and we evaluated the possibilities of urinary nucleic acids of S100A8/A9 as diagnostic and prognostic markers. METHODS: Tissues from 132 CaP patients who underwent prostatectomy or transurethral resection and 90 BPH patients who underwent transurethral prostatectomy were assessed.sd In addition, S100A8 and S100A9 nucleic acid levels were measured in the urine of 283 CaP patients and 363 BPH controls. RESULTS: S100A8 and S100A9 mRNA levels were lower in CaP than BPH tissues (P < 0.001). S100A8 and S100A9 expression was increased in cancer tissues with poorer prognosis. In 69 specimens from prostatectomy patients, S100A8/A9 were the independent predictor of biochemical recurrence (hazard ratio 5.22, 95 % confidence interval 1.800-15.155, P = 0.002). Immunohistochemical staining revealed that BPH tissues stained more strongly for both S100A8 and S100A9 than CaP tissues (P < 0.001). S100A8 and S100A9 urinary nucleic acid levels were lower in CaP than in BPH (P = 0.001 and <0.001, respectively). CONCLUSIONS: S100A8/A9 levels are lower in CaP than in BPH. Both were more highly expressed in patients with aggressive disease and shorter biochemical recurrence-free time. S100A8/A9 urinary cell-free nucleic acid levels correlated positively with expression levels obtained from tissue staining. Therefore, S100A8/A9 measurement in tissues and urine may have diagnostic and prognostic value in CaP.