Diagnostic Urinary Biomarkers Research Articles

Identification of Potential Predictive and Diagnostic Urinary Biomarkers for Acute Rejection in Renal Transplant Recipients: A Proteomics Study

Acute rejection (AR) is one of the main predictors of long-term survival of allograft. The development of noninvasive diagnostic biomarkers of AR is an unmet need for the timely detection. This study aimed to identify novel detective biomarkers of AR by analyzing the urine proteome profile of transplant patients. Forty-two transplant patients including 30 biopsy-proven AR patients (including antibody and T-cell mediated rejection) and 12 transplant patients with stable renal function (control group) were enrolled. Label-free quantification (LFQ) proteomics technique was performed on urine samples. Multivariate statistical analysis was applied for biomarker identification. The ELISA method validated EGF (epidermal growth factor) from the top 10 candidate biomarkers in an independent cohort. Gene ontology and possible pathways were also analyzed. LFQ analysis revealed 453 identified proteins differentially expressed between groups that mainly participated in complement and coagulation pathways and proteolysis. Ten proteins with the highest AUCs (Area under the ROC Curve) were identified as candidate diagnostic biomarkers. Candidate biomarkers were mainly associated with extracellular matrix (ECM) degradation and epithelial-to-mesenchymal transition (EMT). Reduction of urinary EGF measured by ELISA in an independent group confirmed proteomics results. We introduced a unique set of diagnostic urinary biomarkers for AR. Interactions of biomarkers and validation of EGF among biomarker panels revealed that ECM remodeling and EMT might be the consequence of immunological processes in AR. If validated as a panel, the mentioned biomarkers might shed light on the pathogenesis of chronic injury after AR and point out the potential treatment strategies.

Trends in urine biomarker discovery for urothelial bladder cancer: DNA, RNA, or protein?

Urothelial bladder cancer is a complex disease displaying a landscape of heterogenous molecular subtypes, mutation profiles and clinical presentations. Diagnosis and surveillance rely on flexible cystoscopy which has high accuracy, albeit accompanied by a high-cost burden for healthcare providers and discomfort for patients. Advances in “omic” technologies and computational biology have provided insights into the molecular pathogenesis of bladder cancer and provided powerful tools to identify markers for disease detection, risk stratification, and predicting responses to therapy. To date, numerous attempts have been made to discover and validate diagnostic biomarkers that could be deployed as an adjunct to the cystoscopic diagnosis and long-term surveillance of bladder cancer. We report a comprehensive literature analysis using PubMed to assess the changing trends in investigating DNA, RNA, or proteins as diagnostic urinary biomarkers over a period of 5 decades: 1970–2020. A gradual shift has been observed in research away from protein biomarkers to nucleic acids including different classes of RNA, and DNA methylation and mutation markers. Until 2000, publications involving protein biomarker discovery constituted 87% of the total number of research articles with DNA comprising 6% and RNA 7%. Since 2000 the proportion of protein biomarker articles has fallen to 40%, and DNA and RNA studies increased to 32% and 28%, respectively. Clearly research focus, perhaps driven by technological innovation, has shifted from proteins to nucleic acids. We optimistically hypothesise that, following thorough validation, a clinically useful detection test for bladder cancer based on a panel of DNA or RNA markers could become reality within 5–10 years.

Abstract 1309: Distinct molecular subtypes and a high diagnostic urinary biomarker of upper urinary tract urothelial carcinoma

Abstract [Backgrounds] Upper urinary tract urothelial carcinomas (UTUCs) are rare malignancies with a histological similarity to urothelial bladder carcinomas (UBCs). Unlike UBCs, little is known about the genetic basis of UTUCs, for which no useful biomarkers are currently available. [Material & Methods] Surgical specimens and matched normal samples were obtained from 209 UTUC patients with various stages and were subjected to integrated molecular analysis including whole exome/transcriptome sequencing. We also investigated a possibility of UTUC diagnosis using urinary sediment-derived DNA. [Result] Hypermutation (≥20/Mb) was observed in 15 samples (6.7%), of which 12 harbored biallelic mismatch repair genes alterations suggestive of Lynch syndrome. The most frequently affected driver genes included TERT (promoter) (50%), followed by KMT2D (46%), FGFR3 (44%), CDKN2A (44%), and TP53 (36%). Mutually exclusive patterns were observed among alterations in FGFR3, RAS (H-/K-/NRAS), TP53, and MDM2, which covered 93.8% of the entire cohort. Approximately 40% of the samples showed highly complexed karyotype (CK) with frequent aneuploidy and chromothripsis. Based on these findings, UTUC cases were classified into 5 genetic subgroups: hypermutated, FGFR3-mutated, RAS-mutated, TP53-mutated with CK, and other cases with none of these features, which were significantly associated distinct clinical outcomes. While hypermutated and FGFR3 subtypes showed favorable prognosis, the TP53/CK subtype exhibited an aggressive clinical course and a shorter survival. Expression-based molecular classification identified unique gene expression subtypes: clusters 1-5, which showed a strong correlation with the mutation-based classification. Of interest, cluster 3 was highly enriched for TP53/CK and showed high expression of PDL1 and PDL2, suggesting a possible role of checkpoint inhibitions. The sequencing analysis of urinary sediment-derived DNA successfully captured those mutations and copy number alterations (CNAs) that had been detected in the corresponding preoperative samples in 84.4% of the cases (38/45), which was significantly higher than the positive diagnosis in urine cytology (class 4 and 5, 31.1%). Failure of detecting mutations were found in 5 tumors that were associated with severe hydronephrosis, which might prevented tumor-containing urinary flow. None of the post operative (0/34) and control (0/19) samples showed mutations/CNAs, including two with positive cytology (class4), both of which were diagnosed as benign pelvis tumors after surgery. [Conclusion] UTUC are classified into 5 distinct genetic subtypes characterized by unique profiles of gene mutations, expression and clinical outcomes, which help an optimal choice of therapeutics, including novel molecular-targeted drugs and checkpoint-inhibitors. Sequencing urinary sediments is a useful, non-invasive biomarker for precise diagnosis and proper treatment. Citation Format: Yoichi Fujii, Yusuke Sato, Hiromichi Suzuki, Tetsuichi Yoshizato, Kenichi Yoshida, Yuichi Shiraishi, Taketo Kawai, Tohru Nakagawa, Hiroaki Nishimatsu, Toshikazu Nishimatsu, Masashi Sanada, Hideki Makishima, Satoru Miyano, Haruki Kume, Seishi Ogawa. Distinct molecular subtypes and a high diagnostic urinary biomarker of upper urinary tract urothelial carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1309.

PT340 - Distinct molecular subtypes and a high diagnostic urinary biomarker of upper urinary tract urothelial carcinoma

PT340 - Distinct molecular subtypes and a high diagnostic urinary biomarker of upper urinary tract urothelial carcinoma

Novel Urinary Biomarkers for Acute Kidney Injury and Prediction of Clinical Outcomes After Pediatric Cardiac Surgery.

Acute kidney injury (AKI) is a serious complication of pediatric cardiac surgery, with high morbidity and mortality. We aimed to evaluate the perioperative risk factors for AKI, and the validity of novel diagnostic urinary biomarkers after pediatric cardiac surgery. We analyzed 103 consecutive pediatric patients (≤ 18years old), who underwent cardiac surgery. AKI was defined by ≥ 50% increase in serum creatinine levels from baseline. Urinary liver-type fatty acid binding protein (L-FABP) and neutrophil gelatinase-associated lipocalin (NGAL) were measured postoperatively at the intensive care unit (ICU) admission, subsequently at 4, 12, and 24h. Areas under the receiver-operating characteristic curves (AUC) were calculated at each assessment time. AKI had developed in 47 patients (45.6%) by the second postoperative day. Univentricular status, aortic cross-clamping time, and intraoperative fluid balance were independently associated with AKI (p = 0.02, 0.01 and 0.01, respectively). Urinary L-FABP and NGAL were significantly higher in the AKI group at each point (p < 0.05). The predictive abilities of both biomarkers (AUC = 0.78-0.90) at ICU admission and 4h after were especially high. The patients with L-FABP greater than the cutoff value at ICU admission and 4h after ICU admission had significantly longer intubation and hospitalization periods (p < 0.05). Those with elevated NGAL levels at admission, and 4h and 24h after ICU admission, had significantly longer intubation, ICU stay, and hospitalization (p < 0.05). L-FABP and NGAL can be useful biomarkers for detecting early AKI after pediatric cardiac surgery and predicting adverse clinical outcomes.

Patients choose certainty over burden in bladder cancer surveillance

BackgroundDue to the high risk of recurrence of non-muscle invasive bladder cancer, all patients undergo regular cystoscopic surveillance for early detection. As cystoscopy is invasive, costly and increases the burden of the disease considerably, there is significant ongoing research and development into non-invasive urinary biomarker substitutes. This study aims to assess the level of sensitivity required before patients accept a new urinary biomarker.MethodsWe studied the preferences for a hypothetical diagnostic urinary biomarker and compared this to usual care (cystoscopy) at different levels of sensitivity among 437 patients with bladder cancer (354 men and 83 women) from the UK Bladder Cancer Prognosis Programme. A standard gamble approach was used to estimate the minimally acceptable sensitivity (MAS) of the new biomarker. Additionally, non-parametric statistical analyses were performed to investigate the association between surveillance preference and various patient characteristics.ResultsAlmost half of patients (183, 43%) would not replace cystoscopy with a urinary biomarker unless it was 100% sensitive. The median MAS was 99.9999%, and nearly 85% of patients demanded a sensitivity of at least 99% before preferring a urinary biomarker test over cystoscopy. These results were consistent across all patient characteristics and demographic categories.ConclusionsOur results indicate that patients demand urinary biomarkers as sensitive as cystoscopy before they would be willing to forego cystoscopy for bladder cancer surveillance.

Urinary biomarkers reporting weakness and validation failure in Type 2 diabetic nephropathy: systematic review.

For better identification of novel diagnostic urinary biomarker in Type 2 diabetic nephropathy (T2DN), methodological and reporting quality is as important as validity of biomarkers. The aim of this systematic review is to find out the best-reported diagnostic urinary biomarkers study in T2DN based on STARD criteria. We also analyzed the validity of urinary markers in the selected articles those followed STARD criteria. The diagnostic accuracy of urinary biomarkers on T2DN is not conclusive because of the poor reporting quality and differences in adjustment for conventional risk factors. For a proper validation of urinary biomarkers on T2DN, in future large well-designed longitudinal studies, with specific prediction analysis and validation of the biomarkers by adjusting with possible conventional risk factors.

Pediatric sleep-disordered breathing: an update on diagnostic testing.

Recent advances in diagnostic testing for obstructive sleep apnea in children have refined the standard tests while identifying several new tools that hold promise to radically change how we diagnose sleep apnea. Studies have demonstrated that the polysomnogram may be modified to permit home assessment of sleep disturbed breathing in children to ensure more widespread access to the test. Alternately, questionnaires, nocturnal oximetry, and diagnostic urinary biomarkers have shown great promise as both sensitive and specific tools to diagnose sleep apnea in children as well as track the severity of the disease. The gold standard polysomnogram has been refined to permit its application in a modified form at home and for brief examinations in children. This standard has been challenged on several fronts, including questionnaires, nocturnal oximetry, drug-induced sleep endoscopy, and noninvasive urinary biomarkers that may ultimately supplant polysomnography as the gold standard to diagnose obstructive sleep apnea syndrome in children.

Urinary biomarkers for the diagnosis of urothelial bladder cancer

Urothelial bladder cancer is a common cancer associated with considerable burden for both patients and healthcare providers alike. The majority of patients present with non-muscle-invasive bladder cancer (NMIBC) which, although not immediately life-threatening, requires appropriate initial management and long-term surveillance which is both invasive and costly. Accurate diagnostic urinary biomarkers could be transformational in this setting, yet have proved to be a significant challenge to bladder cancer scientists over the last two decades. Such biomarkers would need to represent a range of tumour grades and stages, encompass inter- and intra-tumour heterogeneity, and compete with the current diagnostic gold standard of cystoscopy with a sensitivity and specificity of 85% and 87%, respectively. For the field to move forward in this current exciting era of high-throughput proteomics and genomics, bladder cancer scientists need to find a consensus on the optimal urinary substrate (DNA, RNA, protein, etc) and deliver robust well-designed studies in the correct populations with appropriate statistical input. Issues relating to tumour heterogeneity and anticipatory diagnosis also require considerable thought. The challenge remains unchanged.

Exosomal MicroRNAs Are Diagnostic Biomarkers and Can Mediate Cell–Cell Communication in Renal Cell Carcinoma

BackgroundApart from an invasive biopsy, currently no tools are available to confirm the diagnosis of clear cell renal cell carcinoma (ccRCC); this resulted in approximately 30% of patients being diagnosed with metastatic disease. ObjectiveTo determine whether urinary microRNAs (miRNAs) can serve as biomarkers to confirm the diagnosis of ccRCC. Design, setting, and participantsGlobal miRNA expression was assessed in 28 preoperative urine samples from patients with ccRCC and 18 healthy participants. The independent validation set consisted of 81 ccRCC patients, 24 patients with benign lesions, and 33 healthy participants. We extracted both cell-free and exosomal RNA for miRNA expression analysis using miRNA-specific polymerase chain reaction assays. We also investigated exosomal miRNA secretion in cell line models and performed exosome transfer between RCC and endothelial cell types. Outcome measurements and statistical analysisReceiver operating characteristic analysis was applied to identify the discrimination power of miRNAs. Results and limitationsOverall, miR-126-3p combined with miR-449a or with miR-34b-5p could significantly distinguish ccRCC patients from healthy participants (miR-126-3p–miR-449a: area under the curve [AUC]: 0.84; 95% confidence interval [CI], 0.7620–0.9151; p<0.001; miR-126-3p–miR-34b-5p: AUC: 0.79; 95% CI, 0.7013–0.8815; p<0.001). The combination of miR-126-3p and miR-34b-5p was also able to distinguish small renal masses (pT1a, ≤4cm) from healthy controls (AUC: 0.79; 95% CI, 0.6848–0.8980; p<0.001). Using miR-126-3p and miR-486-5p in combination, we were able to differentiate between benign lesions and ccRCC (AUC: 0.85; 95% CI, 0.7295–0.9615; p<0.01). The expression of a number of miRNAs returned to a level comparable with health after surgery. Kidney cancer cell lines were found to secrete exosomal miR-126-3p, miR-17-5p, miR-21-3p, and miR-25-3p, and these miRNAs were found to be internalized by other cell types. ConclusionsWe identified exosomal miRNAs as potential noninvasive diagnostic urinary biomarkers for ccRCC and provided evidence that miRNAs are secreted by the tumor and can function as a tool for intercellular communication. Patient summaryWe identified urinary microRNAs that can serve as diagnostic biomarkers for clear cell renal cell carcinoma.

Carbonic Anhydrase IX as a Diagnostic Urinary Marker for Urothelial Bladder Cancer

Urinary biomarkers are needed to improve the management and reduce the cost of urothelial bladder cancer (UBC); however, none have been recommended yet for clinical practice. This study evaluated carbonic anhydrase IX (CAIX) as a diagnostic urinary biomarker for UBC. CAIX was analyzed by quantitative polymerase chain reaction in urine samples of 196 patients with UBC and 123 controls with hematuria. Paired samples from urine and tumor tissue were evaluated in 16 cases. Data were validated in 155 independent samples. The sensitivity and specificity of CAIX for UBC detection were 86.2% and 95.1%, respectively (area under the curve [AUC]: 90.5%). There was a significant association of CAIX expression between the paired urine and tumor specimens (p=0.002). CAIX showed a significantly higher predictive accuracy than urinary cytology (90.5% vs 71.7%), specifically in low-grade tumors (90.0% vs 61.8%). CAIX expression decreased with increasing tumor stage and grade. Analyses in an independent validation cohort confirmed the high accuracy of CAIX for diagnosing UBC (AUC: 88.3%). Patient summaryWe evaluated carbonic anhydrase IX (CAIX) as a urinary marker for bladder cancer (BCa) using a large series of patients from a single hospital. We found that urinary CAIX has a high sensitivity and specificity for diagnosing BCa.

Mass spectrometric based approaches in urine metabolomics and biomarker discovery.

Urine metabolomics has recently emerged as a prominent field for the discovery of non-invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non-biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid-liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4°C) and repetitive freeze-thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high-performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)-MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC-MS, other MS-based technologies have been utilized in urine metabolomics. These include direct injection (infusion)-MS, capillary electrophoresis-MS and gas chromatography-MS. In this article, the current progresses of different MS-based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:115-134, 2017.

Analysis of urinary metabolomic profiling for unstable angina pectoris disease based on nuclear magnetic resonance spectroscopy.

(1)H NMR-based urinary metabolic profiling is used for investigating the unstable angina pectoris (UAP) metabolic signatures, in order to find out candidate biomarkers to facilitate medical diagnosis. In this work, 27 urine samples from UAP patients and 20 healthy controls were used. The metabolic profiles of the samples were analyzed by multivariate statistics analysis, including PCA, PLS-DA and OPLS-DA. The PCA analysis exhibited slight separation with R(2)X of 0.681 and Q2 of 0.251, while the PLS-DA (R(2)X = 0.121, R(2)Y = 0.931, and Q(2) = 0.661) and the OPLS-DA (R(2)X = 0.121, R(2)Y = 0.931, Q(2) = 0.653) demonstrated that the model showed good performance. By OPLS-DA, 20 metabolites were identified. A diagnostic model was further constructed using the receiver-operator characteristic (ROC) curves (AUC = 0.953), which exhibited a satisfying sensitivity of 92.6%, specificity of 90% and accuracy of 89.1%. The results demonstrated that the NMR-based metabolomics approach showed good performance in identifying diagnostic urinary biomarkers, providing new insights into the metabolic process related to UAP.

Development and prospective multicenter evaluation of the long noncoding RNA MALAT-1 as a diagnostic urinary biomarker for prostate cancer.

The current strategy for diagnosing prostate cancer (PCa) is mainly based on the serum prostate-specific antigen (PSA) test. However, PSA has low specificity and has led to numerous unnecessary biopsies. We evaluated the effectiveness of urinary metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), a long noncoding RNA, for predicting the risk of PCa before biopsy. The MALAT-1 score was tested in a discovery phase and a multi-center validation phase. The predictive power of the MALAT-1 score was evaluated by the area under receiver operating characteristic (ROC) curve (AUC) and by decision curve analysis. As an independent predictor of PCa, the MALAT-1 score was significantly higher in men with a positive biopsy than in those with a negative biopsy. The ROC analysis showed a higher AUC for the MALAT-1 score (0.670 and 0.742) vs. the total PSA (0.545 and 0.601) and percent free PSA (0.622 and 0.627) in patients with PSA values of 4.0-10 ng/ml. According to the decision curve analysis, using a probability threshold of 25%, the MALAT-1 model would prevent 30.2%-46.5% of unnecessary biopsies in PSA 4-10 ng/ml cohorts, without missing any high-grade cancers. Our results demonstrate that urine MALAT-1 is a promising biomarker for predicting prostate cancer risk.

Standardization of Diagnostic Biomarker Concentrations in Urine: The Hematuria Caveat

Sensitive and specific urinary biomarkers can improve patient outcomes in many diseases through informing early diagnosis. Unfortunately, to date, the accuracy and translation of diagnostic urinary biomarkers into clinical practice has been disappointing. We believe this may be due to inappropriate standardization of diagnostic urinary biomarkers. Our objective was therefore to characterize the effects of standardizing urinary levels of IL-6, IL-8, and VEGF using the commonly applied standards namely urinary creatinine, osmolarity and protein. First, we report results based on the biomarker levels measured in 120 hematuric patients, 80 with pathologically confirmed bladder cancer, 27 with confounding pathologies and 13 in whom no underlying cause for their hematuria was identified, designated “no diagnosis”. Protein levels were related to final diagnostic categories (p = 0.022, ANOVA). Osmolarity (mean = 529 mOsm; median = 528 mOsm) was normally distributed, while creatinine (mean = 10163 µmol/l, median = 9350 µmol/l) and protein (0.3297, 0.1155 mg/ml) distributions were not. When we compared AUROCs for IL-6, IL-8 and VEGF levels, we found that protein standardized levels consistently resulted in the lowest AUROCs. The latter suggests that protein standardization attenuates the “true” differences in biomarker levels across controls and bladder cancer samples. Second, in 72 hematuric patients; 48 bladder cancer and 24 controls, in whom urine samples had been collected on recruitment and at follow-up (median = 11 (1 to 20 months)), we demonstrate that protein levels were approximately 24% lower at follow-up (Bland Altman plots). There was an association between differences in individual biomarkers and differences in protein levels over time, particularly in control patients. Collectively, our findings identify caveats intrinsic to the common practice of protein standardization in biomarker discovery studies conducted on urine, particularly in patients with hematuria.

Identification of Diagnostic Urinary Biomarkers for Acute Kidney Injury

Acute kidney injury (AKI) is an important cause of death among hospitalized patients. The 2 most common causes of AKI are acute tubular necrosis (ATN) and prerenal azotemia (PRA). Appropriate...