Urine as liquid gold: the transcriptional landscape of acute rejection defined by urinary cell mRNA profiling of kidney allograft recipients.

PURPOSE OF REVIEW: Because all functioning nephrons contribute to urine formation, we reasoned that urine would be a suitable substitute to kidney allograft biopsy to discern human kidney allograft status. In view of compelling data that ribonucleic acid (RNA) sequencing outperforms microarray-based profiling, we performed RNA sequencing of urinary cells and kidney allograft biopsies to define the transcriptional landscape of allograft rejection. RECENT FINDINGS: Whole genome transcriptome profiling identified unique and shared gene signatures of acute T cell mediated rejection (TCMR) and antibody mediated rejection (AMR). We found that biopsy rejection signatures are enriched in urinary cells and that the immune cellular landscape is more diverse and enriched in urine compared to biopsies. Towards a patient friendly protocol for urinary cell messenger RNA (mRNA) profiling, we developed a filtration-based protocol for the initial processing of urine at home and demonstrated excellent performance characteristics of the filter- based protocol. SUMMARY: Acute rejection signatures are enriched in urinary cells. Urinary cell mRNA profiles are diagnostic and prognostic of acute rejection and could serve as yardsticks of in-vivo immune status. RNA sequencing yields insights into mechanisms of rejection and helps prioritize therapeutic targets. The filtration protocol for home processing of urine may optimize immune surveillance.

Predictive urinary RNA biomarkers of kidney injury after extracorporeal shock wave lithotripsy.

BACKGROUND: Extracorporeal shock wave lithotripsy (ESWL) is considered one of the best choices for the treatment of various kinds of urinary tract calculi, although it might cause acute kidney injury. OBJECTIVE: To measure the urinary long non-coding RNA-messenger RNA (LncRNA-mRNA) panel before and after ESWL to evaluate post-ESWL renal injury in a reliable and non-invasive method. PATIENTS AND METHODS: The study included 60 patients with renal stones treated with ESWL and 30 healthy volunteers. Voided urine samples were obtained before, 2 h, and 1 day after ESWL. We measured the urinary level of LncRNA (SBF2-AS1, FENDRR-19) and mRNA (GBP1, NLRP3) by real-time qPCR and compared the results with serum creatinine and eGFR. RESULTS: LncRNA (SBF2-AS1, FENDRR-19) and mRNA (GBP1, NLRP3) levels were higher in patients with renal stones when compared with healthy volunteers. They showed a statistically significant increase in the level of LncRNA-mRNA panel in baseline and after ESWL treatment. CONCLUSION: LncRNA (SBF2-AS1, FENDRR-19) and mRNA (GBP1, NLRP3) levels were significantly elevated following ESWL treatment, highlighting the usefulness of urinary biomarkers in identifying patients at higher risk of developing renal injury after ESWL treatment.

Toll-like receptor inflammatory cascade and the development of diabetic kidney disease in children and adolescents with type 1 diabetes.

AIM: This study aimed to evaluate the association of toll-like receptor (TLR) inflammatory cascade with the development of diabetic kidney disease (DKD) in children and adolescents with type 1 diabetes (T1D). METHODS: A total of 49 T1D patients and 49 normoglycaemic (NG) subjects aged 5-20 years old were recruited. TLR2, TLR4, MYD88, NFKB, MCP1/CCL2 and IL18 mRNA expressions were measured in peripheral blood mononuclear cells by reverse transcription-quantitative polymerase chain reaction. Fasting glucose, glycated haemoglobin, serum urea, serum creatinine and urinary albumin-to-creatinine ratio (ACR) were determined. RESULTS: The mRNA expressions of TLR2, TLR4, MYD88 and NFKB were significantly increased in the T1D group compared with the NG group. The mRNA expression levels of MCP1/CCL2 and IL18 were higher in 21 T1D patients (42.9%) (average of MCP1/CCL2: 6.6-fold and IL18: 5.8-fold) than in NG patients. Furthermore, ACR was increased in the T1D group compared with the NG group. CONCLUSION: The increased mRNA expression of TLR2, TLR4, MYD88, NFKB, MCP1/CCL2 and IL18 favours the development of an inflammatory process that may lead to a decline in renal function and consequently DKD in children and adolescents with T1D. This suggests that these genes are early mediators of onset DKD since the beginning of the lives of the paediatric T1D patients.

Urinary mRNA Signatures as Predictors of Renal Function Decline in Patients With Biopsy-Proven Diabetic Kidney Disease.

The clinical manifestations of diabetic kidney disease (DKD) are more heterogeneous than those previously reported, and these observations mandate the need for the recruitment of patients with biopsy-proven DKD in biomarker research. In this study, using the public gene expression omnibus (GEO) repository, we aimed to identify urinary mRNA biomarkers that can predict histological severity and disease progression in patients with DKD in whom the diagnosis and histologic grade has been confirmed by kidney biopsy. We identified 30 DKD-specific mRNA candidates based on the analysis of the GEO datasets. Among these, there were significant alterations in the urinary levels of 17 mRNAs in patients with DKD, compared with healthy controls. Four urinary mRNAs-LYZ, C3, FKBP5, and G6PC-reflected tubulointerstitial inflammation and fibrosis in kidney biopsy and could predict rapid progression to end-stage kidney disease independently of the baseline eGFR (tertile 1 vs. tertile 3; adjusted hazard ratio of 9.68 and 95% confidence interval of 2.85-32.87, p < 0.001). In conclusion, we demonstrated that urinary mRNA signatures have a potential to indicate the pathologic status and predict adverse renal outcomes in patients with DKD.

Detection of Urinary Exosomal HSD11B2 mRNA Expression: A Useful Novel Tool for the Diagnostic Approach of Dysfunctional 11ß-HSD2-Related Hypertension.

Objective: Apparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5αTHF/THE) or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C>G genotype (corresponding to a 221 A>G substitution) in patients with AME and essential hypertension (EH). Aim of the Study: To detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C>G genotype, and hypertension. Methods: In this observational case-control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (ß-2 microglobulin) gene was selected as the reference housekeeping gene. Results: Among family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels (median 169, range 118-220 copies/µl) that progressively decreased in 221 AG heterozygous with hypertension (108, range 92-124 copies/µl), 221 AG heterozygous normotensives (23.35, range 8-38.7 copies/µl), and wild-type 221 AA subjects (5.5, range 4.5-14 copies/µl). Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (p < 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression. Conclusions: HSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C>G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension.

Longitudinal follow-up and performance validation of an mRNA-based urine test (Xpert® Bladder Cancer Monitor ) for surveillance in patients with non-muscle-invasive bladder cancer.

OBJECTIVE: To evaluate the performance of the Xpert Bladder Cancer Monitor (Xpert; Cepheid, Sunnyvale, CA, USA) test as a predictor of tumour recurrence in patients with non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Patients (n = 429) undergoing surveillance for NMIBC underwent Xpert, cytology, and UroVysion testing. Patients with a positive Xpert and a negative cystoscopy result (positive-negative [PN] group, n = 66) and a control group of double negative patients (negative Xpert and cystoscopy results [NN] group) were followed for 12 months (±90 days). RESULTS: Histology-confirmed recurrences were detected in 58 patients (13.5%). Xpert had an overall sensitivity of 60.3% and a specificity of 76.5%. The sensitivity for high-grade (HG) cancer was 87% with a negative predictive value (NPV) of 99%. Urine cytology showed an overall sensitivity of 23.2% (47.6% sensitivity for HG tumours) and a specificity of 88.3%. In the PN group, 32% (n = 21) developed a recurrence within 12 months, 11 of which were HG tumours. In the NN control group, 14% (n = 9) developed a recurrence and only two were HG tumours. The hazard ratio for developing recurrence in the PN group was 2.68 for all tumours and 6.84 for HG cancer. CONCLUSIONS: The Xpert test has a high sensitivity for detecting the recurrence of cancer and a high NPV for excluding HG cancer. In addition, the data suggest that patients with a positive Xpert assay in the setting of negative cystoscopy are at high risk for recurrence and need close surveillance.

Urinary Cell mRNA Profiles Predictive of Human Kidney Allograft Status.

Immune monitoring of kidney allograft recipients and personalized therapeutics may help reach the aspirational goal of "one transplant for life." The invasive kidney biopsy procedure, the diagnostic tool of choice, has become safer and the biopsy classification more refined. Nevertheless, biopsy-associated complications, interobserver variability in biopsy specimen scoring, and costs continue to be significant concerns. The dynamics of the immune repertoire make frequent assessments of allograft status necessary, but repeat biopsies of the kidney are neither practical nor safe. To address the existing challenges, we developed urinary cell mRNA profiling and investigated the diagnostic, prognostic, and predictive accuracy of absolute levels of a hypothesis-based panel of mRNAs encoding immunoregulatory proteins. Enabled by our refinements of the PCR assay and by investigating mechanistic hypotheses, our single-center studies identified urinary cell mRNAs associated with T cell-mediated rejection, antibody-mediated rejection, interstitial fibrosis and tubular atrophy, and BK virus nephropathy. In the multicenter National Institutes of Health Clinical Trials in Organ Transplantation-04, we discovered and validated a urinary cell three-gene signature of T-cell CD3 ε chain mRNA, interferon gamma inducible protein 10 (IP-10) mRNA, and 18s ribosomal RNA that is diagnostic of subclinical acute cellular rejection and acute cellular rejection and prognostic of acute cellular rejection and graft function. The trajectory of the signature score remained flat and below the diagnostic threshold for acute cellular rejection in the patients with no rejection biopsy specimens, whereas a sharp rise was observed during the weeks before the biopsy specimen that showed acute cellular rejection. Our RNA sequencing and bioinformatics identified kidney allograft biopsy specimen gene signatures of acute rejection to be enriched in urinary cells matched to acute rejection biopsy specimens. The urinary cellular landscape was more diverse and more enriched for immune cell types compared with kidney allograft biopsy specimens. Urinary cell mRNA profile-guided clinical trials are needed to evaluate their value compared with current standard of care.

Can repeat biopsy be skipped after initial complete resection of T1 bladder cancer? The role of a novel urinary mRNA biomarker.

OBJECTIVES: To prospectively investigate the role of a urinary mRNA biomarker (Xpert Test) after initial complete resection of T1 bladder cancer (BC) for the prediction of positive repeat biopsy for malignancy. METHODS: Patients who underwent TURBT for NMIBC between September 2018 and April 2020 were included. Patients with benign pathology, incomplete resection, concomitant CIS/upper tract urothelial tumor or muscle invasive BC, were excluded. 2 to 6 weeks after primary TURBT, voided urine sample was retrieved for Xpert analysis and patients were scheduled for repeat biopsy. The primary outcome was to determine the role of positive Xpert test to predict positive repeat biopsy for malignancy. RESULTS: During the study period, 254 patients met the study inclusion criteria of which 61 (24%) patients had recurrent NMIBC. Complete resection was censured by the presence of detrusor muscle in the specimen with documented T1 disease in all study participants. Xpert test was positive in 128 patients; of whom 85 (66.4%) showed positive repeat biopsy (HR=6.2, 95%CI=3.46-9.4, P = 0.002). The sensitivity, specificity, positive and negative predictive values of Xpert test for repeat biopsy were 85.9% (95%CI: 82-89), 72.3% (95%CI: 68-76), 66.4% (95%CI: 62-71) and 88.9% (95%CI: 85-94), respectively. On median (range) follow up of 12(3-25) months, tumor recurrence was encountered in 84 (35%) patients. On multivariate Cox regression analysis, Xpert test was significantly associated with tumor recurrence (HR= 9.7, 95%CI=5-18, P <0.001). CONCLUSIONS: Positive Xpert test after primary complete resection of T1 BC is significantly associated with positive repeat biopsy for malignancy. In addition, Xpert test is an independent predictor of early tumor recurrence. Xpert test might be applied after initial complete resection of NMIBC to minimize unnecessary repeat biopsy with potential saving of healthcare costs and reduction in patient morbidity.

Bladder cancer risk stratification using a urinary mRNA biomarker panel - A path towards cystoscopy triaging.

OBJECTIVES: The risk of bladder cancer (BCa) diagnosis and recurrence necessitates cystoscopy. Improved risk stratification may inform personalized triage and surveillance strategies. We aim to develop a urinary mRNA biomarker panel for risk stratification in patients undergoing BCa screening and surveillance. METHODS AND MATERIALS: Urine samples were collected from patients undergoing cystoscopy for BCa screening or surveillance. In patients who underwent transurethral resection of bladder tumor, urine samples were categorized based on tumor histopathology, size, and focality. Subjects with intermediate and high-risk BCa based on American Urological Association (AUA) guideline for non-muscle invasive bladder cancer were classified as "increased-risk"; those with no cancer and AUA low-risk BCa were classified as "low-risk". Urine was evaluated for ROBO1, WNT5A, CDC42BPB, ABL1, CRH, IGF2, ANXA10, and UPK1B expression. A diagnostic model to detect "increased-risk" BCa was created using forward logistic regression analysis of cycle threshold values. Model validation was performed with ten-fold cross-validation. Sensitivity and specificity for detection of "increased-risk" BCa was determined and net benefit analysis performed. RESULTS: Urine samples (n = 257) were collected from 177 patients (95 screening, 76 surveillance, 6 both). There were 65 diagnoses of BCa (12 low, 22 intermediate, 31 high risk). ROBO1, CRH, and IGF2 expression correlated with "increased-risk" disease yielding sensitivity of 92.5% (95% CI, 84.9%-98.1%) and specificity of 73.5% (95% CI, 67.7-79.9%). The overall calculated standardized net benefit of the model was 0.81 (95%CI, 0.71-0.90). CONCLUSIONS: A 3-marker urinary mRNA panel allows for non-invasive identification of "increased-risk" BCa and with further validation may prove to be a tool to reduce the need for cystoscopies in low-risk patients.

Urinary microRNA and mRNA in Tumors.

Liquid biopsy is gaining importance in the context of analysis of circulating subcellular components, such as exosomes and nucleic acids, and the investigation of biological fluids is increasing because they express features common to the tissue of origin. Particularly, urine has become one of the most attractive biofluids in clinical practice due to its easy collection approach, its availability of large quantities, and its noninvasiveness. Furthermore, a peculiarity is that, compared to serum or plasma, urine is characterized by a simpler composition that improves isolation and identification of biomarkers. Recent studies have been associated with the investigation of mRNAs and microRNAs as potential noninvasive cancer biomarkers in urine, and to date, several approaches for isolating and measuring urinary nucleic acids have been established, despite still developing. This chapter aims at giving some main published evidences on urinary microRNAs and mRNAs, with the intent to consider their potential translational use in clinical practice.

Urinary Sediment mRNA Level of CREBBP and CYBA in Children With Steroid-Resistant Nephrotic Syndrome.

Background: This study aimed to evaluate gene expression patterns in urinary sediment samples of children with steroid-resistant nephrotic syndrome (SRNS). Methods: The messenger RNA (mRNA) levels of 770 immune-related genes were detected using a NanoString nCounter platform. To verify the NanoString results, quantitative analysis of nine gene mRNAs was performed using real-time RT-PCR in more samples. Results: Firstly, compared with the steroid-sensitive nephrotic syndrome (SSNS) group (n=3), significant changes were observed in the mRNA level of 70 genes, including MAP3K14, CYBA, SLC3A2, CREB-binding protein (CREBBP), CD68, forkhead box P1 (FOXP1), CD74, ITGB2, IFI30, and so forth, in the SRNS group (n=3). A total of 129 children with idiopathic nephrotic syndrome (INS), 15 with acute glomerulonephritis, and 6 with immunoglobulin A nephropathy (IgAN) were enrolled to verify the NanoString results. Compared with patients with IgAN, those with INS had significantly lower levels of FOXP1 (P=0.047) and higher levels of CREBBP (P=0.023). Among SSNS, the mRNA level of ITGB2 was significantly lower in the non-relapse group than in the non-frequent relapse and frequent-relapse groups (P=0.006). Compared with the SSNS group, CREBBP was significantly elevated in the SRNS group (P=0.02). Further, CYBA significantly decreased in the SRNS group (P=0.01). The area under the curve (AUC) for CREBBP and CYBA was 0.655 and 0.669, respectively. CREBBP had a sensitivity of 83.3% and a specificity of 49.4% and CYBA had a sensitivity of 58.3% and a specificity of 83.1% to rule out SSNS and SRNS. The diagnosis value was better for CREBBP+CYBA than for CREBBP or CYBA alone, indicating that the combination of CREBBP and CYBA was a more effective biomarker in predicting steroid resistance (AUC=0.666; sensitivity=63.9%; specificity=76.4%). Conclusions: This study was novel in investigating the urinary sediment mRNA level in children with INS using high-throughput NanoString nCounter technology, and 70 genes that may relate to SRNS were found. The results revealed that the urinary sediment mRNA level of ITGB2 was significantly lower in the non-relapse group than in the non-frequent relapse and frequent-relapse groups. Meanwhile, CREBBP was significantly elevated and CYBA was significantly lowered in the SRNS group compared with the SSNS group.

Clinical utility of current biomarkers for prostate cancer detection.

Although prostate-specific antigen (PSA) remains the most used test to detect prostate cancer (PCa), the limited specificity and an elevated rate of overdiagnosis are the main problems associated with PSA testing. Over the last three decades, a large body of evidence has indicated that PSA screening methods for PCa are problematic, although PSA screening significantly reduces PCa-specific mortality. A number of novel biomarkers have been introduced to overcome these limitations of PSA in the clinical setting. These biomarkers have demonstrated an increased ability to select patients for biopsy and identify men at risk for clinically significant PCa. Although a number of assays require further validation, initial data are promising. Forthcoming results will ultimately determine the clinical utility and commercial availability of these assays. Extensive efforts have recently been made to identify and commercialize novel PCa biomarkers for more effective detection of PCa, either alone or in combination with currently available clinical tools. This review highlights the role of existing and promising serum and urinary biomarkers for the detection and prognostication of PCa before prostate biopsy.

Prospective Validation of Clinical Usefulness of a Novel mRNA-based Urine Test (Xpert® Bladder Cancer Monitor) for surveillance in Non Muscle Invasive Bladder Cancer.

OBJECTIVES: To assess the clinical performance characteristics of Xpert Monitor test for recurrence detection during surveillance of patients with non muscle invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Patient with previous history of NMIBC were included in the study. Voided urine specimens were collected for Xpert monitor analysis and cytology. Office cystoscopy was performed for all study participants with in patient biopsy specimen retrieval for positive or suspicious cases. Test characteristics were calculated based on cystoscopy/biopsy results and compared between Xpert and cytology. RESULTS: Between March 2018 and May 2019, 181 patients including 168 (92.8%) males fulfilled the study criteria with median age 61 years, Primary tumors were low, intermediate, high risk in 2.8%, 22.7% and 74.5% of patients respectively. Biopsy proven recurrence was detected in 19 patients (10.4%). Xpert Monitor had a sensitivity of 73.7% with a negative predictive value (NPV) of 96.3%. Xpert Monitor was positive in all cases with high grade tumors (9 patients). Urine cytology showed sensitivity of 47% and an NPV of 93.2%. During follow up surveillance, out of 162 cystoscopy negative patients (CNP), 9.3% developed recurrence within 8 months. Xpert Monitor was found to be an independent predictor of early recurrence in CNP (HR=2.8, 95%CI=1.1-7.2, p=0.01). CONCLUSIONS: Xpert Monitor urine test has a superior diagnostic performance for recurrence detection in NMIBC patients compared to urine cytology. It might be a helpful tool not only for excluding bladder cancer recurrence in those patients, but also for prediction of possible future early recurrence.

Integrated RNA and metabolite profiling of urine liquid biopsies for prostate cancer biomarker discovery.

Sensitive and specific diagnostic and prognostic biomarkers for prostate cancer (PCa) are urgently needed. Urine samples are a non-invasive means to obtain abundant and readily accessible "liquid biopsies". Herein we used urine liquid biopsies to identify and characterize a novel group of urine-enriched RNAs and metabolites in patients with PCa and normal individuals with or without benign prostatic disease. Differentially expressed RNAs were identified in urine samples by deep sequencing and metabolites in urine were measured by mass spectrometry. mRNA and metabolite profiles were distinct in patients with benign and malignant disease. Integrated analysis of urinary gene expression and metabolite signatures unveiled an aberrant glutamate metabolism and tricarboxylic acid (TCA) cycle node in prostate cancer-derived cells. Functional validation supported a role for glutamate metabolism and glutamate oxaloacetate transaminase 1 (GOT1)-dependent redox balance in PCa, which could be exploited for novel biomarkers and therapies. In this study, we discovered cancer-specific changes in urinary RNAs and metabolites, paving the way for the development of sensitive and specific urinary PCa diagnostic biomarkers either alone or in combination. Our methodology was based on single void urine samples (i.e., without prostatic massage). The integrated analysis of metabolomic and transcriptomic data from these liquid biopsies revealed a glutamate metabolism and tricarboxylic acid cycle node that was specific to prostate-derived cancer cells and cancer-specific metabolic changes in urine.

All change in the prostate cancer diagnostic pathway.

Following detection of high levels of serum prostate-specific antigen, many men are advised to have transrectal ultrasound-guided biopsy in an attempt to locate a cancer. This nontargeted approach lacks accuracy and carries a small risk of potentially life-threatening sepsis. Worse still, it can detect clinically insignificant cancer cells, which are unlikely to be the origin of advanced-stage disease. The detection of these indolent cancer cells has led to overdiagnosis, one of the major problems of contemporary medicine, whereby many men with clinically insignificant disease are advised to undergo unnecessary radical surgery or radiotherapy. Advances in imaging and biomarker discovery have led to a revolution in prostate cancer diagnosis, and nontargeted prostate biopsies should become obsolete. In this Perspective article, we describe the current diagnostic pathway for prostate cancer, which relies on nontargeted biopsies, and the problems linked to this pathway. We then discuss the utility of prebiopsy multiparametric MRI and novel tumour markers. Finally, we comment on how the incorporation of these advances into a new diagnostic pathway will affect the current risk-stratification system and explore future challenges.

Urinary cell transcriptomics and acute rejection in human kidney allografts.

BACKGROUNDRNA sequencing (RNA-Seq) is a molecular tool to analyze global transcriptional changes, deduce pathogenic mechanisms, and discover biomarkers. We performed RNA-Seq to investigate gene expression and biological pathways in urinary cells and kidney allograft biopsies during an acute rejection episode and to determine whether urinary cell gene expression patterns are enriched for biopsy transcriptional profiles.METHODSWe performed RNA-Seq of 57 urine samples collected from 53 kidney allograft recipients (patients) with biopsies classified as acute T cell-mediated rejection (TCMR; n = 22), antibody-mediated rejection (AMR; n = 8), or normal/nonspecific changes (No Rejection; n = 27). We also performed RNA-Seq of 49 kidney allograft biopsies from 49 recipients with biopsies classified as TCMR (n = 12), AMR (n = 17), or No Rejection (n = 20). We analyzed RNA-Seq data for differential gene expression, biological pathways, and gene set enrichment across diagnoses and across biospecimens.RESULTSWe identified unique and shared gene signatures associated with biological pathways during an episode of TCMR or AMR compared with No Rejection. Gene Set Enrichment Analysis demonstrated enrichment for TCMR biopsy signature and AMR biopsy signature in TCMR urine and AMR urine, irrespective of whether the biopsy and urine were from the same or different patients. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of immune cell types in urinary cells compared with biopsies.CONCLUSIONSRNA-Seq of urinary cells and biopsies, in addition to identifying enriched gene signatures and pathways associated with TCMR or AMR, revealed genomic changes between TCMR and AMR, as well as between allograft biopsies and urinary cells.

Validation of a 2-gene mRNA urine test for the detection of ≥GG2 prostate cancer in an opportunistic screening population.

BACKGROUND: A 2-gene urine-based molecular test that targets messenger RNAs known to be overexpressed in aggressive prostate cancer (PCa) has been described as a helpful method for detecting clinically significant prostate cancer (grade group [GG] ≥2). We performed an external validation of this test in men undergoing initial prostate biopsy (Bx) within a Spanish opportunistic screening scenario. METHODS: We analyzed archived samples from 492 men who underwent prostate Bx in an opportunistic screening scenario, with prostate-specific antigen (PSA) 3 to 10 ng/mL and/or suspicious digital rectal exploration (DRE) and without previous multi-parametric magnetic resonance imaging (mpMRI). Urinary biomarker measurements were combined with clinical risk factors to determine a risk score, and accuracy for GG ≥ 2 PCa detection was compared with PCA3, European randomized screening in prostate cancer (ERSPC), and prostate biopsy collaborative group (PBCG) risk calculators in a validation workup that included calibration, discrimination, and clinical utility analysis. RESULTS: In our cohort, the detection rates for GG1 and GG ≥ 2 PCa were 20.3% and 14.0%, respectively. The median PSA level was 3.9 ng/mL and 13.4% of subjects had suspicious DRE findings. The median risk score for men with GG ≥ 2 PCa was 21 (interquartile range: 14-28), significantly higher than benign+GG1 PCa (10, 6-18), P < .001, achieving the highest area under the curve among the models tested, 0.749 (95% confidence interval: 0.690-0.807). The urine test was well-calibrated, while ERSPC showed a slight underestimation and PBCG a slight overestimation of risk. Assuming a GG2 non-detection rate of 11% without using mpMRI, use of the urinary biomarker-based clinical model could have helped avoid 37.2% of excess biopsies while delaying the diagnosis of eight patients (1.6% of the entire cohort) with GG ≥ 2 PCa. CONCLUSIONS: In this first evaluation in an opportunistic screening population, the urinary biomarker-based test improved the detection of clinically significant PCa. Facing men with elevated PSA and/or suspicious DRE, it could be a useful tool to help avoid excess initial Bx and to identify patients most likely to benefit from Bx.

A critical appraisal of biomarkers in prostate cancer.

PURPOSE: A number of urine and blood-based biomarker tests have been described for prostate cancer, although to date there has only been a limited exploration of the methodology behind the validation studies that underpin these tests. METHODS: In this review, a selection of commercially available urine and blood-based biomarker tests for prostate cancer are described, and the underlying key validation studies for each test are critically appraised using the Standards for Reporting Diagnostic Accuracy (STARD) 2015 statement. RESULTS: The ExoDx Prostate Intelliscore, SelectMDx, Progensa PCA3, Mi-Prostate Score, 4K Score, and Prostate Health Index (PHI) tests were reviewed. Most of the validation studies supporting these tests perform exploratory analyses to determine cut-off values in a post hoc manner, comprise cohorts that are primarily Caucasian, report receiver operating characteristic curves that combine the biomarker's result with established clinical nomograms and are based on a reference standard (prostate biopsy) that lacks central pathology review. Deficiencies in STARD reporting guidelines include frequent failure to provide a published study protocol, prospective study registration in a registry, a flow diagram, justification for sample size determination, a discussion of adverse events with testing, and information on how missing or indeterminate test results should be managed. CONCLUSIONS: Key validation studies that support many commercially available urine and blood-based biomarkers for prostate cancers have deficiencies in transparency based on STARD reporting guidelines, and limitations in methodology must be considered when deciding when these tests should be applied in clinical practice.

Clinical performance of Xpert Bladder Cancer (BC) Monitor, a mRNA-based urine test, in active surveillance (AS) patients with recurrent non-muscle-invasive bladder cancer (NMIBC): results from the Bladder Cancer Italian Active Surveillance (BIAS) project.

PURPOSE: To investigate the clinical performance of a new mRNA-based urine test, aiming to avoid unnecessary follow-up cystoscopy in patients under active surveillance (AS) for recurrent NMIBC. METHODS: This is a prospective cohort study enrolling patients with history of low-grade (LG) NMIBC, who developed a recurrence during the follow-up and underwent AS. Their urinary samples were analyzed by Xpert BC Monitor (Cepheid, Sunnyvale, CA, USA). The primary endpoint was to investigate if Xpert BC Monitor could avoid unnecessary cystoscopy during the follow-up period. Its sensitivity, specificity, PPVs and NPVs were calculated. A cutoff of 0.4 "linear discriminant analysis" (LDA) was optimized for the AS setting. RESULTS: The cohort consisted of 106 patients with a mean age of 72 ± 9.52 and a median follow-up from AS start of 8.8 (range 0-56.5) months. No statistically significant difference was found for the mean age, smoker status, lesion size, and number of lesions with a cutoff of 0.4. Of 106 patients, 22 (20.8%) were deemed to require treatment because of cystoscopic changes in size and/or number of lesions during the follow-up period. Using a cutoff value of < 0.4, 34 (33.7%) cystoscopies could be avoided due to low LDA value, missing 2/22 (9%) failures, none with high-grade (HG) NMIBC. Further research on larger population remains mandatory before its clinical use. CONCLUSION: Xpert BC Monitor seems to be a reliable assay, which might avoid unnecessary cystoscopies without missing HG NMIBC when its cutoff is optimized for the AS setting.

Urinary podocyte mRNA is a potent biomarker of anti-neutrophil cytoplasmic antibody-associated glomerulonephritis.

BACKGROUND: Anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN) is a critical kidney disease that sometimes results in an unfavorable renal outcome. Cellular crescent formation is a hallmark of ANCA-GN and is associated with renal prognosis, response to treatment, and it was reportedly associated with podocyte detachment. Because there is a need to explore non-invasive biomarkers for the evaluation of ANCA-GN activity, we tested whether urinary podocyte mRNA might be a potent non-invasive biomarker. METHODS: We measured two different types of urinary podocyte mRNA, including podocin mRNA in relation to urine creatinine concentration (U-PodCR) and urinary podocin mRNA in relation to nephrin mRNA (U-PNR), which were reportedly associated with the activity of various glomerular diseases. RESULTS: In ANCA-GN patients (n = 19), we discovered that U-PodCR was positively correlated with the percent of crescent formation until 50% crescent was reached because of podocyte depletion; U-PNR was correlated with the percent of crescent formation in all patients. Furthermore, patients with high levels of urinary podocyte mRNA exhibited a favorable renal outcome compared with the outcomes of patients with low levels of urinary podocyte mRNA. The levels of urinary podocyte mRNA were correlated with the rate of improvement in estimated glomerular filtration rate. CONCLUSIONS: U-PodCR, U-PNR or a combination of these parameters might serve as a non-invasive potential biomarker in patients with ANCA-GN to predict the percent of crescent formation and renal prognosis.