RNA-based urinary assays for non-muscle invasive bladder cancer.

PURPOSE OF REVIEW: To provide an overview of the recent literature on RNA-based molecular urine assays for the diagnosis and surveillance of non-muscle invasive bladder cancer (NMIBC). RECENT FINDINGS: Articles were eligible for inclusion if performance metrics sensitivity, specificity, and negative-predictive value (NPV) were reported or could be calculated. Only prospective studies published between 2020-2022 were included. Five out of fourteen studies addressed the primary diagnostic setting; the proportion of gross hematuria patients in all study populations was >50%. Only one study reported performance metrics within a microscopic hematuria subgroup. This study evaluated Xpert Bladder and reported a sensitivity: 73%, specificity: 84%, NPV: 99%, and PPV: 12%. Ten studies assessed test performance during surveillance for NMIBC. For the detection of high-grade (HG) and high-risk (HR) NMIBC, sensitivity, specificity, NPV, and PPV varied between 78-100%, 64-89%, 97.0-99.7%, and 9.2-39%. SUMMARY: Multiple RNA-based urine assays have been investigated for the detection of urothelial cancer in the primary or surveillance setting. However, studies included within this review have important limitations, hampering the interpretation of study results. As such, performance metrics should be interpreted with caution and further research is required to evaluate the clinical impact of RNA-based urine assays in daily practice.

Development of a Whole-urine, Multiplexed, Next-generation RNA-sequencing Assay for Early Detection of Aggressive Prostate Cancer.

BACKGROUND: Despite biomarker development advances, early detection of aggressive prostate cancer (PCa) remains challenging. We previously developed a clinical-grade urine test (Michigan Prostate Score [MiPS]) for individualized aggressive PCa risk prediction. MiPS combines serum prostate-specific antigen (PSA), the TMPRSS2:ERG (T2:ERG) gene fusion, and PCA3 lncRNA in whole urine after digital rectal examination (DRE). OBJECTIVE: To improve on MiPS with a novel next-generation sequencing (NGS) multibiomarker urine assay for early detection of aggressive PCa. DESIGN, SETTING, AND PARTICIPANTS: Preclinical development and validation of a post-DRE urine RNA NGS assay (Urine Prostate Seq [UPSeq]) assessing 84 PCa transcriptomic biomarkers, including T2:ERG, PCA3, additional PCa fusions/isoforms, mRNAs, lncRNAs, and expressed mutations. Our UPSeq model was trained on 73 patients and validated on a held-out set of 36 patients representing the spectrum of disease (benign to grade group [GG] 5 PCa). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The area under the receiver operating characteristic curve (AUC) of UPSeq was compared with PSA, MiPS, and other existing models/biomarkers for predicting GG ≥3 PCa. RESULTS AND LIMITATIONS: UPSeq demonstrated high analytical accuracy and concordance with MiPS, and was able to detect expressed germline HOXB13 and somatic SPOP mutations. In an extreme design cohort (n = 109; benign/GG 1 vs GG ≥3 PCa, stratified to exclude GG 2 cancer in order to capture signal difference between extreme ends of disease), UPSeq showed differential expression for T2:ERG.T1E4 (1.2 vs 78.8 median normalized reads, p < 0.00001) and PCA3 (1024 vs 2521, p = 0.02), additional T2:ERG splice isoforms, and other candidate biomarkers. Using machine learning, we developed a 15-transcript model on the training set (n = 73) that outperformed serum PSA and sequencing-derived MiPS in predicting GG ≥3 PCa in the held-out validation set (n = 36; AUC 0.82 vs 0.69 and 0.69, respectively). CONCLUSIONS: These results support the potential utility of our novel urine-based RNA NGS assay to supplement PSA for improved early detection of aggressive PCa. PATIENT SUMMARY: We have developed a new urine-based test for the detection of aggressive prostate cancer, which promises improvement upon current biomarker tests.

Reduction of oxidative stress on DNA and RNA in obese patients after Roux-en-Y gastric bypass surgery-An observational cohort study of changes in urinary markers.

Increased oxidative stress in obesity and diabetes is associated with morbidity and mortality risks. Levels of oxidative damage to DNA and RNA can be estimated through measurement of 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) in urine. Both markers have been associated with type 2 diabetes, where especially 8-oxoGuo is prognostic for mortality risk. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery that has considerable effects on bodyweight, hyperglycemia and mortality, might be working through mechanisms that reduce oxidative stress, thereby reducing levels of the urinary markers. We used liquid chromatography coupled with tandem mass spectrometry to analyze the content of 8-oxodG and 8-oxoGuo in urinary samples from 356 obese patients treated with the RYGB-procedure. Mean age (SD) was 44.2 (9.6) years, BMI was 42.1 (5.6) kg/m2. Ninety-six (27%) of the patients had type 2 diabetes. Excretion levels of each marker before and after surgery were compared as estimates of the total 24-hour excretion, using a model based on glomerular filtration rate (calculated from cystatin C, age, height and weight), plasma- and urinary creatinine. The excretion of 8-oxodG increased in the first months after RYGB. For 8-oxoGuo, a gradual decrease was seen. Two years after RYGB and a mean weight loss of 35 kg, decreased hyperglycemia and insulin resistance, excretion levels of both markers were reduced by approximately 12% (P < 0.001). For both markers, mean excretion levels were about 30% lower in the female subgroup (P < 0.0001). Also, in this subgroup, excretion of 8-oxodG was significantly lower in patients with than without diabetes. We conclude, that oxidative damage to nucleic acids, reflected in the excretion of 8-oxodG and 8-oxoGuo, had decreased significantly two years after RYGB-indicating that reduced oxidative stress could be contributing to the many long-term benefits of RYGB-surgery in obesity and type 2 diabetes.

Oncoprotein 18 is necessary for malignant cell proliferation in bladder cancer cells and serves as a G3-specific non-invasive diagnostic marker candidate in urinary RNA.

BACKGROUND: Urine-based diagnostics indicated involvement of oncoprotein 18 (OP18) in bladder cancer. In cell culture models we investigated the role of OP18 for malignant cell growth. METHODS: We analyzed 113 urine samples and investigated two human BCa cell lines as a dual model: RT-4 and ECV-304, which represented differentiated (G1) and poorly differentiated (G3) BCa. We designed specific siRNA for down-regulation of OP18 in both cell lines. Phenotypes were characterized by cell viability, proliferation, and expression of apoptosis-related genes. Besides, sensitivity to cisplatin treatment was evaluated. RESULTS: Analysis of urine samples from patients with urothelial BCa revealed a significant correlation of the RNA-ratio OP18:uroplakin 1A with bladder cancer. High urinary ratios were mainly found in moderately to poorly differentiated tumors (grade G2-3) that were muscle invasive (stage T2-3), whereas samples from patients with more differentiated non-invasive BCa (G1) showed low OP18:UPK1A RNA ratios. Down-regulation of OP18 expression in ECV-304 shifted its phenotype towards G1 state. Further, OP18-directed siRNA induced apoptosis and increased chemo-sensitivity to cisplatin. CONCLUSIONS: This study provides conclusive experimental evidence for the link between OP18-derived RNA as a diagnostic marker for molecular staging of BCa in non-invasive urine-based diagnostics and the patho-mechanistic role of OP18 suggesting this gene as a therapeutic target.

Methodology for the at-home collection of urine samples for prostate cancer detection.

Urine from patients with prostate cancer (PCa) contains gene transcripts that have been used for PCa diagnosis and prognosis. Historically, patient urine samples have been collected after a digital rectal examination of the prostate, which was thought necessary to boost the levels of prostatic secretions in the urine. We herein describe methodology that allows urine to be collected by patients at home and then posted to a laboratory for analysis. RNA yields and quality were comparable to those for post digital rectal examination urine, and there was improved sensitivity for the detection of TMPRSS2:ERG transcripts by RT-PCR. The At-Home collection protocol has opened up the potential to perform large-scale PCa studies without the inconvenience, cost, discomfort and expense of patients having to visit the clinic.

8-Hydroxyguanosine as a possible RNA oxidative modification marker in urine from colorectal cancer patients: Evaluation by ultra performance liquid chromatography-tandem mass spectrometry.

Oxidative RNA damage has been found to be associated with a variety of diseases, and 8-hydroxyguanosine (8-OHG) is a typical marker of oxidative modification of RNA. This guanosine modification is an emerging biomarker for disease detection and determination of 8-OHG in human urine is favored because it is noninvasive to patients. However, due to its poor ionization efficiency in mass spectrometry and trace amount in urine, accurate quantification of this modified nucleoside is still challenging. Herein, a rapid, accurate, sensitive and robust method using solid-phase extraction (SPE) combined with isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for detection of this oxidative RNA modification in human urine. The limit of detection can reach 1.5 fmol and the method exhibits good precision on intra-day (1.8-3.3%) and inter-day (0.6-1.2%) analyses. Satisfactory recovery (87.5-107.2%) at three spiked levels was achieved by using HLB cartridge for urine pretreatment. Using this method, we quantified 8-OHG in urine from 65 colorectal cancer (CRC) patients and 76 healthy volunteers. The measured level of urinary 8-OHG for CRC patients and healthy controls is 1.91 ± 0.63 nmol/mmol creatinine and 1.33 ± 0.35 nmol/mmol creatinine, respectively. We found the content of 8-OHG in urine was raised in CRC patients patients, implying this oxidative RNA modification marker could act as a potential noninvasive indicator for early screening of CRC. In addition, this study will make contributions to the investigations of the influences of oxidative stress on the formation and development of CRC.

Urine Gene Expression Profiles in Bladder Pain Syndrome Patients Treated with Triamcinolone.

BACKGROUND: The pathogenesis of bladder pain syndrome (BPS) remains incompletely defined, and there is no standard treatment for BPS as yet. OBJECTIVE: To gain detailed insight into the disease pathobiology of BPS through comparative gene expression analysis of urine from BPS patients versus control individuals and, furthermore, to determine the efficacy of triamcinolone treatment in BPS patients in terms of the gene expression profiles in urine. DESIGN, SETTING, AND PARTICIPANTS: A prospective pilot study including 21 urine samples from patients with Hunner's lesions (n=6) and controls (n=9) between January and August 2017. INTERVENTION: Triamcinolone treatment of BPS patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Urine samples from BPS patients were collected before (pretreatment group) and 2 wk after triamcinolone treatment (post-treatment group). Gene expression of urine sediment was analyzed using RNA sequencing. Pathways and biological processes in which differentially expressed genes are involved were analyzed. RESULTS AND LIMITATIONS: A total of 3745 genes were found to be differentially expressed between the three groups tested. Gene expression differences between controls and BPS samples (630 differentially expressed genes) were more pronounced than the differences between pre- and post-treatment BPS samples (197 differentially expressed genes). Gen Set Enrichment Analysis showed that differentially expressed genes in BPS patients (pretreatment), compared with controls, were enriched for some functional gene networks associated with several metabolic processes and ribosome biogenesis. The limited number of patients included may not accurately represent the BPS population. CONCLUSIONS: Gene expression profiles of urine sediment are able to discriminate between BPS and control patients. Moreover, we show that triamcinolone induces changes in urine gene expression profiles. PATIENT SUMMARY: In this report, we looked at gene expression profiles of urine sediment from patients with Hunner's lesions, before and after triamcinolone treatment, and control individuals. We found that urine gene expression profiles are able to discriminate Hunner's lesions patients from controls. Furthermore, we report, for the first time, that triamcinolone treatment of patients with Hunner's lesions induces changes in bladder gene expression profiles that can be observed in urine samples.

Urinary nucleic acid oxidation product levels show differential associations with pharmacological treatment in patients with type 2 diabetes.

The relationship between RNA and DNA oxidation and pharmacological treatment has not been systematically investigated in patients with type 2 diabetes (T2D). We aimed to investigate the association between pharmacological treatments and levels of urinary markers of nucleic acid oxidation in T2D patients. Vejle Diabetes Biobank cohort data was nested into nationwide registry data. Multiple logistic regression was used to associate drug usage with risk of high (above median) RNA and DNA oxidation. Data from 2664 T2D patients (64% male, age range: 25-75) were included. Questionnaire-validated lipid lowering drug use was associated with low RNA oxidation (Odds ratio, OR 0.71, 95% CI: [0.59-0.87]). Insulin and non-specific antidiabetic drugs were associated with low DNA oxidation (insulin: OR 0.60, 95% CI [0.49-0.73]). Oral antidiabetics were associated with high DNA oxidation and RNA oxidation (OR 1.30, 95% CI [1.10-1.53] and OR 1.26, 95% CI [1.07-1.29]). Our findings indicate that diabetes-related drugs are associated with RNA and DNA oxidation and further studies are required to determine causality in T2D patients.

Urine RNA Processing in a Clinical Setting: Comparison of 3 Protocols.

OBJECTIVE: The objective of this study was to compare quantitative and qualitative RNA extraction results from clinical voided urine samples between 3 commercially available extraction protocols. METHODS: For phase 1, fresh voided urine samples from 10 female subjects were collected and processed in clinic and transported to the laboratory with cold packs. RNA was purified with 1 of 3 RNA extraction protocols: (1) TRI Reagent Protocol; (2) Absolutely RNA Nanoprep Kit; and (3) ZR Urine RNA Isolation Kit. Real-time polymerase chain reactions (RT-PCR) were performed. As the ZR Urine RNA Isolation Kit provided the highest quality RNA in phase 1, for phase 2, RNA was extracted from 9 additional voided urine specimens using this kit to perform additional qualitative analyses. RESULTS: Median RNA yield was significantly higher with the TRI Reagent Protocol as compared with the other protocols (P = 0.007). However, there was a significantly lower median threshold cycle value from polymerase chain reaction (indicating improved downstream application performance) with the ZR Urine RNA Isolation Kit as compared with the other methods (P = 0.005). In phase 2, the median RNA integrity number of urine RNA was 2.5 (range, 1.6-5.9). CONCLUSIONS: Although other methods may provide a higher quantity of RNA, when using clinical urine samples, the ZR Urine RNA Isolation Kit provided the highest quality of extracted RNA. This kit is especially attractive for the clinical setting because it does not require an initial centrifugation step. The urine RNA obtained with this kit may be useful for polymerase chain reaction but is not likely to be of high enough integrity for RNA sequencing.

RNA oxidation and iron levels in patients with diabetes.

AIM: The urinary biomarker for oxidative stress to RNA, 8-oxo-7,8-dihydro-guanosine (8-oxoGuo) is associated with mortality in patients with type 2 diabetes. Iron has also been linked to diabetes. In individuals with untreated hereditary iron overload it has been observed that 8-oxoGuo was higher compared to controls. In the current study, we hypothesized that 8-oxoGuo was associated with diagnosis of diabetes, and that iron confounded this association. METHODS: Participants from a general Danish population were included in the study (n = 3567). UPLC-MS/MS method was used for 8-oxoGuo (nmol/mmol creatinine) measurement in spot urine. Iron biomarkers included total plasma iron, ferritin, transferrin saturation (TS) and transferrin. RESULTS: 8-oxoGuo was 17% higher in diabetes patients (n = 208) compared to non-diabetes controls. Unadjusted logistic regression model showed an odds ratio of diabetes of 1.38 (95%CI:1.21-1.57, P < 0.0001) per unit increase of 8-oxoGuo. When the model was adjusted for possible confounders the odds ratio was 1.09 (95%CI:0.94-1.26, P = 0.24). When additional adjustment was performed including ferritin, TS, or transferrin, respectively, the OR were 1.14 (95%CI:0.97-1.33, P = 0.09), 1.10 (95%CI: 0.95-1.28, P = 0.18), and 1.17 (95%CI:1.01-1.38, P = 0.04). CONCLUSIONS: Our study indicates that 8-oxoGuo is higher in diabetes patients. The lack of association between 8-oxoGuo and diabetes in the adjusted model may be due to the cross-sectional design including post-treatment bias. Our data did not show consistent effect of all iron biomarkers in relation to diabetes. Most likely, the iron biomarkers were affected by inflammation thus not reflecting true iron levels.

"Mix-to-Go" Silver Colloidal Strategy for Prostate Cancer Molecular Profiling and Risk Prediction.

Molecular profiling via analysis of multiple disease biomarkers is a powerful tool for disease diagnosis and risk prediction. Due to simplicity and minimal instrumentation requirement, colloidal-based colorimetric DNA/RNA assays are attractive for driving molecular profiling toward widespread clinical usage. Still, the reliability and speed of current colorimetric assays need to be further improved upon for eventual clinical use. Herein, we propose a "mix-to-go" colloidal strategy that utilizes the electrostatic attraction between negatively charged target sequences and positively charged silver nanoparticles (AgNPs) to induce aggregation of AgNPs to profile a panel of clinically validated urinary prostate cancer (PCa) RNA biomarkers ( TMPRSS2:ERG, T2:ERG; prostate cancer antigen 3, PCA3; and kallikrein-related peptidase 2, KLK2). Our strategy is unique in inducing a rapid (10 s), unambiguous solution color change in the presence of target sequences, without the additional NP aggregation agents that are used in existing electrostatic-mediated aggregation assays. Our strategy is analytically specific and sensitive for the detection of 105 and 104 target copies by the naked eye and UV-vis spectrophotometry, respectively. Analytical accuracies of our strategy in detecting T2:ERG, PCA3, and KLK2 RNA biomarkers were 95.9%, 97.3%, and 100%, respectively, as validated by quantitative reverse transcription-polymerase chain reaction. To further evaluate clinical molecular profiling performance beyond conventional proof-of-concept demonstration, we tested our colloidal strategy for noninvasive PCa risk prediction of 73 patients, using the urinary RNA biomarker panel comprising of T2:ERG, PCA3, and KLK2. We found that elevated T2:ERG and PCA3 levels were positively associated with high-risk PCa and obtained a corresponding area-under-the-curve values of 0.790 and 0.833 for predicting PCa and high-risk PCa on biopsy, respectively. We believe our "mix-to-go" strategy may serve as a reliable and accessible Ag colloidal-based molecular profiling approach for clinical applications.

Indicator of RNA oxidation in urine for the prediction of mortality in patients with type 2 diabetes and microalbuminuria: A post-hoc analysis of the Steno-2 trial.

OBJECTIVE: The RNA oxidation product, 8-oxo-7,8-dihydroguanosine (8-oxoGuo), has been associated with mortality in patients with type 2 diabetes (T2D). However, the identification and the potential effect of approved treatments decreasing urine 8-oxoGuo level remain unraveled. In the Steno-2 study intensified multifactorial treatment compared with conventional multifactorial treatment reduced mortality in T2D patients with microalbuminuria by 45%. We assessed association between 8-oxoGuo at advanced baseline and total mortality with up to 19.9 years follow-up and from end of intervention to end of follow-up up to (up to 13.9 years). MATERIALS AND METHODS: In 1993, 160 T2D patients with microalbuminuria were included in the Steno-2 trial. Urine samples from baseline were not available, but samples were available from 155 patients (97%) in 1995 (advanced baseline) and from 125 patients (96%) in 2001 (end of intervention). Hazard ratios (HR) for log2-transformed 8-oxoGuo and dichotomized (cut-off at median; low vs. high RNA oxidation) were estimated using Cox regressions. RESULTS: During follow-up of 19.9 years after advanced baseline, 89 died and no association between 8-oxoGuo and mortality was found (p = 0.40). From the end of 7.8 years of intervention and during remaining 13.9 years of observation, 61 died and doubling the urine 8-oxoGuo level was associated with mortality with a HR 3.08 (95% CI [1.86 -5.12]; p < 0.001) after multiple adjustments. Patients with low 8-oxoGuo in the intensified-treatment had the lowest risk of dying compared with high 8-oxoGuo in the conventional-treatment both from advanced baseline onwards, adjusted HR 0.40 (95% CI [0.21 -0.75]; p = 0.004), and from end of intervention onwards, adjusted HR 0.28 (95% CI [0.13 -0.61]; p = 0.001). CONCLUSIONS: In T2D patients with microalbuminuria, high levels of urine 8-oxoGuo after 7.8 years of multifactorial intervention was associated with higher mortality during 13.9 years of post-trial follow-up. Patients with low 8-oxoGuo in the intensified treatment group had the lowest risk of dying.

Diagnostic value of urinary survivin as a biomarker for bladder cancer: a systematic review and meta-analysis of published studies.

OBJECTIVE: This study is a meta-analysis and aims to determine the value of urinary survivin for detecting bladder cancer (BC) on the basis of preceding statistical performance and to compare their diagnostic value. MATERIALS AND METHODS: Considering that the urinary survivin data were from both RNA and protein levels, the key words "bladder cancer" AND "survivin" and "bladder cancer" AND "survivin RNA" were used; and PubMed, Web of Science, and Cochrane Library were systematically searched to identify relevant articles. The methodological quality of each study was assessed by QUADAS-2. Data were analyzed by STATA 12.0 and Meta-disc v.1.4 software package. A random-effects model was used and subgroup analysis was carried out to identify possible sources of heterogeneity. RESULTS: Nine articles for survivin protein test with 789 patients and 684 controls, and 12 articles for survivin RNA test with 880 patients and 922 controls were identified. The results showed that the pooled sensitivity was 0.79 (95% CI 0.73, 0.84), specificity was 0.87 (95% CI 0.79, 0.92) of the survivin protein test for bladder cancer, and the sensitivity and specificity was 0.84 (95% CI 0.79, 0.88) and 0.94 (95% CI 0.89, 0.97) of the survivin RNA test. The AUC of the two approaches was 0.89 (95% CI 0.86, 0.91) and 0.94 (95% CI 0.92, 0.96), respectively. CONCLUSIONS: The survivin protein and survivin RNA both had great potential as biomarkers for BC detection, and survivin RNA showed higher accuracy than survivin protein on BC diagnosis.

Increased oxidative damage of RNA in liver injury caused by hepatitis B virus (HBV) infection.

To evaluate the urinary levels of 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) in liver injury patients with hepatitis B virus (HBV) infection and to explore the relationship between urinary 8-oxo-dGsn or 8-oxo-Gsn and degree of liver damage. We enrolled 138 liver injury patients with HBV infection and 169 age- and sex-matched healthy controls in this study. A sensitive and accurate isotope-diluted liquid chromatograph mass spectrometer/mass spectrometer (LC-MS/MS) method was used to measure the urinary levels of 8-oxo-Gsn and 8-oxo-dGsn. Simultaneously, pathological analysis of liver biopsy tissues was carried out, and immunohistochemistry was carried out for 8-oxo-Guo, 8-oxo-dGuo and MTH1 protein in some liver injury tissues. We analysed the correlation between the degrees of inflammation and fibrosis and levels of 8-oxo-Gsn and 8-oxo-dGsn. We also analysed the levels of urinary 8-oxo-Gsn and 8-oxo-dGsn with clinical data of HBeAg, HBsAg, and HBV genotype and detected the levels of plasma aspartate aminotransferase, alanine aminotransferase (AST), platelet, alkaline phosphatase, prothrombin time (PT) and HBV DNA, and calculated the aspartate amino transferase-to-platelet ratio index (APRI) score. Nonparametric correlations were used to evaluate the correlation between 8-oxo-Gsn, 8-oxo-dGsn or APRI and various laboratory biochemical indicators. Results showed that the levels of urinary 8-oxo-Gsn and 8-oxo-dGsn in patients with liver injury were significantly higher than those of healthy controls (both p < .001). Urinary 8-oxo-Gsn was significantly associated with AST, APRI and PT (p = .013, p = .026 and p = .049). The receiver operating characteristic curves of 8-oxo-Gsn were 0.696 (0.632-0.759) and 0.731 (0.672-0.790) for inflammatory activity and fibrosis, respectively. Patients with higher levels of urinary 8-oxo-Gsn are more likely to have a high degree of fibrosis and urinary 8-oxo-Gsn may have a great potential in assessing liver fibrosis.

Markers of DNA/RNA damage from oxidation as predictors of a registry-based diagnosis of psychiatric illness in type 2 diabetic patients.

Oxidative stress is a potential biological mediator of the higher rates of psychiatric illness (PI) observed after the onset of type 2 diabetes (T2DM). We investigated validated urinary markers of systemic DNA/RNA damage from oxidation (8-oxodG/8-oxoGuo respectively) as predictors of incident PI in a cohort of 1381 newly diagnosed T2DM patients, who were followed prospectively for a total of 19 years after diagnosis. Psychiatric diagnoses were from Danish national registries. Patients were examined at the time of diagnosis and at a 6-year follow-up. At baseline, 8-oxodG was slightly lower in PI vs. non-PI patients, while at 6-year follow-up, 8-oxoGuo was significantly higher in PI patients. Using Cox proportional hazard models, we found that higher levels of 8-oxodG at 6-year follow-up significantly predicted lower incidence of PI after the adjustment for confounders. In a subgroup analysis, this association was most predominant in minor PIs (unipolar depression and anxiety) compared to major PIs such as schizophrenia and bipolar disorder. These observations indicate that higher levels of systemic oxidative stress are not associated with a higher risk of PI after T2DM onset. Only PI patients treated in hospital care were included in the registries, and the conclusion thus only applies to these individuals.

Quantitative RNA Analysis from Urine Using Real Time PCR.

Urine is emerging as a biological fluid suitable to perform liquid biopsy in a minimally invasive manner, a fundamental attribute for prevention and early detection of cancer. Urine biomarkers can be analyzed in voided urine, in urine sediment, and urine supernatant. In the case of urothelial carcinoma, in which tumor cells are in direct contact with urine, the assessment of the levels of biomarkers in the urinary cell fraction appears to be the most promising approach to identify diagnostic and prognostic biomarkers in a noninvasive way. Here, we describe a protocol to collect and process urine samples to obtain urinary exfoliated cells. Furthermore, we describe the methodology to isolate RNA from urinary cells and to quantify gene expression levels from these urinary cells.

Urinary RNA-based biomarkers for prostate cancer detection.

Prostate cancer (PCa) is the commonest malignancy in the male population worldwide. Serum prostate specific antigen (PSA) test is the most important biomarker for the detection, follow-up and therapeutic monitoring of PCa. Defects in PSA specificity have elicited research for new biomarkers to improve early diagnosis and avoid false-positive results. This review evaluates urinary RNA-based biomarkers. Urine is a versatile body fluid for non-invasive biomarker detection in case of urological malignancies. The importance of RNA-based biomarkers has been demonstrated by the current use of PCA3, a long non coding RNA biomarker already approved by the Food and Drugs Administration. Through the years, other urinary RNA biomarkers have been evaluated, including the well-known TMPRSS2:ERG transcript, as well as many messenger RNAs, long non coding RNAs and micro-RNA. Validation of a specific urinary RNA-based marker or an algorithm of different biomarkers levels as diagnostic markers for PCa could be useful to avoid unnecessary prostate biopsies.

Association Between Combined TMPRSS2:ERG and PCA3 RNA Urinary Testing and Detection of Aggressive Prostate Cancer.

IMPORTANCE: Potential survival benefits from treating aggressive (Gleason score, ≥7) early-stage prostate cancer are undermined by harms from unnecessary prostate biopsy and overdiagnosis of indolent disease. OBJECTIVE: To evaluate the a priori primary hypothesis that combined measurement of PCA3 and TMPRSS2:ERG (T2:ERG) RNA in the urine after digital rectal examination would improve specificity over measurement of prostate-specific antigen alone for detecting cancer with Gleason score of 7 or higher. As a secondary objective, to evaluate the potential effect of such urine RNA testing on health care costs. DESIGN, SETTING, AND PARTICIPANTS: Prospective, multicenter diagnostic evaluation and validation in academic and community-based ambulatory urology clinics. Participants were a referred sample of men presenting for first-time prostate biopsy without preexisting prostate cancer: 516 eligible participants from among 748 prospective cohort participants in the developmental cohort and 561 eligible participants from 928 in the validation cohort. INTERVENTIONS/EXPOSURES: Urinary PCA3 and T2:ERG RNA measurement before prostate biopsy. MAIN OUTCOMES AND MEASURES: Presence of prostate cancer having Gleason score of 7 or higher on prostate biopsy. Pathology testing was blinded to urine assay results. In the developmental cohort, a multiplex decision algorithm was constructed using urine RNA assays to optimize specificity while maintaining 95% sensitivity for predicting aggressive prostate cancer at initial biopsy. Findings were validated in a separate multicenter cohort via prespecified analysis, blinded per prospective-specimen-collection, retrospective-blinded-evaluation (PRoBE) criteria. Cost effects of the urinary testing strategy were evaluated by modeling observed biopsy results and previously reported treatment outcomes. RESULTS: Among the 516 men in the developmental cohort (mean age, 62 years; range, 33-85 years) combining testing of urinary T2:ERG and PCA3 at thresholds that preserved 95% sensitivity for detecting aggressive prostate cancer improved specificity from 18% to 39%. Among the 561 men in the validation cohort (mean age, 62 years; range, 27-86 years), analysis confirmed improvement in specificity (from 17% to 33%; lower bound of 1-sided 95% CI, 0.73%; prespecified 1-sided P = .04), while high sensitivity (93%) was preserved for aggressive prostate cancer detection. Forty-two percent of unnecessary prostate biopsies would have been averted by using the urine assay results to select men for biopsy. Cost analysis suggested that this urinary testing algorithm to restrict prostate biopsy has greater potential cost-benefit in younger men. CONCLUSIONS AND RELEVANCE: Combined urinary testing for T2:ERG and PCA3 can avert unnecessary biopsy while retaining robust sensitivity for detecting aggressive prostate cancer with consequent potential health care cost savings.

The urinary microbiome and its contribution to lower urinary tract symptoms; ICI-RS 2015.

AIMS: The microbiome is the term used for the symbiotic microbial colonisation of healthy organs. Studies have found bacterial identifiers within voided urine which is apparently sterile on conventional laboratory culture, and accordingly there may be health and disease implications. METHODS: The International Consultation on Incontinence Research Society (ICI-RS) established a literature review and expert consensus discussion focussed on the increasing awareness of the urinary microbiome, and potential research priorities. RESULTS: The consensus considered the discrepancy between findings of conventional clinical microbiology methods, which generally rely on culture parameters predisposed towards certain "expected" organisms. Discrepancy between selective culture and RNA sequencing to study species-specific 16S ribosomal RNA is increasingly clear, and highlights the possibility that protective or harmful bacteria may be overlooked where microbiological methods are selective. There are now strong signals of the existence of a "core" urinary microbiome for the human urinary tract, particularly emerging with ageing. The consensus reviewed the potential relationship between a patient's microbiome and lower urinary tract dysfunction, whether low-count bacteriuria may be clinically significant and mechanisms which could associate micro-organisms with lower urinary tract symptoms. CONCLUSIONS: Key research priorities identified include the need to establish the scope of microbiome across the range of normality and clinical presentations, and gain consensus on testing protocols. Proteomics to study enzymatic and other functions may be necessary, since different bacteria may have overlapping phenotype. Longitudinal studies into risk factors for exposure, cumulative risk, and emergence of disease need to undertaken. Neurourol. Urodynam. 36:850-853, 2017. © 2017 Wiley Periodicals, Inc.

Clinical Utility of Cxbladder for the Diagnosis of Urothelial Carcinoma.

INTRODUCTION: This study aimed to demonstrate the clinical utility of non-invasive multigene Cxbladder urine tests in reducing the overall number of diagnostic tests and invasive procedures used in the clinical evaluation of patients presenting with microhematuria, a key symptom of urothelial carcinoma (UC). There is a belief that using non-invasive molecular diagnostic tests in patients with hematuria may lead to patients undergoing unnecessary and costly invasive procedures that can cause adverse events and decrease patient quality of life. The objective of this study was to determine whether or not this was the case, using Cxbladder. METHODS: Data from 396 patient-by-urologist interactions generated 792 decision points from a standardized cohort of 33 patients evaluated by 12 urologists. Participant physicians recommended a selection of tests and procedures based on referral data, then reviewed and amended their recommendations in the context of diagnostic information from Cxbladder used in the Triage and Triage and Detect clinical modalities. RESULTS: All urologists changed their diagnostic behavior in at least one patient case with the addition of Cxbladder results. The total number of diagnostic procedures was reduced by 5% and 25% following disclosure of results from Cxbladder in the Triage and the Triage and Detect modalities, respectively. The total number of requested invasive procedures was reduced from 425 at referral to 379 (-11%) and 292 (-31%) following disclosure of Cxbladder information in the Triage and Triage and Detect modalities, respectively. CONCLUSIONS: Urologists made compelling changes to their clinical decision-making when they were provided with Cxbladder results for patients presenting with hematuria. Cxbladder provides an increase in clinical utility by focusing the use of invasive diagnostic procedures to appropriate patients, reducing both the total number and number of invasive procedures used in the clinical management of patients with hematuria, thereby improving the diagnostic experience and outcomes for patients. FUNDING: Pacific Edge Ltd.