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Renal cell carcinoma (RCC) is the most common malignant tumor in the urinary system, accounting for 80 % to 90 % for all renal malignancies. Traditional diagnostic methods like magnetic resonance imaging (MRI) and computed tomography (CT) lack the sensitivity and specificity as they lack specific biomarkers. These limitations impede effective monitoring of tumor recurrence. This study aims to employ Attenuated Total Reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy, an optical technology sensitive to molecular groups, to analyze the potential optical biomarkers in urine and plasma samples from RCC patients pre- and post-surgery. The results reveal distinctive spectral information from both plasma and urine samples. Post-surgery urine spectra exhibit complexity compared to plasma, showing reduced content at 1072 cm -1 , 1347 cm -1 and 1654 cm -1 bands, while increased content at 1112 cm -1 , 1143 cm -1 , 1447 cm -1 , 3334 cm -1 and 3420 cm -1 bands. Utilizing machine learning models such as eXtreme Gradient Boosting (XGBoost), support vector machine (SVM), partial least squares (PLS), and artificial neural network (ANN), the study evaluated plasma and urine samples pre- and post-surgery. Remarkably, the XGBoost method excelled in distinguishing between tumor conditions and recovery, achieving an impressive AUC value of 0.99. These results underscore the potential of ATR-FTIR technology in identifying RCC optical biomarkers, with XGBoost showing promise as a valuable screening tool for RCC recurrence diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aromatic caninurine formamase (AFMID) is an enzyme involved in the tryptophan pathway, metabolizing N-formylkynurenine to kynurenine. AFMID had been found significantly downregulated in clear cell renal cell carcinoma (ccRCC) in both tissue and urine samples. Although ccRCC is characterized by a typical Warburg-like phenotype, mitochondrial dysfunction, and elevated fat deposition, it is unknown whether AFMID plays a role in tumorigenesis and the development of ccRCC. In the present study, AFMID overexpression had inhibitory effects for ccRCC cells, decreasing the rate of cell proliferation. Quantitative proteomics showed that AFMID overexpression altered cellular signaling pathways involved in cell growth and cellular metabolism pathways, including lipid metabolism and inositol phosphate metabolism. Further urine proteomic analysis indicated that cellular function dysfunction with AFMID overexpression could be reflected in the urine. The activity of predicted upregulators DDX58, TREX1, TGFB1, SMARCA4, and TNF in ccRCC cells and urine showed opposing change trends. Potential urinary biomarkers were tentatively discovered and further validated using an independent cohort. The protein panel of APOC3, UMOD, and CILP achieved an AUC value of 0.862 for the training cohort and 0.883 for the validation cohort. The present study is of significance in terms of highlighting various aspects of pathway changes associated with AFMID enzymes, discovering potential specific biomarkers for potential patient diagnosis, and therapeutic targeting.

Laminin, fibronectin, and collagen IV are pivotal extracellular matrix (ECM) components. The ECM environment governs the fundamental properties of tumors, including proliferation, vascularization, and invasion. Given the critical role of cell-matrix adhesion in malignant tumor progression, we hypothesize that the concentrations of these proteins may be altered in the plasma of patients with clear cell renal cell carcinoma (ccRCC). This study aimed to evaluate the serum, urine, and tissue levels of laminin-5, collagen IV, and fibronectin among a control group and ccRCC patients, with the latter divided into stages T1-T2 and T3-T4 according to the TNM classification. We included 60 patients with histopathologically confirmed ccRCC and 26 patients diagnosed with chronic cystitis or benign prostatic hyperplasia (BPH). Collagen IV, laminin-5, and fibronectin were detected using Surface Plasmon Resonance Imaging biosensors. Significant differences were observed between the control group and ccRCC patients, as well as between the T1-T2 and T3-T4 subgroups. Levels were generally higher in plasma and tissue for fibronectin and collagen IV in ccRCC patients and lower for laminin. The ROC (Receiver operating characteristic) analysis yielded satisfactory results for differentiating between ccRCC patients and controls (AUC 0.84-0.93), with statistical significance for both fibronectin and laminin in plasma and urine. Analysis between the T1-T2 and T3-T4 groups revealed interesting findings for all examined substances in plasma (AUC 0.8-0.95). The results suggest a positive correlation between fibronectin and collagen levels and ccRCC staging, while laminin shows a negative correlation, implying a potential protective role. The relationship between plasma and urine concentrations of these biomarkers may be instrumental for tumor detection and staging, thereby streamlining therapeutic decision-making.

Renal cell carcinoma (RCC) is the sixth most common cancer in men and is often asymptomatic, leading to incidental detection in advanced disease stages that are associated with aggressive histology and poorer outcomes. Various cancer biomarkers are found in urine samples from patients with RCC. In this study, we propose to investigate the use of Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) on dried urine samples for distinguishing RCC. We analyzed dried urine samples from 49 patients with RCC, confirmed by histopathology, and 39 healthy donors using ATR-FTIR spectroscopy. The vibrational bands of the dried urine were identified by comparing them with spectra from dried artificial urine, individual urine components, and dried artificial urine spiked with urine components. Urea dominated all spectra, but smaller intensity peaks, corresponding to creatinine, phosphate, and uric acid, were also identified. Statistically significant differences between the FTIR spectra of the two groups were obtained only for creatinine, with lower intensities for RCC cases. The discrimination of RCC was performed through Principal Component Analysis combined with Linear Discriminant Analysis (PCA-LDA) and Support Vector Machine (SVM). Using PCA-LDA, we achieved a higher discrimination accuracy (82%) (using only six Principal Components to avoid overfitting), as compared to SVM (76%). Our results demonstrate the potential of urine ATR-FTIR combined with machine learning techniques for RCC discrimination. However, further studies, especially of other urological diseases, must validate this approach.

Renal cell carcinoma (RCC) is a substantial pathology of the urinary system with a growing prevalence rate. However, current clinical methods have limitations for managing RCC due to the heterogeneity manifestations of the disease. Metabolic analyses are regarded as a preferred noninvasive approach in clinics, which can substantially benefit the characterization of RCC. This study constructs a nanoparticle-enhanced laser desorption ionization mass spectrometry (NELDI MS) to analyze metabolic fingerprints of renal tumors (n = 456) and healthy controls (n = 200). The classification models yielded the areas under curves (AUC) of 0.938 (95% confidence interval (CI), 0.884-0.967) for distinguishing renal tumors from healthy controls, 0.850 for differentiating malignant from benign tumors (95% CI, 0.821-0.915), and 0.925-0.932 for classifying subtypes of RCC (95% CI, 0.821-0.915). For the early stage of RCC subtypes, the averaged diagnostic sensitivity of 90.5% and specificity of 91.3% in the test set is achieved. Metabolic biomarkers are identified as the potential indicator for subtype diagnosis (p < 0.05). To validate the prognostic performance, a predictive model for RCC participants and achieve the prediction of disease (p = 0.003) is constructed. The study provides a promising prospect for applying metabolic analytical tools for RCC characterization., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Introduction: Renal cell carcinoma is one of the ten more common malignant tumors worldwide, with a high incidence and mortality rate. Kidney cancer frequently presents at an advanced stage, and it is almost invariably fatal. Much progress has been made in identifying molecular targets for therapy in the hope of improving survival rates, but still, we have no good markers for early detection or progression of the disease. Von Hippel Lindau syndrome (VHL) is an autosomal dominant cancer hereditary syndrome in which affected individuals are at risk of developing bilateral and multifocal renal cell carcinomas (RCC) as well as other tumors. These patients provide an ideal platform to investigate the potential of urinary exosomal miRNA biomarkers in the early development of ccRCC, as these patients are regularly imaged and tumors are actively monitored until the tumor reaches 3 cm before surgical excision. This allows for pre- and post-surgical urine collection and comparison to excised tumor tissues. Studying different biomarkers in urine can provide comprehensive molecular profiling available to patients and physicians and can be a great source of additional tumor genetic information., Methods: Pre- and postoperative urine samples were obtained from a cohort of VHL patients undergoing surveillance and surgical excision of ccRCCs, and exosomes were extracted. MicroRNA-Seq analysis was performed on miRNA extracted from both urine-derived exosomes and FFPE material from excised ccRCCs., Results: MicroRNA-Seq analysis highlighted a significant difference in the urinary exosome-derived miRNA expression profiles between VHL patients and normal control individuals. This included decreased expression of the miR-320 family, such as miR-320a, known to be decreased in sporadic ccRCC and suppressed by the HIF1α transcription factor activated by the loss of the VHL gene. MiR-542-5p represented a potential marker of VHL-associated ccRCC that was lowly expressed in normal control urinary exosomes, significantly increased in the preoperative urinary exosomes of tumor-bearing VHL patients, and subsequently reduced to normal levels of expression after tumor excision. In concordance with this, the expression of miR-542-5p was increased in the VHL-associated ccRCC in comparison to the normal kidney., Conclusions: This study shows the potential for miRNA profiling of exosomes from readily available biofluids to both distinguish VHL patient urine from normal control urine microRNAs and to provide biomarkers for the presence of VHL syndrome-associated ccRCC. Further validation studies are necessary to demonstrate the utility of urinary exosome-derived miRNAs as biomarkers in kidney cancer.

Metabolomics generates complex data necessitating advanced computational methods for generating biological insight. While machine learning (ML) is promising, the challenges of selecting the best algorithms and tuning hyperparameters, particularly for nonexperts, remain. Automated machine learning (AutoML) can streamline this process; however, the issue of interpretability could persist. This research introduces a unified pipeline that combines AutoML with explainable AI (XAI) techniques to optimize metabolomics analysis. We tested our approach on two data sets: renal cell carcinoma (RCC) urine metabolomics and ovarian cancer (OC) serum metabolomics. AutoML, using Auto-sklearn, surpassed standalone ML algorithms like SVM and k-Nearest Neighbors in differentiating between RCC and healthy controls, as well as OC patients and those with other gynecological cancers. The effectiveness of Auto-sklearn is highlighted by its AUC scores of 0.97 for RCC and 0.85 for OC, obtained from the unseen test sets. Importantly, on most of the metrics considered, Auto-sklearn demonstrated a better classification performance, leveraging a mix of algorithms and ensemble techniques. Shapley Additive Explanations (SHAP) provided a global ranking of feature importance, identifying dibutylamine and ganglioside GM(d34:1) as the top discriminative metabolites for RCC and OC, respectively. Waterfall plots offered local explanations by illustrating the influence of each metabolite on individual predictions. Dependence plots spotlighted metabolite interactions, such as the connection between hippuric acid and one of its derivatives in RCC, and between GM3(d34:1) and GM3(18:1&#95;16:0) in OC, hinting at potential mechanistic relationships. Through decision plots, a detailed error analysis was conducted, contrasting feature importance for correctly versus incorrectly classified samples. In essence, our pipeline emphasizes the importance of harmonizing AutoML and XAI, facilitating both simplified ML application and improved interpretability in metabolomics data science.

Background: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC with high rates of metastasis. Targeted therapies such as tyrosine kinase and checkpoint inhibitors have improved treatment success, but therapy-related side effects and tumor recurrence remain a challenge. As a result, ccRCC still have a high mortality rate. Early detection before metastasis has great potential to improve outcomes, but no suitable biomarker specific for ccRCC is available so far. Therefore, molecular biomarkers derived from body fluids have been investigated over the past decade. Among them, RNAs from urine-derived extracellular vesicles (EVs) are very promising., Methods: RNA was extracted from urine-derived EVs from a cohort of 78 subjects (54 ccRCC patients, 24 urolithiasis controls). RNA-seq was performed on the discovery cohort, a subset of the whole cohort (47 ccRCC, 16 urolithiasis). Reads were then mapped to the genome, and expression was quantified based on 100 nt long contiguous genomic regions. Cluster analysis and differential region expression analysis were performed with adjustment for age and gender. The candidate biomarkers were validated by qPCR in the entire cohort. Receiver operating characteristic, area under the curve and odds ratios were used to evaluate the diagnostic potential of the models., Results: An initial cluster analysis of RNA-seq expression data showed separation by the subjects' gender, but not by tumor status. Therefore, the following analyses were done, adjusting for gender and age. The regions differentially expressed between ccRCC and urolithiasis patients mainly overlapped with small nucleolar RNAs (snoRNAs). The differential expression of four snoRNAs (SNORD99, SNORD22, SNORD26, SNORA50C) was validated by quantitative PCR. Confounder-adjusted regression models were then used to classify the validation cohort into ccRCC and tumor-free subjects. Corresponding accuracies ranged from 0.654 to 0.744. Models combining multiple genes and the risk factors obesity and hypertension showed improved diagnostic performance with an accuracy of up to 0.811 for SNORD99 and SNORA50C (p = 0.0091)., Conclusions: Our study uncovered four previously unrecognized snoRNA biomarkers from urine-derived EVs, advancing the search for a robust, easy-to-use ccRCC screening method., (© 2024. The Author(s).)

Active surveillance (AS) is the best strategy for small renal masses (SRMs) management; however, reliable methods for early detection and disease aggressiveness prediction are urgently needed. The aim of the present study was to validate DNA methylation biomarkers for non-invasive SRM detection and prognosis. The levels of methylated genes TFAP2B , TAC1 , PCDH8 , ZNF677 , FLRT2, and FBN2 were evaluated in 165 serial urine samples prospectively collected from 39 patients diagnosed with SRM, specifically renal cell carcinoma (RCC), before and during the AS via quantitative methylation-specific polymerase chain reaction. Voided urine samples from 92 asymptomatic volunteers were used as the control. Significantly higher methylated TFAP2B , TAC1 , PCDH8 , ZNF677, and FLRT2 levels and/or frequencies were detected in SRM patients' urine samples as compared to the control. The highest diagnostic power (AUC = 0.74) was observed for the four biomarkers panel with 92% sensitivity and 52% specificity. Methylated PCDH8 level positively correlated with SRM size at diagnosis, while TFAP2B had the opposite effect and was related to SRM progression. To sum up, SRMs contribute significantly to the amount of methylated DNA detectable in urine, which might be used for very early RCC detection. Moreover, PCDH8 and TFAP2B methylation have the potential to be prognostic biomarkers for SRMs.

Objective: Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor characterized by the highest mortality rate of the genitourinary cancers, and, therefore, new diagnostic and/or prognostic biomarkers are urgently needed., Methods: Based on genome-wide DNA methylation profiling in 11 pairs of ccRCC and non-cancerous renal tissues (NRT), the methylation at regulatory regions of ZNF677, FBN2, PCDH8, TFAP2B, TAC1, and FLRT2 was analyzed in 168 renal tissues and 307 urine samples using qualitative and quantitative methylation-specific PCR (MSP)., Results: Significantly higher methylation frequencies for all genes were found in ccRCC tissues compared to NRT (33-60% vs. 0-11%). The best diagnostic performance demonstrated a panel of ZNF677, FBN2, PCDH8, TFAP2B & TAC1 with 82% sensitivity and 96% specificity. Hypermethylation of ZNF677 and PCDH8 in the tissue samples was significantly related to numerous adverse clinicopathologic parameters. For the urine-based ccRCC detection, the highest diagnostic power (AUC = 0.78) was observed for a panel of ZNF677 & PCDH8 (with or without FBN2 or FLRT2) with 69-78% sensitivity and 69-80% specificity, albeit with lower values in the validation cohort. Besides, methylation of PCDH8 was significantly related to higher tumor stage and fat invasion in the study and validation cohorts. Moreover, PCDH8 was strongly predictive for OS (HR, 5.7; 95% CI 1.16-28.12), and its prognostic power considerably increased in combination with ZNF677 (HR, 12.5; 95% CI 1.47-105.58)., Conclusion: In summary, our study revealed novel, potentially promising DNA methylation biomarkers of ccRCC with the possibility to be applied for non-invasive urine-based ccRCC detection and follow-up., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

MN/CA9 is a cell surface glycoprotein and a tumor-associated antigen. It plays a crucial role in the regulation of cell proliferation and oncogenesis. There is no ideal tumor marker currently available for renal cell carcinoma (RCC) with sufficient sensitivity and specificity. Therefore, we studied MN/CA9 gene expression in the tumor tissue, apparently normal kidney tissue, preoperative blood, and urine samples of patients with RCC. We included thirty cases of renal tumors (26 RCC and 4 benign tumors) in the study. We applied an RT-PCR assay for MN/CA9 gene expression to 26 RCC kidney tumor samples and four benign kidney tumor tissue samples. We also evaluated MN/CA9 gene expression in preoperative blood and urine samples of 15 of these cases. Additionally, thirty-five grossly normal renal tissue samples, including 21 from kidneys with RCC, were also evaluated for gene expression. The RT-PCR analysis revealed that twenty-one out of 26 RCC tissue samples showed MN/CA9 gene expression compared to three out of 35 non-malignant renal tissue samples (p < 0.05). Two out of four benign renal tissue samples also expressed this gene. We also observed MN/CA9 gene expression in nine out of 15 blood samples and four out of 15 urine samples. All patients with urinary MN/CA9 gene expression showed expression in blood and tumor tissue samples. We found a correlation in terms of MN/CA9 expression between blood and tumor tissue samples of RCC patients as those who exhibit MN/CA9 expression in blood were also positive at the tumor tissue levels. The difference in MN/CA9 gene expression in tumor tissue, blood, and urine samples in relation to the stage of the disease, nuclear grade, and histological cell-type was not statistically significant. However, all the three patients who had metastatic RCC had MN/CA9 gene expression in their blood. The existence of a tumor-associated antigen such as MN/CA9 may present a possible target for molecular diagnosis and management of RCC., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

To improve treatment outcomes in real practice, useful biomarkers are desired when predicting postoperative recurrence for renal cell carcinoma (RCC). We collected data from patients who underwent definitive surgery for RCC and for benign urological tumor at our department between November 2016 and December 2019. We evaluated the differences in pre- and postoperative urinary metabolites with our precise quantitative method and identified predictive factors for RCC recurrence. Additionally, to clarify the significance of metabolites, we measured the intracellular metabolite concentration of three RCC cell lines. Among the 56 patients with RCC, nine had a recurrence (16.0%). When comparing 27 patients with T1a RCC and 10 with benign tumor, a significant difference was observed between pre- and postoperative concentrations among 10 urinary metabolites. In these 10 metabolites, multiple logistic regression analysis identified five metabolites (lactic acid, glycine, 2-hydroxyglutarate, succinic acid, and kynurenic acid) as factors to build our recurrence prediction model. The values of area under the receiver operating characteristic curve, sensitivity, and specificity in this predictive model were 0.894%, 88.9%, and 88.0%, respectively. When stratified into low and high risk groups of recurrence based on this model, we found a significant drop of recurrence-free survival rates among the high risk group. In in vitro studies, intracellular metabolite concentrations of metastatic tumor cell lines were much higher than those of primary tumor cell lines. By using our quantitative evaluation of urinary metabolites, we could predict postoperative recurrence with high sensitivity and specificity. Urinary metabolites could be noninvasive biomarkers to improve patient outcome., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Renal cancer represents the 7th most common tumor worldwide, affecting 400,000 people annually. This malignancy, which is the third most frequent cancer among urological diseases, displays a completely different prognosis if the tumor is detected in the early stages or advance phases. Unfortunately, more than 50% of renal cancers are discovered incidentally, with a consistent percentage of cases where the tumor remains clinically silent till the metastatic process is established. In day-to-day clinical practice, no available predictive biomarkers exist, and the existent imaging diagnostic techniques harbor several gaps in terms of diagnosis and prognosis. In the last decade, many efforts have been reported to detect new predictive molecular biomarkers using liquid biopsies, which are less invasive in comparison to renal biopsy. However, until now, there has been no clear evidence that a liquid biopsy biomarker could be relevant to the creation of a precise and tailored medical management in these oncological patients, even though circulating RNA biomarkers remain among the most promising. Given the idea that liquid biopsies will play a future key role in the management of these patients, in the present review, we summarize the current state of circulating RNA (miRNA, lncRNAs, and circRNAs) as possible biomarkers of renal cancer presence and aggressiveness in patients.

Background: Recently, our group showed that Vim3 is overexpressed in tissue samples of renal oncocytomas and Mxi-2 in clear cell renal carcinoma (ccRCC). The mechanism leading to the truncation of both proteins is known and involves with two miRs, both detectable in urine. Since the analysis of miRs is time-consuming, our aim was to identify the truncated proteins in urine instead. Furthermore, urine samples from small renal masses (SRMs) (n = 45, <4 cm) were analyzed to get a pre-surgical differentiation of the cancer subtypes., Methods: Urines were accessed from the urological biobank (n = 350). Proteins were isolated from urine samples, and Western blots were performed. Each sample was analyzed with ELISA for the expression of Vim3 and Mxi-2. A lateral flow assay was established. For the detection of SRMs, the miRs were isolated and qRT-PCR was performed., Results: A significant increase of Vim3 in urines from patients with oncocytoma (n = 20) was detectable with ELISA compared to all other subtypes of RCCs (chromophobe (n = 50), papillary (n = 40), ccRCC (n = 200), and controls (n = 40) (***p < 0.0001)). Mxi-2 was predominantly overexpressed in ccRCCs (***p < 0.0001). Lateral flow assay of Vim3 and Mxi-2 shows two bands in the case of oncocytoma and ccRCC indicating the specificity of this test. For SRMs, an overexpression of miR-15a/Mxi2 was detectable in urine samples from ccRCC and chromoRCC patients. In contrast to that, miR-498/Vim3 were predominantly overexpressed in oncocytoma patients., Conclusion: Both proteins (Vim3 and Mxi-2) were detectable in patients' urines and can be used for the non-invasive differentiation of kidney cancers., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Purpose of Review: We aim to summarize the current state of art about the possible use of biomarkers for predicting renal cell carcinoma (RCC) recurrence after curative treatment. In addition, we aim to provide a snapshot about the clinical implication of biomarkers use for follow-up planification., Recent Findings: A wide variety of biomarkers have been proposed. RCC biomarkers have been individuated in tumoral tissue, blood, and urine. A variety of molecules, including proteins, DNA, and RNA, warrant a good accuracy for RCC recurrence and progression prediction. Their use in prediction models might warrant a better patients' risk stratification. Future prognostic models will probably include a combination of classical features (tumor grade, stage, etc.) and novel biomarkers. Such models might allow a more accurate treatment and follow-up planification.

Introduction: Biomarkers for the diagnosis and monitoring treatment response of kidney cancer are urgently needed. Neutrophil gelatinase-associated lipocalin (NGAL) is a relevant urinary biomarker for the diagnosis of a wide variety of acute and chronic kidney diseases. Its potential utility as a prognostic marker of kidney cancer is largely unknown and, therefore, was the subject of this investigation., Materials and Methods: A retrospective study was done on 50 kidney tumor patients (urine samples prospectively collected before nephrectomy between 2004 and 2012, stored at Biobank Resource Center). The specificity, sensitivity and the predictive value of NGAL were determined for progression-free and disease-specific survival after nephrectomy in renal cell carcinoma (particularly, the clear cell renal cell carcinoma (ccRCC)). Urinary NGAL concentration (u-NGAL) was determined by CMIA technique (ARCHITECT® urine NGAL essay/ABBOTT ® )., Results: Out of the 50 kidney tumor patients, 40 had clear cell carcinoma with a median u-NGAL excretion of 1.4 (IQR: 5.76) ng/mg urinary creatinine (Ucr). u-NGAL was correlated to tumor stage (p = 0.005), and Fuhrman grade (p = 0.0002). Multivariate Cox regression analysis showed a significant association between u-NGAL excretion and clear cell renal cell carcinoma progression free survival and disease specific survival (p = 0.002; p = 0.0001)., Conclusions: Urinary NGAL was significantly associated with the stage and the grade of kidney cancer. u-NGAL excretion could be considered as a potential biomarker to identify ccRCC patients with the more pejorative outcomes.

Renal cell carcinoma is a very aggressive and often fatal disease for which there are no specific biomarkers found to date. The purpose of this work was to find features that differentiate urine metabolic profiles of healthy people and cancer patients. Laser desorption/ionization mass spectrometry on gold nanostructures-based techniques were used for the metabolic analysis of urine of 50 patients with kidney cancer. Comparison with data from 50 healthy volunteers led to the discovery of several compounds that may be considered potential renal cell carcinoma (RCC) biomarkers. Statistical analysis of data allowed for the discovery of m/z values that had the greatest impact on group differentiation. A database search enabled the assignment of signals for the most promising 15 features among them: serine, heptanol, 3-methylene-indolenine, 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate, phosphodimethylethanolamine, 4-methoxyphenylacetic acid, N-acetylglutamine, 3,5-dihydroxyphenylvaleric acid, hydroxyhexanoylglycine, valyl-leucine, leucyl-histidine, oleamide, 9,12,13-trihydroxyoctadecenoic acid, stearidonyl carnitine and squalene. Differences of metabolite profiles of human urine could be identified by gold nanoparticle-enhanced target (AuNPET) LDI MS method and used for the detection of renal cancer.

The lack of symptoms at the early stages of clear cell renal cell carcinoma (ccRCC) allows the tumour to metastasize, leading to a dramatic reduction in patient survival. Therefore, we studied and set up a method based on urinary microRNAs (miRNAs) for the diagnosis of ccRCC. First, miRNA expression in ccRCC specimens and kidney tissues from healthy subjects (HSs) was investigated through analysis of data banks and validated by comparing expression of miRNAs in ccRCC and adjacent non-cancerous kidney tissue specimens by RT-qPCR. Subsequently, we developed an algorithm to establish which miRNAs are more likely to be found in the urine of ccRCC patients that indicated miR-122, miR-1271, and miR-15b as potential interesting markers. The evaluation of their levels and three internal controls in the urine of 13 patients and 14 HSs resulted in the development of a score (7p-urinary score) to evaluate the presence of ccRCC in patients. The resulting area under the Receiver Operating Characteristic (ROC) curve, sensitivity, and specificity were equal to 0.96, 100% (95% CI 75-100%), and 86% (95% CI 57-98%), respectively. In conclusion, our study provides a proof of concept that combining the expression values of some urinary miRNAs might be useful in the diagnosis of ccRCC.

Purpose: Human Kidney Injury Molecule-1 (hKIM-1) was proposed as urinary biomarker of renal cell carcinoma (RCC). The aim of the study was to validate urinary hKIM-1 as a biomarker of RCC., Material and Methods: Forty-six participants were enrolled into the study, including 30 patients with clear-cell or papillary RCC and 16 matched patients in the comparison group. Preoperative urinary hKIM-1 levels were measured using commercially available ELISA kit and normalized to urinary creatinine levels., Results: The concentrations of urinary hKIM-1 normalized to urinary creatinine in patients with RCC and comparison group did not differ significantly (1.35 vs. 1.32 ng/mg creatinine, p=.25). There was also no difference in urinary hKIM-1 concentration regarding stage or grade of renal cancer. Additional analysis of patients without chronic kidney disease (defined as eGFR ≥60mL/min/1.73m²) also did not reveal significant difference in urinary hKIM-1 concentrations between the groups (1.54 vs. 1.37; p=.47)., Conclusion: Results of our study do not confirm recent suggestions that urinary hKIM-1 may be a biomarker of RCC.

Background: The rising incidence of renal cell carcinomas (RCC) constitutes a significant challenge owing to risk of overtreatment. Because aberrant microRNA (miR) promoter methylation contributes to cancer development, we investigated whether altered miR-30a-5p expression associates with DNA promoter methylation and evaluated the usefulness as clear cell RCC (ccRCC) diagnostic and prognostic markers., Methods: Genome-wide methylome and RNA sequencing data from a set of ccRCC and normal tissue samples from The Cancer Genome Atlas (TCGA) database were integrated to identify candidate CpG loci involved in cancer onset. MiR-30a-5p expression and promoter methylation were quantitatively assessed by PCR in a tissue set (Cohort #1) and urine sets (Cohorts #2 and 3) from IPOPorto and Homburg University Hospital. Non-parametric tests were used for comparing continuous variables. MiR-30a-5p promoter methylation (miR-30a-5p me ) performance as diagnostic (receiver operator characteristics [ROC] - validity estimates) and prognostic [metastasis-free (MFS) and disease-specific survival (DSS)] biomarker was further validated in urine samples from ccRCC patients by Kaplan Meier curves (with log rank) and both univariable and multivariable analysis., Results: Two significant hypermethylated CpG loci in TCGA ccRCC samples, correlating with miR-30a-5p transcriptional downregulation, were disclosed. MiR-30a-5p me in ccRCC tissues was confirmed in an independent patient's cohort of IPOPorto and associated with shorter time to relapse. In urine samples, miR-30a-5p me levels identified cancer both in testing and validation cohorts, with 83% sensitivity/53% specificity and 63% sensitivity/67% specificity, respectively. Moreover, higher miR-30a-5p me levels independently predicted metastatic dissemination and survival., Conclusion: To the best of our knowledge, this is the first study validating the diagnostic and prognostic potential of miR-30a-5p me for ccRCC in urine samples, providing new insights for its clinical usefulness as non-invasive cancer biomarker.

Renal cell carcinoma (RCC) is frequently diagnosed incidentally as an early-stage small renal mass (SRM; pT1a, ≤4 cm). Overtreatment of patients with benign or clinically indolent SRMs is increasingly common and has resulted in a recent shift in treatment recommendations. There are currently no available biomarkers that can accurately predict clinical behavior. Therefore, we set out to identify early biomarkers of RCC progression. We employed a quantitative label-free liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomics approach and targeted parallel-reaction monitoring to identify and validate early, noninvasive urinary biomarkers for RCC-SRMs. In total, we evaluated 115 urine samples, including 33 renal oncocytoma (≤4 cm) cases, 30 progressive and 26 nonprogressive clear cell RCC (ccRCC)-SRM cases, in addition to 26 healthy controls. We identified six proteins, which displayed significantly elevated expression in clear cell RCC-SRMs (ccRCC-SRMs) relative to healthy controls. Proteins C12ORF49 and EHD4 showed significantly elevated expression in ccRCC-SRMs compared to renal oncocytoma (≤4 cm). Additionally, proteins EPS8L2, CHMP2A, PDCD6IP, CNDP2 and CEACAM1 displayed significantly elevated expression in progressive relative to nonprogressive ccRCC-SRMs. A two-protein signature (EPS8L2 and CCT6A) showed significant discriminatory ability (areas under the curve: 0.81, 95% CI: 0.70-0.93) in distinguishing progressive from nonprogressive ccRCC-SRMs. Patients (Stage I-IV) with EPS8L2 and CCT6A mRNA alterations showed significantly shorter overall survival (p = 1.407 × 10 -6 ) compared to patients with no alterations. Our in-depth proteomic analysis identified novel biomarkers for early-stage RCC-SRMs. Pretreatment characterization of urinary proteins may provide insight into early RCC progression and could potentially help assign patients to appropriate management strategies., (© 2019 UICC.)

Purpose of Review: Renal cell carcinoma (RCC) is the third most common urologic malignancy. First symptoms are usually unspecific and belated causing the stages to be high when diagnosed. As compensation, incidental detection of RCC by abdominal imaging techniques for other medical purposes is a reality that favours a decrease in the stage of new diagnosed tumours. Therefore, identifying novel predictive biomarkers for diagnosis, progression and prognosis of RCC is fundamental., Recent Findings: To date, several studies have demonstrated that microRNAs (miRNAs) play a role in the particular scenario of urologic tumors, and alterations at miRNA level are involved in the initiation, progression and metastases formation of renal cancer. In the present review, we have summarized the up‑to‑date preliminary clinical works on the role of urinary miRNA profiling in RCC, including an evaluation of its value as a potential biomarker for diagnosis, prognosis and follow up of RCC patients.

Background: Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established., Methods: Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine., Results: Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings., Conclusions: These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.

Background: Renal cell carcinoma (RCC) is often detected incidentally as a small renal mass (SRM; pT1a, ≤4 cm). It is clinically challenging to predict progression in patients with SRMs. This is largely due to the recent recognition of clinically progressive and non-progressive RCC-SRMs. It is critical to accurately stratify SRM patients according to risk to avoid unnecessary treatment. This is especially significant for elderly and infirm patients, where the risk of surgery outweighs mortality from SRMs., Methods: We employed a qRT-PCR array-based approach and targeted qRT-PCR to identify and validate early, non-invasive diagnostic and prognostic biomarkers of RCC-SRMs. In total, we evaluated eighty urine samples, including 30 renal oncocytoma (≤4 cm) cases, 26 progressive and 24 non-progressive clear cell RCC-SRM (ccRCC-SRM) cases., Results: We identified nine urinary miRNAs which displayed significantly elevated expression in ccRCC-SRMs (pT1a; ≤4 cm) relative to renal oncocytoma (≤4 cm). Additionally, miR-328-3p displayed significantly down-regulated expression in progressive relative to non-progressive ccRCC-SRMs. Patients with elevated miR-328-3p expression had significantly longer overall survival (HR = 0.29, 95% CI = 0.08-1.03, p = 0.042) compared to patients with low miR-328-3p expression. We also found no significant association between miR-328-3p expression levels and gender, age, laterality, tumor size, or grade, suggesting that miR-328-3p is an independent prognostic biomarker., Conclusions: Our in-depth miRNA profiling approach identified novel biomarkers for early-stage ccRCC-SRMs. Pretreatment characterization of urinary miRNAs may provide insight into early RCC progression and could potentially aid clinical decision-making, improving patient management and reducing overtreatment., (Copyright © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: MicroRNAs (miRNAs) are emerging as promising molecules in the diagnosis, prognosis and treatment of urological tumours. Recently, our group performed two independent studies highlighting that miR-210-3p may be a useful biomarker not only for diagnosis but also for post-surgery clear cell Renal Cell Carcinoma (ccRCC) management., Objective: The aim of this study is to further explore the effectiveness of miRNA as non-invasive biomarker for clinical outcomes and ccRCC response to the treatment., Methods: We analyzed miR-210-3p levels in neoplastic and healthy tissue and in urine specimens collected at surgery and during follow-up of 21 ccRCC patients by RTqPCR., Results: Firstly, we confirmed that the expression of miR-210-3p was upregulated in tumor tissues and in urine samples of analyzed cohort. Of note is that miR-210-3p expression was significantly reduced in urine samples from disease-free patients during follow-up (from 3 to 12 months) compared to the baseline levels observed at the time of surgery. In a small subgroup of patients presenting metastatic progression (such as bone, intestinal or lung metastasis), the urine levels of miR-210-3p correlated with responsiveness to the therapy., Conclusions: This pilot study highlights the relevance of secreted miR-210-3p as powerful non-invasive prognostic and predictive biomarker for the evaluation of clinical outcomes and treatment response during ccRCC follow up.

Renal cell carcinoma (RCC) is often diagnosed incidentally as a small renal mass (SRM; pT1a, ≤4 cm). Increasing concerns surrounding the overtreatment of patients with benign or clinically silent SRMs has resulted in a recent shift in treatment recommendations, especially in elderly and infirm patients. There are currently no biomarkers that can predict progression. We used a quantitative label-free liquid chromatography-tandem mass spectrometry peptidomics approach and targeted parallel-reaction monitoring to identify early, noninvasive diagnostic and prognostic biomarkers for early-stage RCC-SRMs. In total, 115 urine samples, including 33 renal oncocytoma (≤4 cm) cases, 30 progressive and 26 nonprogressive clear cell RCC-SRM cases, and 26 healthy controls were evaluated. Nine endogenous peptides that displayed significantly elevated expression in clear cell RCC-SRMs relative to healthy controls were identified. Peptides NVINGGSHAGNKLAMQEF, VNVDEVGGEALGRL, and VVAGVANALAHKYH showed significantly elevated expression in clear cell RCC-SRMs relative to renal oncocytoma. Additionally, peptides SHTSDSDVPSGVTEVVVKL and IVDNNILFLGKVNRP displayed significantly elevated expression in progressive relative to nonprogressive clear cell RCC-SRMs. Peptide SHTSDSDVPSGVTEVVVKL showed the most significant discriminatory utility (area under the curve, 0.76; 95% CI, 0.62-0.90; P = 0.0027). Patients with elevated SHTSDSDVPSGVTEVVVKL expression had significantly shorter overall survival (hazard ratio, 4.13; 95% CI, 1.09-15.65; P = 0.024) compared to patients with low expression. Pretreatment characterization of urinary peptides can provide insight into early RCC progression and may aid clinical decision-making and improve disease management., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Despite rapid advances in diagnostic and therapeutic medicine, renal cell carcinoma (RCC) continues to cause significant morbidity and mortality in patients. While there has been a shift towards earlier detection, approximately 16% of patients present with metastatic disease at the time of diagnosis. Kidney injury molecule-1 (KIM-1) is a glycoprotein that has been shown to be a robust and reliable biomarker of acute proximal tubular injury. As KIM-1 is mainly expressed in RCC derived from the proximal tubules, it is a reliable marker to differentiate between proximal tubular primary RCC and distal nephron primary RCC. Several studies have investigated urinary KIM-1 (uKIM-1) in RCC and demonstrated that it is a sensitive and specific marker for detecting localized RCC, as patients had markedly reduced uKIM-1 levels following nephrectomy, with uKIM-1 levels correlating with tumor size and grade. In addition, levels of KIM-1 present in plasma have also shown utility as a biomarker of RCC with levels being elevated in RCC cases at least 5 years before diagnosis. This review focuses on a progressive understanding of KIM-1 in the diagnosis of RCC using biopsies, urine, and plasma samples, and it will also provide some insight into potential roles of KIM-1 in the growth and spread of RCC.

Exosome-derived miRNAs are regarded as biomarkers for the diagnosis and prognosis of many human cancers. However, its function in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, differentially expressed miRNAs from urinal exosomes were identified using next-generation sequencing (NGS) and verified using urine samples of ccRCC patients and healthy donors. Then, the exosomes were analysed in early-stage ccRCC patients, healthy individuals and patients suffering from other urinary system cancers. Thereafter, the target gene of the miRNA was detected. Its biological function was investigated in vitro and in vivo. The results showed that miR-30c-5p could be amplified in a stable manner. Its expression pattern was significantly different only between ccRCC patients and healthy control individuals, but not compared with that of other urinary system cancers, which indicated its specificity for ccRCC. Additionally, the overexpression of miR-30c-5p inhibited ccRCC progression in vitro and in vivo. Heat-shock protein 5 (HSPA5) was found to be a direct target gene of miR-30c-5p. The depletion of HSPA5 caused by miR-30c-5p inhibition reversed the promoting effect of ccRCC growth. In conclusion, urinary exosomal miR-30c-5p acts as a potential diagnostic biomarker of early-stage ccRCC and may be able to modulate the expression of HSPA5, which is correlated with the progression of ccRCC., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Wilms' tumor is the most common type of childhood kidney cancer. To improve risk stratification and identify novel therapeutic targets for patients with Wilms' tumor, we used high-resolution mass spectrometry proteomics to identify urine tumor markers associated with Wilms' tumor relapse. We determined the urine proteomes at diagnosis of 49 patients with Wilms' tumor, non-Wilms' tumor renal tumors, and age-matched controls, leading to the quantitation of 6520 urine proteins. Supervised analysis revealed specific urine markers of renal rhabdoid tumors, kidney clear cell sarcomas, renal cell carcinomas as well as those detected in patients with cured and relapsed Wilms' tumor. In particular, urine prohibitin was significantly elevated at diagnosis in patients with relapsed as compared with cured Wilms' tumor. In a validation cohort of 139 patients, a specific urine prohibitin ELISA demonstrated that prohibitin concentrations greater than 998 ng/mL at diagnosis were significantly associated with ultimate Wilms' tumor relapse. Immunohistochemical analysis revealed that prohibitin was highly expressed in primary Wilms' tumor specimens and associated with disease stage. Using functional genetic experiments, we found that prohibitin was required for the growth and survival of Wilms' tumor cells. Overexpression of prohibitin was sufficient to block intrinsic mitochondrial apoptosis and to cause resistance to diverse chemotherapy drugs, at least in part by dysregulating factors that control apoptotic cytochrome c release from mitochondrial cristae. Thus, urine prohibitin may improve therapy stratification, noninvasive monitoring of treatment response, and early disease detection. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin dysregulation may offer improved therapies for patients with Wilms' and other relapsed or refractory tumors.

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early-stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC-TFE3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)-204-5p levels in urinary exosomes of 40-week-old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA-204-5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR-204-5p-containing exosomes. Notably, we also observed increased miR-204-5p levels in urinary exosomes in 20-week-old renal PRCC-TFE3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40-week-old Tg mice, suggesting that miR-204-5p increases follow expression of constitutively active TFE3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR-204-5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC-TFE3 fusion gene. These findings suggest that miR-204-5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Objective: To evaluate the role of urine aquaporin 1 and perilipin 2 as biomarkers adjunct to renal mass biopsy in guiding the management of patients with small renal masses., Methods: Preoperative aquaporin 1 and perilipin 2 levels in 57 patients with small renal masses undergoing partial nephrectomy were analyzed and compared with postoperative tumor histology. An algorithm was created utilizing aquaporin 1 and perilipin 2 in conjunction with renal mass biopsy. Cut-off values were implemented to maximize biomarker sensitivity and specificity. Renal mass biopsy utilization and intervention were then compared with rates in traditional renal mass biopsy algorithms., Results: All clear cell and papillary renal cell carcinomas were correctly identified and assigned to the treatment path. All benign lesions were correctly sorted to a confirmatory renal mass biopsy path. Two chromophobe masses did not have elevated aquaporin 1 and perilipin 2, and would require renal mass biopsy. Compared with protocols that call for all small renal masses to be biopsied, confirmatory renal mass biopsy could have been safely avoided in 74% of patients with elevated aquaporin 1 and perilipin 2. Compared with protocols that do not utilize renal mass biopsy, surgical intervention would have been avoided in 23% of patients with benign masses., Conclusions: Aquaporin 1 and perilipin 2 possess high sensitivity and specificity for detecting clear cell and papillary renal cell carcinoma. Use of these markers might compliment renal mass biopsy in the characterization of small renal masses., (© 2018 The Japanese Urological Association.)

Objective: This study aims to observe the changes and diagnostic values of urinary trace elements in patients with renal cell carcinoma (RCC)., Methods: A total of 28 RCC patients that had been performed radical surgery for more than 3 years were included into this case-control study; meanwhile, thirty healthy volunteers without kidney diseases were set as the control group over the same period. The levels of various urinary trace elements in both groups were measured, and the results were performed the significance analysis and pattern recognition analysis using with partial least square discriminant analysis and Fisher analysis., Results: The significance analysis showed that compared with the control group, the case group exhibited significantly reduced levels of Mg (26.3 mg/L for the case group vs. 52.1 mg/L for the control group, P < 0.05), V (72.9 μg/L for the case group vs. 110.1 μg/L for the control group, P < 0.05), Mo (59.6 μg/L for the case group vs. 261.7 μg/L for the control group, P < 0.05), and Sn (4.5 μg/L for the case group vs. 27.3 μg/L for the control group, P < 0.05) while significantly increased Cd (25.0 μg/L for the case group vs. 15.5 μg/L for the control group, P < 0.05). The accuracy of the discriminant function established by the Fisher analysis was 91.4%., Conclusions: Patients with RCC exhibit differences in such urinary trace elements as Mg, V, Mo, Sn, and Cd with healthy populations, and the discriminant accuracy is high., Competing Interests: None

Some urine-based molecular markers can be used effectively in certain clinical situations, but so far none of them have made it into the daily routine. For this purpose, clinical validation in large multicentre prospective studies with defined endpoints is lacking, whereby the cost effectiveness should also be a criterion of the evaluation. This also applies to the many promising "research parameters", which were usually only examined in small groups of patients. The uniform standardization of molecular biological methods is the first step that makes a comparison possible. Current impediments to conducting such studies include elaborate analytics and their still high costs. In summary, the development that has occurred-from the uroscopy to the information that a drop of urine is able to provide today-gives an optimistic outlook on the future: the old idea (of comprehensive urine diagnostics) in new bottles (with new analysis methods?) maybe yet be realized.

In this study, we compared different types of biomolecular markers in kidney cancer patients and in normal healthy controls, using fluorescence emission spectra and synchronous fluorescence excitation spectra. We were able to provide an accurate classification of the spectral features of kidney cancer patients relative to that of normal controls, in terms of the concentration ratios of biomolecules (viz., tryptophan, NADH, FAD, basic porphyrin, and acidic porphyrin) based on the intensity of their spectral peaks. The specificity and sensitivity of the method were 90%. The rationale of our current approach is to evolve an innovative protocol for the spectral characterization of in vitro optical analyses suitable for both small clinics and hospitals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Over the last decade, the improvement in molecular techniques and the acquisition of genomic information has transformed and increased the quality of patient care and our knowledge of diseases. Areas covered: Protein expression levels in immunohistochemistry and molecular biomarkers are reported for their ability to predict recurrence, progression, development of metastases, or patient survival. In particular, for renal cell carcinoma, we take into consideration the biomarkers applicable to immunohistochemistry and with molecular and genetic analyses. In urothelial carcinoma, there is great interest in the possibility of distinguishing the basal vs. luminal subtypes and to acquire deeper insight into the tumor biology through examining exosomes in urine and biomarkers in the serum. In prostate cancer, single gene expression and multiple gene expression classifiers are reviewed as a tool to distinguish indolent vs. aggressive disease. Expert commentary: The genomic information along with the application of ancillary techniques allow the definition of a neoplasia not only by its morphology but also by its biological signature. This continuous increase in knowledge will result in a better comprehension of oncogenesis, development of targeted therapies and optimizing decision-making processes related to patient care.

Our recent study found that high urinary total arsenic levels were associated with renal cell carcinoma (RCC). Recent studies demonstrated that low circulating adiponectin was related to RCC. The aim of the present study was to explore the relationship between adiponectin gene (ADIPOQ) polymorphisms and RCC and investigate whether individuals with an ADIPOQ risk genotype, obesity, and high urinary total arsenic levels have a modified odds ratio (OR) of RCC. A total of 389 RCC patients and 389 age- and sex-matched controls were recruited between November 2006 and December 2012 in Taiwan. Image-guided biopsy or surgical resection of renal tumors was performed to pathologically verify RCC. Genomic DNA was used to examine the genotypes of the ADIPOQ rs182052, ADIPOQ rs2241766, ADIPOQ rs1501299, and ADIPOQ rs1063539 SNPs by PCR-RFLP. HPLC-HG-AAS was used to measure the concentrations of urinary arsenic species. Participants with the ADIPOQ rs182052 G/A+A/A genotype had a significantly higher OR of RCC compared with those with the ADIPOQ rs182052 G/G genotype. The OR (95% confidence interval [CI]) was 1.70 (1.23-2.36). The OR of RCC for the combined effect of high urinary total arsenic levels and obesity, which was dose-dependent, in individuals with the ADIPOQ rs182052 G/A+A/A genotype was 9.33 (3.85-22.62). The present study found significant combined effects of obesity and the ADIPOQ rs182052 G/A+A/A genotype on the arsenic-related risk of RCC in a population with low arsenic exposure. Arsenic exposure, obesity, and the ADIPOQ rs182052 polymorphism could be predictors of a higher OR of RCC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Renal cell carcinoma (RCC) is the most prevalent and lethal malignancy of the kidney. Despite all the efforts made, no tissue biomarker is currently used in the clinical management of patients with kidney cancer. A search for possible biomarkers in urine for clear cell renal cell carcinoma (ccRCC) has been conducted. Non-targeted metabolomic analyses were performed on paired samples of surgically removed renal cancer and normal tissue, as well as on urine samples. Extracts were analyzed by liquid chromatography/high-resolution mass spectrometry (LC-HRMS). Hydroxybutyrylcarnitine, decanoylcarnitine, propanoylcarnitine, carnitine, dodecanoylcarnitine, and norepinephrine sulfate were found in much higher concentrations in both cancer tissues (compared with the paired normal tissue) and in urine of cancer patients (compared with control urine). In contrast, riboflavin and acetylaspartylglutamate (NAAG) were present at significantly higher concentrations both in normal kidney tissue as well as in urine samples of healthy persons. This preliminary study resulted in the identification of several compounds that may be considered potential clear cell renal carcinoma biomarkers. Graphical abstract PLS-DA plot based on LC-MS data for normal and cancer human tissue samples. The aim of this work was the identification of up- and downregulated compounds that could potentially serve as renal cancer biomarkers.

Introduction: Currently, there is no accurate diagnostic molecular biomarker for renal cell carcinoma (RCC). The aim of this study was to assess the expression of microRNA-15a (miR-15a) in urine of patients with RCC and to evaluate its potential as a diagnostic molecular biomarker., Materials and Methods: In total, 67 patients with solid renal tumors were enrolled: clear-cell RCC (ccRCC, n = 22), papillary RCC (pRCC, n = 16), chromophobe RCC (chRCC, n = 14), oncocytoma (n = 8), papillary adenoma (n = 2) and angiomyolipoma (n = 5). MiRNA-15a expression levels measurement in urine were performed using qPCR. Urine of 15 healthy volunteers without kidney pathology was used as control., Results: We observed a difference in mean miR-15a expression values in groups of pre-operative patients with RCC, benign renal tumors and healthy persons (2.50E-01 ± 2.72E-01 vs 1.32E-03 ± 3.90E-03 vs 3.36E-07 ± 1.04E-07 RFU, respectively, p < 0.01). There was no difference in miR-15a expression between ccRCC, pRCC and chRCC (p > 0.05). Direct association between RCC size and miR-15a expression values was obtained (Pearson correlation coefficient-0.873). On the 8th day after nephrectomy, mean expression value in patients with RCC decreased by 99.53% (p < 0.01). MiR-15a expression differentiated RCC from benign renal tumors with 98.1% specificity, 100% sensitivity at a cut-off value of 5.00E-06 RFU, with AUC-0.955., Conclusions: MiR-15a expression measured in urine may be used as diagnostic molecular biomarker for RCC.

Urinary profiling datasets, previously acquired by capillary electrophoresis coupled to mass-spectrometry were investigated to identify a general urinary marker pattern for detection of solid tumors by targeting common systemic events associated with tumor-related inflammation. A total of 2,055 urinary profiles were analyzed, derived from a) a cancer group of patients (n = 969) with bladder, prostate, and pancreatic cancers, renal cell carcinoma, and cholangiocarcinoma and b) a control group of patients with benign diseases (n = 556), inflammatory diseases (n = 199) and healthy individuals (n = 331). Statistical analysis was conducted in a discovery set of 676 cancer cases and 744 controls. 193 peptides differing at statistically significant levels between cases and controls were selected and combined to a multi-dimensional marker pattern using support vector machine algorithms. Independent validation in a set of 635 patients (293 cancer cases and 342 controls) showed an AUC of 0.82. Inclusion of age as independent variable, significantly increased the AUC value to 0.85. Among the identified peptides were mucins, fibrinogen and collagen fragments. Further studies are planned to assess the pattern value to monitor patients for tumor recurrence. In this proof-of-concept study, a general tumor marker pattern was developed to detect cancer based on shared biomarkers, likely indicative of cancer-related features.

Context: Von Hippel-Lindau disease (VHLD) is a rare inherited neoplastic syndrome. Among all the VHLD-associated tumors, clear cell renal cell carcinoma (ccRCC) is the major cause of death., Objective: The aim of this paper is the discovery of new non-invasive biomarker for the monitoring of VHLD patients., Materials and Methods: We compared the urinary proteome of VHLD patients, ccRCC patients and healthy volunteers., Results: Among all differentially expressed proteins, alpha-1-antitrypsin (A1AT) and APOH (beta-2-glycoprotein-1) are strongly over-abundant only in the urine of VHLD patients with a history of ccRCC., Discussion and Conclusion: A1AT and APOH could be promising non-invasive biomarkers.

The objective of this prospective follow-up trial was to ascertain whether the urinary kidney injury molecule-1 (uKIM-1) associates with tumor tissue (tKIM-1) expression and with the pathological characteristics of clear renal cell carcinoma (cRCC) in radically nephrectomized (RN) and/or in partially nephrectomized (PN) patients with cRCC, pre- and postoperatively. This clinical study included 40 patients subjected to RN/PN (cRCC group) and 30 healthy volunteers (control group). Urinary KIM-1 was determined by ELISA TIM-1/KIM-1 kit and normalized by urinary creatinine. Immunohistochemical staining (monoclonal anti-human anti-TIM-1/KIM-1/HAVCR antibody) was used for semiquantitative analysis of the tKIM-1 expression and expressed as a score (% KIM-1 positively stained tubules). Both markers were interpreted in terms of the tumor characteristics comprising tumor size, Fuhrman grade, pathological (pT) stage, tumor/nodes/metastasis (TNM) stage, lymphovascular invasion and type of surgery RN/PN. Preoperative uKIM-1 was significantly higher in the cRCC group compared to controls, such as uKIM-1 was statistically higher in RN than in PN patients. Postoperatively, uKIM-1 decreased to control values. Expression of tKIM-1 was documented in all nephrectomized patients. Significant associations were achieved between uKIM-1 and tKIM-1 and with considered tumor characteristics, especially with tumor size and grade. Based on the accomplished associations, we found uKIM-1 as a highly sensitive marker for cRCC diagnosis. The clinical trial registration number: 1110-2012.

Objective To evaluate the application of weak cation exchange (WCX) magnetic bead-based Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in detecting differentially expressed proteins in the urine of renal clear cell carcinoma (RCCC) and its value in the early diagnosis of RCCC.Methods Eleven newly diagnosed patients (10 males and 1 female, aged 46-78, mean 63 years) of renal clear cell carcinoma by biopsy and 10 healthy volunteers (all males, aged 25-32, mean 29.7 years) were enrolled in this study. Urine samples of the RCCC patients and healthy controls were collected in the morning. Weak cation exchange (WCX) bead-based MALDI-TOF MS technique was applied in detecting differential protein peaks in the urine of RCCC. ClinProTools2.2 software was utilized to determine the characteristic proteins in the urine of RCCC patients for the predictive model of RCCC. Results The technique identified 160 protein peaks in the urine that were different between RCCC patients and health controls; and among them, there was one peak (molecular weight of 2221.71 Da) with statistical significance (P=0.0304). With genetic algorithms and the support vector machine, we screened out 13 characteristic protein peaks for the predictive model. Conclusions The application of WCX magnetic bead-based MALDI-TOF MS in detecting differentially expressed proteins in urine may have potential value for the early diagnosis of RCCC.

The analysis of volatile organic compounds (VOCs) emanating from biological samples appears as one of the most promising approaches in metabolomics for the study of diseases, namely cancer. In fact, it offers advantages, such as non-invasiveness and robustness for high-throughput applications. The purpose of this work was to study the urinary volatile metabolic profile of patients with renal cell carcinoma (RCC) (n = 30) and controls (n = 37) with the aim of identifying a potential specific urinary volatile pattern as a non-invasive strategy to detect RCC. Moreover, the effect of some confounding factors such as age, gender, smoking habits and body mass index was evaluated as well as the ability of urinary VOCs to discriminate RCC subtypes and stages. A headspace solid-phase microextraction/gas chromatography-mass spectrometry-based method was performed, followed by multivariate data analysis. A variable selection method was applied to reduce the impact of potential redundant and noisy chromatographic variables, and all models were validated by Monte Carlo cross-validation and permutation tests. Regarding the effect of RCC on the urine VOCs composition, a panel of 21 VOCs descriptive of RCC was defined, capable of discriminating RCC patients from controls in principal component analysis. Discriminant VOCs were further individually validated in two independent samples sets (nine RCC patients and 12 controls, seven RCC patients with diabetes mellitus type 2) by univariate statistical analysis. Two VOCs were found consistently and significantly altered between RCC and controls (2-oxopropanal and, according to identification using NIST14, 2,5,8-trimethyl-1,2,3,4-tetrahydronaphthalene-1-ol), strongly suggesting enhanced potential as RCC biomarkers. Gender, smoking habits and body mass index showed negligible and age-only minimal effects on the urinary VOCs, compared to the deviations resultant from the disease. Moreover, in this cohort, the urinary volatilome did not show ability to discriminate RCC stages and histological subtypes. The results validated the value of urinary volatilome for the detection of RCC and advanced with the identification of potential RCC urinary biomarkers., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Clear cell Renal Cell Carcinoma (ccRCC) causes over 13,000 deaths each year, and about 20,000 new cases/year in Europe. In most cases, the causes are unknown and, most importantly, there are no reliable biomarkers for the diagnosis and prognosis of this disease. The search for sensitive biomarkers for early diagnosis and prognosis of clear cell Renal Cell Carcinoma (ccRCC) is currently a fast growing field. We carried out proteomics analysis of 93 urinary samples of healthy subjects (HS) and patients affected by ccRCC, prostate cancer (PCa) and chronic kidney disease (CKD), that was able to successfully distinguish each group.The most significant candidate biomarker was identified by mass spectrometry as Raf Kinase Inhibitor Protein (RKIP), a key regulator of cell signaling, already described in several cancer types as a metastasis suppressor. By combining ELISA, immunoblotting and tissue microarray, we demonstrated that, in ccRCC, urinary excretion of RKIP and its phosphorylated form (p-RKIP) reflected the tissue expression of these putative biomarkers. Baseline urinary RKIP, evaluated in an independent cohort of 56 ccRCC patients and 28 HS, successfully distinguished both groups and, most importantly, a cut-off value of 10 ng/mg/g Pr/uCr enabled a highly accurate prediction of Cancer-specific survival and Progression-free survival. Furthermore, p-RKIP was totally undetectable in both tissue and urine samples of ccRCC, showing a great potential for diagnostics purposes.Our data indicate that urinary RKIP encompasses both the unphosphorylated and the phosphorylated form and that their combined evaluation can help in the diagnosis and prognosis of ccRCC.

Background: Recent findings show that cell-free miRNAs are stable in biological fluid. Urine provides an alternative to blood serum or plasma as a potential source of tumor biomarkers. MiR-210 is proven to be overexpressed in patients with a clear cell renal cell carcinoma (ccRCC). The purpose of this pilot study was to examine the urinary cell-free miR-210 as a potential tool of liquid biopsy for ccRCC., Methods: Overall, 75 patients with a ccRCC and 45 control subjects without a cancer were included in this study. Urine samples were centrifuged twice and the cell-free urine supernanants were stored in -80°C until use. A total of 350µl cell-free urine was used for the extraction of total RNA. The expression levels of these miRNAs were performed by using quantitative RT-PCR., Results: The level of urinary cell-free miR-210 was significantly higher in patients with ccRCC than in control subjects (P<0.001). The urinary cell-free miR-210 yielded the areas under the receiver operating characteristics curve of 0.76 in discriminating the patients with ccRCC from the control subjects with a sensitivity of 57.8% and a specificity of 80.0%. Moreover, the expression level of urinary cell-free miRNA-210 was significantly decreased in the patients a week after surgery (P<0.001)., Conclusion: Urinary cell-free miR-210 may be used as a potential tool of liquid biopsy for ccRCC diagnosis. Further studies are needed to confirm our results., (Copyright © 2017. Published by Elsevier Inc.)

Context: By 2020 the estimated incidence of genitourinary (GU) cancers (prostate, bladder, and kidney) will be over 2 million worldwide and responsible for ∼800 000 deaths. Current diagnosis and monitoring methods of GU cancer patients are often invasive and/or lack sensitivity and specificity. Given the utility of blood-based cell-free nucleic acid (cfNA) biomarkers, the development of urinary cfNA biomarkers may improve the sensitivity of urine assays utilizing urine sediment for GU cancers. This review of urinary cfNA in GU cancers identifies the current stage of research, potential clinical utility, and the next steps needed to enter clinical use., Objective: To critically evaluate the literature of urinary cfNA in GU cancers for clinical utility in diagnosis, screening, and precision medicine. Furthermore, the strategy for future efforts to discover potential new urinary cfNA biomarkers will be described., Evidence Acquisition: A PubMed database (2006 to current) search was performed according to Preferred Reporting Items for Systemic Review and Meta-analysis using key Medical Subject Headings terms. Additional studies were obtained by cross-referencing from the literature., Evidence Synthesis: The collective research publications in urinary cfNA of GU cancers present a promising alternative liquid biopsy approach compared with blood biopsies and urine sediment, particularly for early-stage GU diseases., Conclusions: Urinary cfNA as a liquid biopsy holds potential for a more sensitive alternative to blood biopsies and urine sediment-based tests for clinical use in GU cancers. Not only does urinary cfNA offer advantages including the potential for more frequent testing, monitoring, and home use, but also has applications in early-stage GU cancers., Patient Summary: In this review, we evaluated the current status of urinary cell-free nucleic acid in genitourinary cancers. We identified the potential advantages of urinary cell-free nucleic acid over blood and urine sediment and its clinical use in genitourinary cancer., (Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3% of adult malignancies. P-Element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a new class of naturally occurring, short non-coding RNAs involved in silencing of transposable elements and in sequence-specific chromatin modifications. There were preliminary data published indicating that piR-823 expression is deregulated in circulating tumor cells and tumor tissue in gastric and kidney cancer, respectively., Patients and Methods: In our study, we analyzed piR-823 levels in 588 biological specimens: tumor tissue (N=153), adjacent renal parenchyma (N=121), blood serum (N=178) and urine (N=20) of patients undergoing nephrectomy for RCC; and in blood serum (N=101) and urine (N=15) of matched healthy controls. Expression levels of piR-823 were determined in all biological specimens by quantitative real-time polymerase chain reaction, compared in patients and controls, and correlated with clinicopathological features of RCC., Results: We identified a significant down-regulation of piR-823 in tumor tissue [p<0.0001, area under the curve (AUC)=0.7945]. On the contrary in blood serum and urine, the expression of piR-823 was significantly higher in patients with RCC compared to healthy individuals (p=0.0005, AUC=0.6264 and p=0.0157, AUC=0.7433, respectively). We further observed higher levels of piR-823 in tumor tissue to be associated with shorter disease-free survival of patients (p=0.0186) and a trend for higher piR-823 levels in serum to be associated with advanced clinical stages of RCC (p=0.0691). There were no other significant associations of piR-823 levels in any type of biological specimen with clinicopathological features of RCC., Conclusion: piR-823 is down-regulated in tumor tissue, but positively correlated with worse outcome, indicating its complex role in RCC pathogenesis. In blood serum, piR-823 is up-regulated, but with unsatisfactory analytical performance. Preliminary data indicate the promising diagnostic utility of urinary piR-823 in patients with RCC., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

RCC usually develops and progresses asymptomatically and, when detected, it is frequently at advanced stages and metastatic, entailing a dismal prognosis. Therefore, there is an obvious demand for new strategies enabling an earlier diagnosis. The importance of metabolic rearrangements for carcinogenesis unlocked a new approach for cancer research, catalyzing the increased use of metabolomics. The present study aimed the NMR metabolic profiling of RCC in urine samples from a cohort of RCC patients (n = 42) and controls (n = 49). The methodology entailed variable selection of the spectra in tandem with multivariate analysis and validation procedures. The retrieval of a disease signature was preceded by a systematic evaluation of the impacts of subject age, gender, BMI, and smoking habits. The impact of confounders on the urine metabolomics profile of this population is residual compared to that of RCC. A 32-metabolite/resonance signature descriptive of RCC was unveiled, successfully distinguishing RCC patients from controls in principal component analysis. This work demonstrates the value of a systematic metabolomics workflow for the identification of robust urinary metabolic biomarkers of RCC. Future studies should entail the validation of the 32-metabolite/resonance signature found for RCC in independent cohorts, as well as biological validation of the putative hypotheses advanced.

Objective: Non-muscle-invasive bladder cancer (NMIBC) comprises a wide spectrum of tumors with different behaviors and prognoses. It follows that the surveillance for these tumors should be adapted according to the risks of recurrence and progression and should be dynamic in design., Methods and Materials: Medline search was conducted from 1980 to 2016 using a combination of MeSH and keyword terms. The highest available evidence was reviewed to define different risk groups in NMIBC. The performance of different follow-up tools such as urine cytology, cystoscopy, and upper tract imaging in detecting bladder carcinoma was assessed. Different commercially available urinary markers were investigated to determine whether such markers would contribute to the surveillance of patients with NMIBC. A follow-up scheme based on the early evidence is proposed., Results: A risk-based approach is paramount. Cystoscopy and cytology are recommended to be done at 3 months following transurethral resection of bladder tumor. For low-risk tumors, annual cystoscopy alone is sufficient; no upper tract evaluations or cytology is needed except at diagnosis. High-risk tumors should be followed up with a more intense schedule: cystoscopy every 3 months for 2 years, 6 months for 2 years, and then annually, with cytology at frequent intervals, and imaging for upper tract evaluation at 1 year and then every 2 years. Intermediate-risk tumors should be subclassified as per the International Bladder Cancer Group recommendations and when associated with 3 or more of the following findings (multiple tumors, size≥3cm, early recurrence<1 year, frequent recurrences>1 per year) then a surveillance strategy similar to that of high risk should be followed. Several urine markers were more sensitive than cytology in the detection of NMIBC; however, these tests are still costly, require specialized laboratories, and do not replace cystoscopy. Until better and cheaper markers are available, their routine use has not been integrated in the follow-up recommendation of current guidelines., Conclusions: Surveillance of NMIBC should follow a risk-adapted approach, with a combination of cystoscopy, cytology, and upper tract imaging. The aim of this approach is to minimize the therapeutic burden of a disease with high recurrence rates without missing progressing tumors. When designing a diagnostic pathway, first-line diagnostic imaging tests should have high sensitivity to ensure disease positives are included in the test population for further investigation. Second-line investigations should be highly specific, to ensure false-positives are minimized., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Prior studies have shown that 26% to 34% of patients with suspected renal cancers have a glomerular filtration rate less than 60 ml/minute/1.73 m(2) but limited information exists regarding proteinuria. We investigated the extent of proteinuria in patients with renal tumors to determine the impact on the classification and progression of chronic kidney disease., Materials and Methods: Among 1,622 patients evaluated between 1999 and 2014, 1,016 had preoperative creatinine and proteinuria measurements available. Patients were classified according to the risk of chronic kidney disease progression into low, moderately increased, high and very high risk groups according to 2012 KDIGO guidelines. Predictors of risk group and chronic kidney disease progression were analyzed using univariable and multivariate models., Results: Before treatment 32% had a glomerular filtration rate less than 60 ml/minute/1.73 m(2). Preoperative proteinuria was present in 22%. Proteinuria was detected in 30% with a reduced glomerular filtration rate and 18% with a normal glomerular filtration rate. Among the 44% at increased risk for chronic kidney disease progression 24%, 12% and 8% were at moderately increased, high and very high risk, respectively. The presence of proteinuria also reclassified 25% with stage III chronic kidney disease as high or very high risk. KDIGO classification predicted renal functional decline, which occurred in 2.2%, 4.4%, 9.4% and 34.6% at 3 years in low, moderately increased, high and very high risk categories, respectively. Predictors of KDIGO group included age and tumor size (each p <0.001), and the main predictors of renal functional decline were KDIGO group, tumor size and radical nephrectomy (each p <0.0001)., Conclusions: Identification of chronic kidney disease using only glomerular filtration rate left 18% of patients undiagnosed. The assessment of glomerular filtration rate and proteinuria classified patients according to risk of chronic kidney disease progression, identifying 44% to be at increased risk. As proteinuria predicted renal functional decline, we advocate for routine evaluation before treatment., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)