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Colorectal cancer (CRC) is a common malignant tumor in the gastrointestinal tract. Changes in amino acid metabolites have been implicated in tumorigenesis and disease progression. Biomarkers on the basis of chiral amino acids, especially D-amino acids, have not been established for early diagnosis of CRC. Quantification of chiral amino acids, especially very low concentrations of endogenous D-amino acids, is technically challenging. We report here the quantification of L- and D-amino acids in urine samples collected from 115 CRC patients and 155 healthy volunteers, using an improved method. The method of chiral labeling, liquid chromatography, and tandem mass spectrometry enabled separation and detection of 28 amino acids (14 L-amino acids, 13 D-amino acids and Gly). Orthogonal partial least squares discriminant analysis identified 14 targeted variables among these chiral amino acids that distinguished the CRC from the healthy controls. Binary logistic regression analysis revealed that D-α-aminobutyric acid (D-AABA), L-alanine (L-Ala), D-alanine (D-Ala), D-glutamine (D-Gln) and D-serine (D-Ser) could be potential biomarkers for CRC. A receiver operating characteristic curve analysis of combined multi-variables contributed to an area under the curve (AUC) of 0.995 with 98.3 % sensitivity and 96.8 % specificity. A model constructed with D-AABA, D-Ala, D-Gln, and D-Ser achieved an AUC of 0.988, indicating important contributions of D-amino acids to the association with CRC. Further analysis also demonstrated that the metabolic aberration was associated with age and the development of CRC, D-methionine (D-Met) was decreased in CRC patients with age over 50, and D/L-Gln in patients at stage IV was higher than patients at stage I. This study provides the signature of D-amino acids in urine samples and offers a promising strategy for developing non-invasive diagnosis of CRC., 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.)

Gliomas, particularly glioblastomas (GBMs), pose significant challenges due to their aggressiveness and poor prognosis. Early detection through biomarkers is critical for improving outcomes. This study aimed to identify novel biomarkers for gliomas, particularly GBMs, using chiral amino acid profiling. We used chiral amino acid analysis to measure amino acid L- and D-isomer levels in resected tissues (tumor and non-tumor), blood, and urine from 33 patients with primary gliomas and 24 healthy volunteers. The levels of D-amino acid oxidase (DAO), a D-amino acid-degrading enzyme, were evaluated to investigate the D-amino acid metabolism in brain tissue. The GBM mouse model was created by transplanting GBM cells into the brain to confirm whether gliomas affect blood and urine chiral amino acid profiles. We also assessed whether D-amino acids produced by GBM cells are involved in cell proliferation. D-asparagine (D-Asn) levels were higher and DAO expression was lower in glioma than in non-glioma tissues. Blood and urinary D-Asn levels were lower in patients with GBM than in healthy volunteers (p < 0.001), increasing after GBM removal (p < 0.05). Urinary D-Asn levels differentiated between healthy volunteers and patients with GBM (area under the curve: 0.93, sensitivity: 0.88, specificity: 0.92). GBM mouse model validated the decrease of urinary D-Asn in GBM. GBM cells used D-Asn for cell proliferation. Gliomas induce alterations in chiral amino acid profiles, affecting blood and urine levels. Urinary D-Asn emerges as a promising diagnostic biomarker for gliomas, reflecting tumor presence and severity., (© 2024. The Author(s).)

Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancer and a major cause of cancer-related mortality worldwide. Small extracellular vesicles (sEVs) are heterogeneous populations of membrane-structured vesicles that can be found in many biological fluids and are currently considered as a potential source of disease-associated biomarkers for diagnosis. The purpose of this study was to define the proteomic and phosphoproteomic landscape of urinary sEVs in patients with HCC. Mass spectrometry-based methods were used to detect the global proteome and phosphoproteome profiles of sEVs isolated by differential ultracentrifugation. Label-free quantitation analysis showed that 348 differentially expressed proteins (DEPs) and 548 differentially expressed phosphoproteins (DEPPs) were identified in the HCC group. Among them, multiple phosphoproteins related to HCC, including HSP90AA1, IQGAP1, MTOR, and PRKCA, were shown to be upregulated in the HCC group. Pathway enrichment analysis indicated that the upregulated DEPPs participate in the regulation of autophagy, proteoglycans in cancer, and the MAPK/mTOR/Rap1 signaling pathway. Furthermore, kinase-substrate enrichment analysis revealed activation of MTOR, AKT1, MAP2Ks, and MAPKs family kinases in HCC-derived sEVs, indicating that dysregulation of the MAPK and mTOR signaling pathways may be the primary sEV-mediated molecular mechanisms involved in the development and progression of HCC. This study demonstrated that urinary sEVs are enriched in proteomic and phosphoproteomic signatures that could be further explored for their potential use in early HCC diagnostic and therapeutic applications.

Monitoring drug efficacy is significant in the current concept of companion diagnostics in metastatic breast cancer. Trastuzumab, a drug targeting human epidermal growth factor receptor 2 (HER2), is an effective treatment for metastatic breast cancer. However, some patients develop resistance to this therapy; therefore, monitoring its efficacy is essential. Here, we describe a deep learning-assisted monitoring of trastuzumab efficacy based on a surface-enhanced Raman spectroscopy (SERS) immunoassay against HER2-overexpressing mouse urinary exosomes. Individual Raman reporters bearing the desired SERS tag and exosome capture substrate were prepared for the SERS immunoassay; SERS tag signals were collected to prepare deep learning training data. Using this deep learning algorithm, various complicated mixtures of SERS tags were successfully quantified and classified. Exosomal antigen levels of five types of cell-derived exosomes were determined using SERS-deep learning analysis and compared with those obtained via quantitative reverse transcription polymerase chain reaction and western blot analysis. Finally, drug efficacy was monitored via SERS-deep learning analysis using urinary exosomes from trastuzumab-treated mice. Use of this monitoring system should allow proactive responses to any treatment-resistant issues., 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.)

Purpose: Nonsmokers account for 10% to 13% of all lung cancer cases in the United States. Etiology is attributed to multiple risk factors including exposure to secondhand smoking, asbestos, environmental pollution, and radon, but these exposures are not within the current eligibility criteria for early lung cancer screening by low-dose CT (LDCT)., Experimental Design: Urine samples were collected from two independent cohorts comprising 846 participants (exploratory cohort) and 505 participants (validation cohort). The cancer urinary biomarkers, creatine riboside (CR) and N-acetylneuraminic acid (NANA), were analyzed and quantified using liquid chromatography-mass spectrometry to determine if nonsmoker cases can be distinguished from sex and age-matched controls in comparison with tobacco smoker cases and controls, potentially leading to more precise eligibility criteria for LDCT screening., Results: Urinary levels of CR and NANA were significantly higher and comparable in nonsmokers and tobacco smoker cases than population controls in both cohorts. Receiver operating characteristic analysis for combined CR and NANA levels in nonsmokers of the exploratory cohort resulted in better predictive performance with the AUC of 0.94, whereas the validation cohort nonsmokers had an AUC of 0.80. Kaplan-Meier survival curves showed that high levels of CR and NANA were associated with increased cancer-specific death in nonsmokers as well as tobacco smoker cases in both cohorts., Conclusions: Measuring CR and NANA in urine liquid biopsies could identify nonsmokers at high risk for lung cancer as candidates for LDCT screening and warrant prospective studies of these biomarkers., (©2024 The Authors; Published by the American Association for Cancer Research.)

Nuclear matrix protein 22 (NMP22) is one of the most important tumor markers of bladder cancer and is significantly elevated in the urine of bladder cancer patients. Therefore, in this work, a highly sensitive ratiometric electrochemical immunosensor was constructed to detect NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs composites. ZIF-8 had a large surface area and good adsorption ability. Multi-Walled Carbon Nanotubes (MWCNTs) can optimize the electrical conductivity of ZIF-8, so that the electrode surface of ferrocene (Fc) obtains a stable and strong electrochemical signal. In addition, AuPt-MB provided another strong detection signal methylene blue (MB) while immobilizing the secondary antibody (Ab 2 ) through Au-N and Pt-N bonds. A ratiometric electrochemical sensor was formed based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB, which showed a great linear connection between I MB / I Fc and the logarithmic concentration of NMP22 with a detection limit of 3.33 fg mL -1 (S/N = 3) under optimized specifications in the concentration interval of 0.01 pg mL -1 to 1000 ng mL -1 . In addition, the ratiometric immunosensor showed good selectivity and stability.

Prostate-Specific Antigen (PSA) based screening of prostate cancer (PCa) needs refinement. The aim of this study was the identification of urinary biomarkers to predict the Prostate Imaging-Reporting and Data System (PI-RADS) score and the presence of PCa prior to prostate biopsy. Urine samples from patients with elevated PSA were collected prior to prostate biopsy (cohort = 99). The re-analysis of mass spectrometry data from 45 samples was performed to identify urinary biomarkers to predict the PI-RADS score and the presence of PCa. The most promising candidates, i.e. SPARC-like protein 1 (SPARCL1), Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), Alpha-1-microglobulin/bikunin precursor (AMBP), keratin 13 (KRT13), cluster of differentiation 99 (CD99) and hornerin (HRNR), were quantified by ELISA and validated in an independent cohort of 54 samples. Various biomarker combinations showed the ability to predict the PI-RADS score (AUC = 0.79). In combination with the PI-RADS score, the biomarkers improve the detection of prostate carcinoma-free men (AUC = 0.89) and of those with clinically significant PCa (AUC = 0.93). We have uncovered the potential of urinary biomarkers for a test that allows a more stringent prioritization of mpMRI use and improves the decision criteria for prostate biopsy, minimizing patient burden by decreasing the number of unnecessary prostate biopsies., (© 2024. The Author(s).)

Current diagnostic methods for canine urothelial carcinoma (UC) are technically challenging or can lack specificity, hence there is a need for novel biomarkers of UC. To this end, we analysed the microRNA (miRNA) cargo of extracellular vesicles (EVs) from urine samples of dogs with UC to identify candidate miRNA biomarkers. Urine was fractionated using ultrafiltration combined with size-exclusion chromatography and small RNA sequencing analysis was performed on both the EV enriched and (EV free) protein fractions. A greater number of candidate miRNA biomarkers were detected in the EV fraction than the protein fraction, and further validation using droplet digital PCR (ddPCR) was performed on the EV enriched fraction of a second cohort of dogs with UC which indicated that miR-182, miR-221 and miR-222 were significantly overrepresented in dogs with UC when compared with healthy dogs and dogs with urinary tract infections. Pathway analysis confirmed that these three miRNAs are involved in cancer. In addition, their potential downstream gene targets were predicted and PIK3R1, a well-known oncogene is likely to be a shared target between miRNA-182 and miRNA-221/222. In summary, this study highlights the potential of urinary EV-associated miRNAs as a source of biomarkers for the diagnosis of canine UC., (© 2024. The Author(s).)

In the quest for efficient tumor diagnosis via liquid biopsy, extracellular vesicles (EVs) have shown promise as a source of potential biomarkers. This study addresses the gap in biomarker efficacy for predicting clinically significant prostate cancer (csPCa) between the Western and Chinese populations. We developed a urinary extracellular vesicles-based prostate score (EPS) model, utilizing the EXODUS technique for EV isolation from 598 patients and incorporating gene expressions of FOXA1, PCA3, and KLK3. Our findings reveal that the EPS model surpasses prostate-specific antigen (PSA) testing in diagnostic accuracy within a training cohort of 234 patients, achieving an area under the curve (AUC) of 0.730 compared to 0.659 for PSA (p = 0.018). Similarly, in a validation cohort of 101 men, the EPS model achieved an AUC of 0.749, which was significantly better than PSA's 0.577 (p < 0.001). Our model has demonstrated a potential reduction in unnecessary prostate biopsies by 26%, with only a 3% miss rate for csPCa cases, indicating its effectiveness in the Chinese population., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Background: This study investigated the use of urinary exosomal mRNA as a potential biomarker for the early detection of prostate cancer (PCa)., Methods: Next-generation sequencing was utilized to analyze exosomal RNA from 10 individuals with confirmed PCa and 10 individuals without cancer. Subsequent validation through qRT-PCR in a larger sample of 43 PCa patients and 92 healthy controls revealed distinct mRNA signatures associated with PCa., Results: Notably, mRNAs for RAB5B, WWP1, HIST2H2BF, ZFY, MARK2, PASK, RBM10, and NRSN2 showed promise as diagnostic markers, with AUC values between 0.799 and 0.906 and significance p values. Combining RAB5B and WWP1 in an exoRNA diagnostic model outperformed traditional PSA tests, achieving an AUC of 0.923, 81.4% sensitivity, and 89.1% specificity., Conclusions: These findings highlight the potential of urinary exosomal mRNA profiling, particularly focusing on RAB5B and WWP1, as a valuable strategy for improving the early detection of PCa., (© 2024. The Author(s).)

Bladder cancer (BC) represents a prevalent malignancy in North America and Europe, posing significant health burdens. The identification of a reliable biomarker for early BC detection is imperative to enhance prognostic outcomes. Our aim for this study is to determine the diagnostic accuracy and potential clinical utility of Angiogenin/Ribonuclease 5 (ANG/RNase 5) as a biomarker for detection of BC. A systematic literature search across multiple databases up to March 20, 2024, was conducted. CMA 3.7 and Meta-disk 1.4 were used to analyze specificity, sensitivity, AUC, DOR, LR+, LR-, Q*index, and SROC for ANG as a urinary biomarker in BC patients. Publication bias was assessed using Egger's regression asymmetry and Begg's rank correlation tests. Additional diagnosing analyses were performed using Python programming language version 3.12.1. In this meta-analysis of seven case-control studies comprising 1,051 participants (576 cases and 481 controls), pooled sensitivity was 0.701 (95 % CI: 0.662-0.738), specificity was 0.787 (95 % CI: 0.752-0.819), LR + was 3.582 (95 % CI: 2.260-5.676), LR- was 0.398 (95 % CI: 0.327-0.485), and DOR was 10.637 (95 % CI: 6.106-18.529). The AUC and Q* index values were 0.823 and 0.756, respectively. Both Begg and Mazumdar Rank Correlation Test (p = 0.229) and Egger's Test of the Intercept (p = 0.135) revealed no significant evidence of publication bias. Our meta-analysis confirms ANG/RNase 5 as a reliable biomarker for early bladder cancer detection, showing strong diagnostic accuracy and no publication bias., 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 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: After accounting for smoking history, lung cancer incidence is greater in African Americans than Whites. In the multiethnic cohort, total nicotine equivalents (TNE) are higher in African Americans than Whites at similar reported cigarettes per day. Greater toxicant uptake per cigarette may contribute to the greater lung cancer risk of African Americans., Methods: In a nested case-control lung cancer study within the Southern Community Cohort, smoking-related biomarkers were measured in 259 cases and 503 controls (40% White; 56% African American). TNE, the trans-3-hydroxycotinine/cotinine ratio, 4-(methylnitrosamino)-1-3-(pyridyl)-1-butanol (NNAL), mercapturic acid metabolites of volatile organic compounds, phenanthrene metabolites, cadmium (Cd), and (Z)-7-(1R,2R,3R,5S)-3,5-dihydroxy-2-[(E,3S)-3-hydroxyoct-1-enyl]cyclopenyl]hept-5-enoic acid were quantified in urine. Unconditional logistic regression was used to estimate the ORs and 95% confidence intervals (CI) for each biomarker and lung cancer risk., Results: TNE, NNAL, and Cd were higher in cases than controls (adjusted for age, race, sex, body mass index, and cigarettes per day). Among cases, these levels were higher in African Americans compared with Whites. After accounting for age, sex, body mass index, and pack-years, a one-SD increase in log-TNE (OR = 1.30; 95% CI, 1.10-1.54) and log-NNAL (OR = 1.27; 95% CI, 1.03-1.58 with TNE adjustment) was associated with lung cancer risk. In this study, in which NNAL concentration is relatively high, the association for log-TNE was attenuated after adjustment for log-NNAL., Conclusions: Smoking-related biomarkers provide additional information for lung cancer risk in smokers beyond smoking pack-years., Impact: Urinary NNAL, TNE, and Cd concentrations in current smokers, particularly African American smokers, may be useful for predicting lung cancer risk., (©2024 American Association for Cancer Research.)

Background: We aimed to identify plasma and urinary metabolites related to colorectal cancer (CRC) risk and elucidate their mediator role in the associations between modifiable risk factors and CRC., Methods: Metabolite quantitative trait loci were derived from 2 published metabolomics genome-wide association studies, and summary-level data were extracted for 651 plasma metabolites and 208 urinary metabolites. Genetic associations with CRC were obtained from a large-scale genome-wide association study meta-analysis (100 204 cases, 154 587 controls) and the FinnGen cohort (4957 cases, 304 197 controls). Mendelian randomization and colocalization analyses were performed to evaluate the causal roles of metabolites in CRC. Druggability evaluation was employed to prioritize potential therapeutic targets. Multivariable Mendelian randomization and mediation estimation were conducted to elucidate the mediating effects of metabolites on the associations between modifiable risk factors and CRC., Results: The study identified 30 plasma metabolites and 4 urinary metabolites for CRC. Plasma sphingomyelin and urinary lactose, which were positively associated with CRC risk, could be modulated by drug interventions (ie, olipudase alfa, tilactase). Thirteen modifiable risk factors were associated with 9 metabolites, and 8 of these modifiable risk factors were associated with CRC risk. These 9 metabolites mediated the effect of modifiable risk factors (Actinobacteria, body mass index, waist to hip ratio, fasting insulin, smoking initiation) on CRC., Conclusion: This study identified key metabolite biomarkers associated with CRC and elucidated their mediator roles in the associations between modifiable risk factors and CRC. These findings provide new insights into the etiology and potential therapeutic targets for CRC and the etiological pathways of modifiable environmental factors with CRC., (© The Author(s) 2024. Published by Oxford University Press.)

Purpose of Review: This review sought to define the emerging roles of urinary tumor DNA (utDNA) for diagnosis, monitoring, and treatment of bladder cancer. Building from early landmark studies the focus is on recent studies, highlighting how utDNA could aid personalized care., Recent Findings: Recent research underscores the potential for utDNA to be the premiere biomarker in bladder cancer due to the constant interface between urine and tumor. Many studies find utDNA to be more informative than other biomarkers in bladder cancer, especially in early stages of disease. Points of emphasis include superior sensitivity over traditional urine cytology, broad genomic and epigenetic insights, and the potential for non-invasive, real-time analysis of tumor biology. utDNA shows promise for improving all phases of bladder cancer care, paving the way for personalized treatment strategies. Building from current research, future comprehensive clinical trials will validate utDNA's clinical utility, potentially revolutionizing bladder cancer management., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A plethora of urine markers for the management of patients with bladder cancer has been developed and studied in the past. However, the clinical impact of urine testing on patient management remains obscure. The goal of this manuscript is to identify scenarios for the potential use of molecular urine markers in the follow-up of patients with high-risk non-muscle-invasive BC (NMIBC) and estimate potential risks and benefits. Information on the course of disease of patients with high-risk NMIBC and performance data of a point-of-care test (UBC rapid™), an MCM-5 directed ELISA (ADXBLADDER™), and 2 additional novel assays targeting alterations of mRNA expression and DNA methylation (Xpert bladder cancer monitor™, Epicheck™) were retrieved from high-quality trials and/or meta-analyses. In addition, the sensitivity of white light cystoscopy (WLC) and the impact of a urine marker result on the performance of WLC were estimated based on fluorescence cystoscopy data and information from the CeFub trial. This information was applied to different scenarios in patient follow-up and sensitivity, estimated number of cystoscopies, and the numbers needed to diagnose were calculated. The sensitivity of guideline-based regular follow-up (SOC) at 1 year was calculated at 96%. For different marker-supported strategies sensitivities ranging from 77% to 97.9% were estimated. Calculations suggest that several strategies are effective for the SOC. While for the SOC 24.6 WLCs were required to diagnose 1 tumor recurrence (NND), this NND dropped below 5 in some marker-supported strategies. Based on the results of this simulation, a marker-supported follow-up of patients with HR NMIBC is safe and offers the option to significantly reduce the number of WLCs. Further research focusing on prospective randomized trials is needed to finally find a way to implement urine markers into clinical decision-making., Competing Interests: Declaration of competing interest Thorsten Ecke is trialist for Concile GmbH, Germany. Florian Roghmann is trialist for Cepheid, CA, USA. Bas W.G. van. Rhijn is consultant for QED Therapeutics, CA, USA and Incyte International Biosciences, DE, USA. Arnulf Stenzl is trialist and consultant for Arquer Ltd, UK, Cepheid, CA, USA and Nucleix Inc., Israel. J. Alfred Witjes is trialist and consultant for Arquer Ltd, UK, Cepheid, CA, USA and Nucleix Inc., Israel. Peter J. Goebell is trialist for Cepheid, CA, USA. Jorgen Bjerggaard Jensen is trialist and consultant for Cepheid, CA, USA and Nucleix Inc., Israel. Bernd J. Schmitz-Dräger is trialist and consultant for Arquer Ltd, UK, Cepheid, CA, USA, Concile GmbH, Germany and Nucleix Inc., Israel. Remaining authors have no conflict of interest to disclose., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: Aberrant glycosylation of proteins is an important hallmark in multiple cancers. Prostate-specific membrane antigen (PSMA), a highly glycosylated protein with 10 N-linked glycosylation sites, is an Food and Drug Administration approved theranostic for prostate cancer. However, glycosylation changes in PSMA that are associated with prostate cancer disease progression have not been fully characterized., Methods: We investigated whether urinary PSMA sialylation correlate with high-grade prostate cancer. Urine samples were collected from men after digital rectal examination (DRE) before prostate biopsy. Lectin-antibody enzyme-linked immunoassay was used to quantify α2,3-sialyl PSMA in post-DRE urine samples from subjects with benign prostate tumors, Grade Group 1 prostate cancer and those with Grade Group ≥2 disease., Results: There are significant increases in α2,3-sialylated PSMA in patients with Grade Group ≥2 disease compared to benign (p = 0.0009) and those with Grade Group 1 disease (p = 0.0063). There were no significant differences in α2,3-sialyl PSMA levels between Grade Group 1 and benign prostate tumors (p = 0.7947)., Conclusions: Our study shows that there are significant differences in the abundance of α2,3-sialylated PSMA in post-DRE urines from disease stratified prostate cancer patients, and the increase is correlated with progression and disease severity. The detection of increased PSMA sialyation in post-DRE urines from patients with higher Grade Group ≥2 disease states provides novel untapped potential for the development of prognostic biomarkers for prostate cancer. Specifically, quantitation of α2,3-sialylated PSMA shows potential for discriminating between benign to intermediate grade disease, which is a significant clinical challenge in staging and risk stratification of prostate cancer., (© 2024 The Authors. The Prostate published by Wiley Periodicals LLC.)

Purpose: T1 bladder cancer is known for its high progression and recurrence rates. Identifying aggressive tumours at the non-muscle-invasive stage is crucial to allow early interventions and subsequently increase patient survival. This study aimed to investigate the potential of the cubilin/myeloperoxidase (CUBN/MPO) ratio as a high-grade T1 bladder cancer biomarker., Methods: Urine samples were collected from 30 patients who underwent transurethral resection of the tumour with high-grade T1 bladder cancer (June 2015 to December 2019) before surgery. The urinary proteome was analysed using high-resolution mass spectrometry and the CUBN/MPO ratio was calculated. The primary outcome was the recurrence during the follow-up (around 31.5 months after resection). Univariate Cox regression and Kaplan-Meier curves were used for data analysis., Results: Patients with a low CUBN/MPO ratio exhibited upregulated MPO and/or downregulated CUBN. This group of patients had a higher incidence of disease recurrence and progression. Low CUBN/MPO ratio was significantly associated with a higher likelihood of recurrence, progression, and death. It is worth noting that this study was exploratory and conducted on a small sample size, so further research is needed to validate these findings in larger cohorts., Conclusion: This study highlights the potential of the CUBN/MPO ratio as a prognostic biomarker for high-grade T1 bladder cancer., (© 2024. The Author(s).)

High-grade serous ovarian cancer (HGSOC) is one of the most common histologic types of ovarian cancer. The purpose of this study was to identify potential prognostic biomarkers in urine specimens from patients with HGSOC. First, 56 urine samples with information on relapse-free survival (RFS) months were collected and classified into good prognosis (RFS ≥ 12 months) and poor prognosis (RFS < 12 months) groups. Next, data-independent acquisition (DIA)-based mass spectrometry (MS) analysis was combined with MSFragger-DIA workflow to identify potential prognostic biomarkers in a discovery set (n = 31). With the aid of parallel reaction monitoring (PRM) analysis, four candidate biomarkers (ANXA1, G6PI, SPB3, and SPRR3) were finally validated in both the discovery set and an independent validation set (n = 25). Subsequent RFS and Cox regression analyses confirmed the utility of these candidate biomarkers as independent prognostic factors affecting RFS in patients with HGSOC. Regression models were constructed to predict the 12-month RFS rate, with area under the receiver operating characteristic curve (AUC) values ranging from 0.847 to 0.905. Overall, candidate prognostic biomarkers were identified in urine specimens from patients with HGSOC and prediction models for the 12-month RFS rate constructed. SIGNIFICANCE: OC is one of the leading causes of death due to gynecological malignancies. HGSOC constitutes one of the most common histologic types of OC with aggressive characteristics, accounting for the majority of advanced cases. In cases where patients with advanced HGSOC potentially face high risk of unfavorable prognosis or disease advancement within a 12-month period, intensive medical monitoring is necessary. In the era of precision cancer medicine, accurate prediction of prognosis or 12-month RFS rate is critical for distinguishing patient groups requiring heightened surveillance. Patients could significantly benefit from timely modifications to treatment regimens based on the outcomes of clinical monitoring. Urine is an ideal resource for disease surveillance purposes due to its easy accessibility. Furthermore, molecules excreted in urine are less complex and more stable than those in other liquid samples. In the current study, we identified candidate prognostic biomarkers in urine specimens from patients with HGSOC and constructed prediction models for the 12-month RFS rate., Competing Interests: Declaration of competing interest There are no potential conflicts of interest to declare. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: This study aims to explore novel microRNAs in urine for screening and predicting clinical characteristics in pancreatic cancer (PC) patients using a microRNA array-based approach., Methods: We used the Toray ® 3D-Gene microRNA array-based approach to compare urinary levels between PC patients and healthy volunteers., Results: (1) Four oncogenic microRNAs (miR-744-5p, miR-572, miR-210-3p, and miR-575) that were highly upregulated in the urine of PC patients compared to healthy individuals were identified by comprehensive microRNA array analysis. (2) Test-scale analysis by quantitative RT-PCR for each group of 20 cases showed that miR-210-3p was significantly upregulated in the urine of PC patients compared to healthy individuals (P = 0.009). (3) Validation analysis (58 PC patients and 35 healthy individuals) confirmed that miR-210-3p was significantly upregulated in the urine of PC patients compared to healthy individuals (P < 0.001, area under the receiver operating characteristic curve = 0.79, sensitivity: 0.828, specificity: 0.743). We differentiated PC patients into invasive ductal carcinoma (IDCa) and intraductal papillary mucinous carcinoma (IPMC) groups. In addition to urinary miR-210-3p levels being upregulated in IDCa over healthy individuals (P = 0.009), urinary miR-210-3p levels were also elevated in IPMC over healthy individuals (P = 0.0018). Urinary miR-210-3p can differentiate IPMC from healthy individuals by a cutoff of 8.02 with an AUC value of 0.762, sensitivity of 94%, and specificity of 63%. (4) To test whether urinary miR210-3p levels reflected plasma miR-210-3p levels, we examined the correlation between urinary and plasma levels. Spearman's correlation analysis showed a moderate positive correlation (ρ = 0.64, P = 0.005) between miR-210-3p expression in plasma and urine., Conclusions: Urinary miR-210-3p is a promising, non-invasive diagnostic biomarker of PC, including IPMC., Trial Registration: Not applicable., (© 2024. The Author(s).)

Objectives: Urinary sex hormones are investigated as potential biomarkers for the early detection of breast cancer, aiming to evaluate their relevance and applicability, in combination with supervised machine-learning data analysis, toward the ultimate goal of extensive screening., Methods: Sex hormones were determined on urine samples collected from 250 post-menopausal women (65 healthy - 185 with breast cancer, recruited among the clinical patients of Candiolo Cancer Institute FPO-IRCCS (Torino, Italy). Two analytical procedures based on UHPLC-MS/HRMS were developed and comprehensively validated to quantify 20 free and conjugated sex hormones from urine samples. The quantitative data were processed by seven machine learning algorithms. The efficiency of the resulting models was compared., Results: Among the tested models aimed to relate urinary estrogen and androgen levels and the occurrence of breast cancer, Random Forest (RF) proved to underscore all the other supervised classification approaches, including Partial Least Squares - Discriminant Analysis (PLS-DA), in terms of effectiveness and robustness. The final optimized model built on only five biomarkers (testosterone-sulphate, alpha-estradiol, 4-methoxyestradiol, DHEA-sulphate, and epitestosterone-sulphate) achieved an approximate 98% diagnostic accuracy on replicated validation sets. To balance the less-represented population of healthy women, a Synthetic Minority Oversampling TEchnique (SMOTE) data oversampling approach was applied., Conclusions: By means of tunable hyperparameters optimization, the RF algorithm showed great potential for early breast cancer detection, as it provides clear biomarkers ranking and their relative efficiency, allowing to ground the final diagnostic model on a restricted selection five steroid biomarkers only, as desirable for noninvasive tests with wide screening purposes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marco Vincenti reports financial support was provided by CRT Foundation. Marco Vincenti reports financial support was provided by Italian Ministry of Education, Universities, and Research. If there are other authors, they 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 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Background: Urothelial carcinoma (UC) is the second most common urological malignancy. Despite numerous molecular markers have been evaluated during the past decades, no urothelial markers for diagnosis and recurrence monitoring have shown consistent clinical utility., Methods: The methylation level of tissue samples from public database and clinical collected were analyzed. Patients with UC and benign diseases of the urinary system (BUD) were enrolled to establish TAGMe (TAG of Methylation) assessment in a training cohort (n = 567) using restriction enzyme-based bisulfite-free qPCR. The performance of TAGMe assessment was further verified in the validation cohort (n = 198). Urine samples from 57 UC patients undergoing postoperative surveillance were collected monthly for six months after surgery to assess the TAGMe methylation., Results: We identified TAGMe as a potentially novel Universal-Cancer-Only Methylation (UCOM) marker was hypermethylated in multi-type cancers and investigated its application in UC. Restriction enzyme-based bisulfite-free qPCR was used for detection, and the results of which were consistent with gold standard pyrosequencing. Importantly, hypermethylated TAGMe showed excellent sensitivity of 88.9% (95% CI: 81.4-94.1%) and specificity of 90.0% (95% CI: 81.9-95.3%) in efficiently distinguishing UC from BUD patients in urine and also performed well in different clinical scenarios of UC. Moreover, the abnormality of TAGMe as an indicator of recurrence might precede clinical recurrence by three months to one year, which provided an invaluable time window for timely and effective intervention to prevent UC upstaging., Conclusion: TAGMe assessment based on a novel single target in urine is effective and easy to perform in UC diagnosis and recurrence monitoring, which may reduce the burden of cystoscopy. Trial registration ChiCTR2100052507. Registered on 30 October 2021., (© 2024. The Author(s).)

Purpose: Bladder cancer (BLCA) is a major cancer of the genitourinary system. Although cystoscopy is the standard protocol for diagnosing BLCA clinically, this procedure is invasive and expensive. Several urine-based markers for BLCA have been identified and investigated, but none has shown sufficient sensitivity and specificity. These observations underscore the importance of discovering novel BLCA biomarkers and developing a noninvasive method for detection of BLCA. Exploring the cancer secretome is a good starting point for the development of noninvasive biomarkers for cancer diagnosis., Experimental Design: In this study, we established a comprehensive secretome dataset of five representative BLCA cell lines, BFTC905, TSGH8301, 5637, MGH-U1, and MGH-U4, by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Expression of BLCA-specific secreted proteins at the transcription level was evaluated using the Oncomine cancer microarray database., Results: The expressions of four candidates-COMT, EWSR1, FUSIP1, and TNPO2-were further validated in clinical human specimens. Immunohistochemical analyses confirmed that transportin-2 was highly expressed in tumor cells relative to adjacent noncancerous cells in clinical tissue specimens from BLCA patients, and was significantly elevated in BLCA urine compared with that in urine samples from aged-matched hernia patients (controls)., Conclusions: Collectively, our findings suggest TNPO2 as a potential noninvasive tumor-stage or grade discriminator for BLCA management., (© 2024 Wiley‐VCH GmbH.)

Background: Currently utilized serum tumor markers and fecal immunochemical tests do not have sufficient diagnostic power for colorectal cancer (CRC) due to their low sensitivities. To establish non-invasive urinary protein biomarkers for early CRC diagnosis, we performed stepwise analyses employing urine samples from CRCs and healthy controls (HCs)., Methods: Among 474 urine samples, 363 age- and sex-matched participants (188 HCs, 175 stage 0-III CRCs) were randomly divided into discovery (16 HCs, 16 CRCs), training (110 HCs, 110 CRCs), and validation (62 HCs, 49 CRCs) cohorts., Results: Of the 23 urinary protein candidates comprehensively identified from mass spectrometry in the discovery cohort, urinary levels of dipeptidase 1 (uDPEP1) and Trefoil factor1 (uTFF1) were the two most significant diagnostic biomarkers for CRC in both training and validation cohorts using enzyme-linked immunosorbent assays. A urinary biomarker panel comprising uDPEP1 and uTFF1 significantly distinguished CRCs from HCs, showing area under the curves of 0.825-0.956 for stage 0-III CRC and 0.792-0.852 for stage 0/I CRC. uDPEP1 and uTFF1 also significantly distinguished colorectal adenoma (CRA) patients from HCs, with uDPEP1 and uTFF1 increasing significantly in the order of HCs, CRA patients, and CRC patients. Moreover, expression levels of DPEP1 and TFF1 were also significantly higher in the serum and tumor tissues of CRC, compared to HCs and normal tissues, respectively., Conclusions: This study established a promising and non-invasive urinary protein biomarker panel, which enables the early detection of CRC with high sensitivity., (© 2024. Japanese Society of Gastroenterology.)

Bladder cancer (BCa) stands as prevalent malignancy of the urinary system globally, especially among men. The clinical classification of BCa into non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) is crucial for prognosis and treatment decisions. However, challenges persist in current diagnostic methods like Urine cytopathology that shows poor sensitivity therefore compromising on accurately diagnosing and monitoring BCa. In recent years, research has emphasized the importance of identifying urine and blood-based specific biomarkers for BCa that can enable early and precise diagnosis, effective tumor classification, and monitoring. The convenient proximity of urine with the urinary bladder epithelium makes urine a good source of noninvasive biomarkers, in particular urinary EVs because of the packaged existence of tumor-associated molecules. Therefore, the review assesses the potential of urinary extracellular vesicles (uEVs) as noninvasive biomarkers for BCa. We have elaborately reviewed and discussed the research that delves into the role of urinary EVs in the context of BCa diagnosis and classification. Extensive research has been dedicated to investigating differential microRNA (miRNA) expressions, with the goal of establishing distinct, noninvasive biomarkers for BCa. The identification of such biomarkers has the potential to revolutionize early detection, risk stratification, therapeutic interventions, and ultimately, the long-term prognosis of BCa patients. Despite notable advancements, inconsistencies persist in the biomarkers identified, methodologies employed, and study populations. This review meticulously compiles reported miRNA biomarkers, critically assessing the variability and discrepancies observed in existing research. By synthesizing these findings, the article aims to direct future studies toward a more cohesive and dependable approach in BCa biomarker identification, fostering progress in patient care and management., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bladder cancer (BC) poses high morbidity and mortality, with urinary exosomal microRNA (miR)-21 showing potential value in its diagnosis and prognosis, and we probed its specific role. We prospectively selected 116 BC patients and 116 healthy volunteers as the BC and control groups, respectively. BC urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 relative expression levels were assessed. The correlations between clinical indexes and urinary exosomal miR-21, prognostic value of miR-21, and diagnostic value of the five candidate miRNAs, urine cytology, and miRNA joint diagnostic panel for BC and urinary exosomal miR-21, miR-4454, and urine cytology for Ta-T1 and T2-T4 stage BC were analyzed. Urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 were highly expressed in BC patients. miR-146a-5p, miR-93-5p, miR-663b, miR-21, miR-4454, miRNA combined diagnostic panel, and urine cytology had certain diagnostic value for BC, with miR-21, miR-4454, and miRNA co-diagnostic panel showing the highest diagnostic value. Collectively, urinary exosomal miR-21 was closely related to Tumor-Node-Metastasis staging and grading in BC patients. Urinary exosomal miR-21 had high diagnostic value for BC and Ta-T1 and T2-T4 stage BC, and had high predictive value for BC poor prognosis, providing an effective indicator for the occurrence, development, and prognostic assessment of BC., (© 2024 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

The current noninvasive diagnostic approaches for detecting bladder cancer (BC) often exhibit limited clinical performance, especially for the initial diagnosis. This study aims to evaluate the validity of a streamlined urine-based PENK methylation test called EarlyTect BCD in detecting BC in patients with hematuria scheduled for cystoscopy in Korean and American populations. The test seamlessly integrates two steps, linear target enrichment and quantitative methylation-specific PCR within a single closed tube. The detection limitation of the test was approximately two genome copies of methylated PENK per milliliter of urine. In the retrospective training set (n = 105), an optimal cutoff value was determined to distinguish BC from non-BC, resulting in a sensitivity of 87.3% and a specificity of 95.2%. In the prospective validation set (n = 210, 122 Korean and 88 American patients), the overall sensitivity for detecting all stages of BC was 81.0%, with a specificity of 91.5% and an area under the curve value of 0.889. There was no significant difference between the two groups. The test achieved a sensitivity of 100% in detecting high-grade Ta and higher stages of BC. The negative predictive value of the test was 97.7%, and the positive predictive value was 51.5%. The findings of this study demonstrate that EarlyTect BCD is a highly effective noninvasive diagnostic tool for identifying BC among patients with hematuria., Competing Interests: Disclosure Statement T.J.O., Y.S., M.A.W., and S.A. are employees and shareholders of Genomictree, Inc.; B.-R.B., C.S.I., S.K., J.J.L., S.B., and S.A. are employees of Promis Diagnostics, Inc.; and J.J.L. and S.A. are shareholders of Promis Diagnostics Inc., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. All rights reserved.)

Low accuracy of diagnosing prostate cancer (PCa) was easily caused by only assaying single prostate specific antigen (PSA) biomarker. Although conventional reported methods for simultaneous detection of two specific PCa biomarkers could improve the diagnostic efficiency and accuracy, low detection sensitivity restrained their use in extreme early-stage PCa clinical assay applications. In order to overcome above drawbacks, this paper herein proposed a multiplexed dual optical microfibers separately functionalized with gold nanorods (GNRs) and Au nanobipyramids (Au NBPs) nanointerfaces with strong localized surface plasmon resonance (LSPR) effects. The sensors could simultaneously detect PSA protein biomarker and long noncoding RNA prostate cancer antigen 3 (lncRNA PCA3) with ultrahigh sensitivity and remarkable specificity. Consequently, the proposed dual optical microfibers multiplexed biosensors could detect the PSA protein and lncRNA PCA3 with ultra-low limit-of-detections (LODs) of 3.97 × 10 -15  mol/L and 1.56 × 10 -14  mol/L in pure phosphorus buffer solution (PBS), respectively, in which the obtained LODs were three orders of magnitude lower than existed state-of-the-art PCa assay technologies. Additionally, the sensors could discriminate target components from complicated physiological environment, that showing noticeable biosensing specificity of the sensors. With good performances of the sensors, they could successfully assay PSA and lncRNA PCA3 in undiluted human serum and urine simultaneously, respectively. Consequently, our proposed multiplexed sensors could real-time high-sensitivity simultaneously detect complicated human samples, that providing a novel valuable approach for the high-accurate diagnosis of early-stage PCa individuals., 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.)

Prostate cancer (PCa) is a common male malignancy and early diagnosis is crucial for successful treatment. The current study aims to validate results from a pilot study that demonstrated an inverse association between urine tyrosine and tryptophan levels and the severity of PCa. This study comprised a cohort of 97 patients with benign prostatic hyperplasia, 93 patients diagnosed with localized PCa, 75 patients diagnosed with locally advanced PCa, and 68 patients diagnosed with metastatic PCa. The tyrosine and tryptophan levels in the samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and electrochemical sensors in accordance with the pilot to maintain uniformity for accurately evaluating the data. One-way ANOVA with post Tukey test as well as the Wilcoxon Rank Sum Test were performed. Analyzing 333 patients across PCa stages with consistent methods, we observed no significant differences in tyrosine and tryptophan levels between PCa patients and controls, finally rejecting the use of tyrosine and tryptophan as PCa biomarkers. We did, however, verify the strong correlation between the urinary concentrations of tyrosine and tryptophan found in the pilot study., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alatraktchi owns PreDiagnose (supplier of screen-printed sensors). This had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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.)

This study aimed to confirm urinary protein fragments in relation to the presence of pancreatic ductal adenocarcinoma (PDAC) via a C-terminal proteomics strategy using exploratory and validation cohorts. Urinary fragments were examined by iTRAQ-labelling of tryptic peptides and concentrations of C-terminal fragments were evaluated. Only the urinary CD276 fragment showed a fold change (FC) of > 1.5 with a significant difference of P < 0.01 between healthy (H) and PDAC participants in both the exploratory (H, n = 42; PDAC, n = 39) and validation cohorts (H, n = 36; resectable PDAC, n = 28). The sensitivity and specificity of the CD276 fragment for diagnosing resectable PDAC were 75% and 89%, respectively, in the validation cohort. Postoperative urinary levels of the CD276 fragment were low as compared to those before surgery (n = 18, P < 0.01). Comprehensive C-terminus proteomics identified an increase in the urinary CD276 fragment level as a feature of patients with PDAC. The urinary CD276 fragment is a potential biomarker for detecting resectable PDAC., (© 2024. The Author(s).)

Extracellular vesicle (EV) molecular phenotyping offers enormous opportunities for cancer diagnostics. However, the majority of the associated studies adopted biomarker-based unimodal analysis to achieve cancer diagnosis, which has high false positives and low precision. Herein, we report a multimodal platform for the high-precision diagnosis of bladder cancer (BCa) through a multispectral 3D DNA machine in combination with a multimodal machine learning (ML) algorithm. The DNA machine was constructed using magnetic microparticles (MNPs) functionalized with aptamers that specifically identify the target of interest, i.e., five protein markers on bladder-cancer-derived urinary EVs (uEVs). The aptamers were hybridized with DNA-stabilized silver nanoclusters (DNA/AgNCs) and a G-quadruplex/hemin complex to form a sensing module. Such a DNA machine ensured multispectral detection of protein markers by fluorescence (FL), inductively coupled plasma mass spectrometry (ICP-MS), and UV-vis absorption (Abs). The obtained data sets then underwent uni- or multimodal ML for BCa diagnosis to compare the analytical performance. In this study, urine samples were obtained from our prospective cohort ( n = 45). Our analytical results showed that the 3D DNA machine provided a detection limit of 9.2 × 10 3 particles mL -1 with a linear range of 4 × 10 4 to 5 × 10 7 particles mL -1 for uEVs. Moreover, the multimodal data fusion model exhibited an accuracy of 95.0%, a precision of 93.1%, and a recall rate of 93.2% on average, while those of the three types of unimodal models were no more than 91%. The elevated diagnosis precision by using the present fusion platform offers a perspective approach to diminishing the rate of misdiagnosis and overtreatment of BCa.

Urine is a complex biofluid that reflects both overall physiologic state and the state of the genitourinary tissues through which it passes. It contains both secreted proteins and proteins encapsulated in tissue-derived extracellular vesicles (EVs). To understand the population variability and clinical utility of urine, we quantified the secreted and EV proteomes from 190 men, including a subset with prostate cancer. We demonstrate that a simple protocol enriches prostatic proteins in urine. Secreted and EV proteins arise from different subcellular compartments. Urinary EVs are faithful surrogates of tissue proteomes, but secreted proteins in urine or cell line EVs are not. The urinary proteome is longitudinally stable over several years. It can accurately and non-invasively distinguish malignant from benign prostatic lesions and can risk-stratify prostate tumors. This resource quantifies the complexity of the urinary proteome and reveals the synergistic value of secreted and EV proteomes for translational and biomarker studies., (© 2024. The Author(s).)

An untargeted metabolomic study identified four potential lung cancer diagnostic biomarkers in human urine. One of the potential biomarkers was an unidentified feature possessing a m / z value of 561+. "561+" was isolated from human urine and tentatively identified as 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol glucuronide with unknown C24,25 stereochemistry using 1 H NMR and mass spectrometry. In a prior report, the C24,25 stereochemistry of the aglycone, 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol, was found to be 24 S ,25 R through GC analysis of the acetonide-TMS derivative. An authentic sample was prepared and found not to have the same stereochemistry as "561+". To identify the C24,25 stereochemistry, four C24,C25 diastereoisomeric alcohols of 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol were prepared from chiral amino acids. Using an LCMS method, the C24,C25 stereochemistry of the "561+" aglycone was determined to be 24 R ,25 S . With the correct aglycone in hand, it was coupled with glucuronic acid to complete the first reported synthesis of 27-nor-5β-cholestane-3α,7α,12α,24 R ,25S pentol glucuronide. Deuterium labeled 27-nor-5β-cholestane-3α,7α,12α,24 R ,25 S pentol was also synthesized for use as an internal standard for MS quantitation.

Bladder cancer (BCa) is the predominant malignancy of the urinary system. Herein, a comprehensive urine proteomic feature was initially established for the noninvasive diagnosis and recurrence monitoring of bladder cancer. 279 cases (63 primary BCa, 87 nontumor controls (NT), 73 relapsed BCa (BCR), and 56 nonrelapsed BCa (BCNR)) were collected to screen urinary protein biomarkers. 4761 and 3668 proteins were qualified and quantified by DDA and sequential window acquisition of all theoretical mass spectra (SWATH-MS) analysis in two discovery sets, respectively. Upregulated proteins were validated by multiple reaction monitoring (MRM) in two independent combined sets. Using the multi-support vector machine-recursive feature elimination (mSVM-RFE) algorithm, a model comprising 13 proteins exhibited good performance between BCa and NT with an AUC of 0.821 (95% CI: 0.675-0.967), 90.9% sensitivity (95% CI: 72.7-100%), and 73.3% specificity (95% CI: 53.3-93.3%) in the diagnosis test set. Meanwhile, an 11-marker classifier significantly distinguished BCR from BCNR with 75.0% sensitivity (95% CI: 50.0-100%), 81.8% specificity (95% CI: 54.5-100%), and an AUC of 0.784 (95% CI: 0.609-0.959) in the test cohort for relapse surveillance. Notably, six proteins (SPR, AK1, CD2AP, ADGRF1, GMPS, and C8A) of 24 markers were newly reported. This paper reveals novel urinary protein biomarkers for BCa and offers new theoretical insights into the pathogenesis of bladder cancer (data identifier PXD044896).

To investigate extracellular vesicles (EVs), biomarkers for predicting lymph node invasion (LNI) in patients with high-risk prostate cancer (HRPCa), plasma, and/or urine samples were prospectively collected from 45 patients with prostate cancer (PCa) and five with benign prostatic hyperplasia (BPH). Small RNA sequencing was performed to identify miRNAs in the EVs. All patients with PCa underwent radical prostatectomy and extended pelvic lymph node dissection. Differentially expressed miRNAs were identified in patients with and without pathologically-verified LNI. The candidate miRNAs were validated in low-risk prostate cancer (LRPCa) and BPH. Four miRNA species (e.g., miR-126-3p) and three miRNA species (e.g., miR-27a-3p) were more abundant in urinary and plasma EVs, respectively, of patients with PCa. None of these miRNA species were shared between urinary and plasma EVs. miR-126-3p was significantly more abundant in patients with HR PCa with LNI than in those without (P = 0.018). miR-126-3p was significantly more abundant in the urinary EVs of patients with HRPCa than in those with LRPCa (P = 0.017) and BPH (P = 0.011). In conclusion, urinary EVs-derived miR-126-3p may serve as a good biomarker for predicting LNI in patients with HRPCa., (© 2024. The Author(s).)

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.

Purpose: There is significant interest in identifying complete responders to neoadjuvant chemotherapy (NAC) before radical cystectomy (RC) to potentially avoid removal of a pathologically benign bladder. However, clinical restaging after NAC is highly inaccurate. The objective of this study was to develop a next-generation sequencing-based molecular assay using urine to enhance clinical staging of patients with bladder cancer., Methods: Urine samples from 20 and 44 patients with bladder cancer undergoing RC were prospectively collected for retrospective analysis for molecular correlate analysis from two clinical trials, respectively. The first cohort was used to benchmark the assay, and the second was used to determine the performance characteristics of the test as it correlates to responder status as measured by pathologic examination., Results: First, to benchmark the assay, known mutations identified in the tissue (M T ) of patients from the Accelerated Methotrexate, Vinblastine, Doxorubicin, Cisplatin trial (ClinicalTrials.gov identifier: NCT01611662, n = 16) and a cohort from University of California-San Francisco (n = 4) were cross referenced against mutation profiles from urine (M U ). We then determined the correlation between M U persistence and residual disease in pre-RC urine samples from a second prospective clinical trial (The pT0 trial; ClinicalTrials.gov identifier: NCT02968732). Residual M U status correlated strongly with residual disease status (pT0 trial; n = 44; P = .0092) when M U from urine supernatant and urine pellet were assessed separately and analyzed in tandem. The sensitivity, specificity, PPV, and NPV were 91%, 50%, 86%, and 63% respectively, with an overall accuracy of 82% for this second cohort., Conclusion: M U are representative of M T and thus can be used to enhance clinical staging of urothelial carcinoma. Urine biopsy may be used as a reliable tool that can be further developed to identify complete response to NAC in anticipation of safe RC avoidance.

Prostate cancer is the second most common cancer in men across the world. Prior to PSA testing, men usually presented with locally advanced disease detected on digital rectal exam or with metastatic disease. PSA ushered in the era of serum biomarkers for prostate cancer. It has taken over three decades to refine the role of PSA in prostate cancer detection. The lack of specificity has spurred research into finding better, readily obtainable biomarkers with high sensitivity and specificity. The trick is to find the prostate cancers that are a threat, not the ones that aren't. Over the last decade and more, many biomarkers have been proposed and tested (HK-2, Pro-PSA, PCA3, TMPRSS2:ERG fusion transcripts, miRNA, just to name a few) but we still await that magical combination of a readily available, reproducible, and hopefully inexpensive biomarker with high sensitivity and specificity. The authors describe the use of a peptide labeled fluorophore for the VPAC1 receptors that are expressed on malignant prostate cancer cells shed in the urine. After initial feasibility work, the authors collected urine from 318 men with lower urinary tract symptoms and a PSA > 4. The patients underwent prostate biopsy yielding Grade Group 2 or higher prostate cancer in 158 patients. One hundred fifty-four or those patients with cancer had a positive result for the biomarker. The sensitivity of the test was 100%, the specificity was 97.56%, positive predictive value was 97.47%, and negative predictive value was 100%.1 These are impressive numbers for a urine biomarker (or any biomarker). This work is certainly promising, BUT, we have seen promising early data on many biomarkers. In this study, the mean PSA in the cancer group was 34.53 ng/mL versus 9.41 in the control (negative) group. Since patients with infection were excluded, the significantly different PSA levels seemed to be selecting the cancers as well. Time and follow up will determine if the "negative biopsy" controls were truly negative. Can the technique and these results be reproduced? The true test will be how this biomarker consistently performs across a broader population of men with a lower, more homogenous PSA elevation. I will eagerly await results of continued study of this promising biomarker for prostate cancer.

Diagnosis and stratification of prostate cancer (PCa) patients using the prostate-specific antigen (PSA) test is challenging. Extracellular vesicles (EVs), as a new star of liquid biopsy, has attracted interest to complement inaccurate PSA screening and invasiveness of tissue biopsy. In this study, a panel of potential small EV (sEV) protein biomarkers is identified from PCa cell lines using label-free LC-MS/MS proteomics. These biomarkers underwent further validation with plasma and urine samples from different PCa stages through parallel reaction monitoring-based targeted proteomics, western blotting, and ELISA. Additionally, a tissue microarray containing cancerous and noncancerous tissues is screened to provide additional evidence of selected sEV proteins associated with cancer origin. Results indicate that sEV protein LAMB1 is highly expressed in human plasma of metastatic PCa patients compared with localised PCa patients and control subjects, while sEV protein Histone H4 is highly expressed in human urine of high-risk PCa patients compared to low-risk PCa patients and control subjects. These two sEV proteins demonstrate higher specificity and sensitivity than the PSA test and show promise for metastatic PCa diagnosis, progression monitoring, and risk stratification., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background: Urine is a promising biological fluid for prostate cancer (PCa) diagnostics due to its non-invasive collection and wide range of biomarkers. The aim of this study was to assess the role of urinary PSA (uPSA) and urinary Zinc (uZinc) as biomarkers for the diagnosis of PCa in combination with routine parameters of standard of care (SOC - blood PSA, abnormal DRE, age) and MRI in patients candidates for prostate biopsy., Methods: Urine samples after prostatic massages were collected from men with suspected PCa scheduled for prostate biopsy. Quantification of uPSA was performed by ECLIA platform and confirmed by ELISA assay, while uZinc measurement was evaluated by ICP-MS and confirmed by colorimetric in vitro assay. Six multivariate logistic regression analysis were performed to assess diagnostic performance of uPSA and uZinc (urine), SOC and MRI alone, and combination of MRI+SOC, MRI+urine and SOC+MRI+urine. The discriminative power of the logistic models was assessed by calculating the area under the receiver operating characteristic (ROC) curves (AUC)., Results: Two hundred thirty-eight patients were included in the analysis; 145 of them were diagnosed with PCa. Urine test showed a better discrimination of HS from CP, in respect of uPSA and uZinc alone, both for PCa of any grade and Gleason Score ≥7 (4+3) (AUC 0.804 and 0.823 respectively). ROC curve combining SOC+MRI+urine showed an AUC=0.882, that is statistically different from SOC or MRI alone, or MRI+SOC (P=0.0001, P=0.0001, and P=0.008 respectively). PCa risk algorithm designed considering SOC+MRI+urine results in potential reduction of 57% of unnecessary biopsies compared to the current standard parameters., Conclusions: The loss of uPSA and Zinc production and secretion during neoplastic transformation of the prostate could potentially represent a hallmark of PCa. Its combination with age, PSA and DRE, as well as with mpMRI could represent an interesting approach to improve the diagnostic accuracy of PCa.

Importance: Benefits of prostate cancer (PCa) screening with prostate-specific antigen (PSA) alone are largely offset by excess negative biopsies and overdetection of indolent cancers resulting from the poor specificity of PSA for high-grade PCa (ie, grade group [GG] 2 or greater)., Objective: To develop a multiplex urinary panel for high-grade PCa and validate its external performance relative to current guideline-endorsed biomarkers., Design, Setting, and Participants: RNA sequencing analysis of 58 724 genes identified 54 markers of PCa, including 17 markers uniquely overexpressed by high-grade cancers. Gene expression and clinical factors were modeled in a new urinary test for high-grade PCa (MyProstateScore 2.0 [MPS2]). Optimal models were developed in parallel without prostate volume (MPS2) and with prostate volume (MPS2+). The locked models underwent blinded external validation in a prospective National Cancer Institute trial cohort. Data were collected from January 2008 to December 2020, and data were analyzed from November 2022 to November 2023., Exposure: Protocolized blood and urine collection and transrectal ultrasound-guided systematic prostate biopsy., Main Outcomes and Measures: Multiple biomarker tests were assessed in the validation cohort, including serum PSA alone, the Prostate Cancer Prevention Trial risk calculator, and the Prostate Health Index (PHI) as well as derived multiplex 2-gene and 3-gene models, the original 2-gene MPS test, and the 18-gene MPS2 models. Under a testing approach with 95% sensitivity for PCa of GG 2 or greater, measures of diagnostic accuracy and clinical consequences of testing were calculated. Cancers of GG 3 or greater were assessed secondarily., Results: Of 761 men included in the development cohort, the median (IQR) age was 63 (58-68) years, and the median (IQR) PSA level was 5.6 (4.6-7.2) ng/mL; of 743 men included in the validation cohort, the median (IQR) age was 62 (57-68) years, and the median (IQR) PSA level was 5.6 (4.1-8.0) ng/mL. In the validation cohort, 151 (20.3%) had high-grade PCa on biopsy. Area under the receiver operating characteristic curve values were 0.60 using PSA alone, 0.66 using the risk calculator, 0.77 using PHI, 0.76 using the derived multiplex 2-gene model, 0.72 using the derived multiplex 3-gene model, and 0.74 using the original MPS model compared with 0.81 using the MPS2 model and 0.82 using the MPS2+ model. At 95% sensitivity, the MPS2 model would have reduced unnecessary biopsies performed in the initial biopsy population (range for other tests, 15% to 30%; range for MPS2, 35% to 42%) and repeat biopsy population (range for other tests, 9% to 21%; range for MPS2, 46% to 51%). Across pertinent subgroups, the MPS2 models had negative predictive values of 95% to 99% for cancers of GG 2 or greater and of 99% for cancers of GG 3 or greater., Conclusions and Relevance: In this study, a new 18-gene PCa test had higher diagnostic accuracy for high-grade PCa relative to existing biomarker tests. Clinically, use of this test would have meaningfully reduced unnecessary biopsies performed while maintaining highly sensitive detection of high-grade cancers. These data support use of this new PCa biomarker test in patients with elevated PSA levels to reduce the potential harms of PCa screening while preserving its long-term benefits.

Although colorectal cancer diagnosed at an early stage shows high curability, methods simultaneously possessing point-of-care testing ability and high sensitivity are limited. Here, an orally deliverable biomarker-activatable probe (termed as HATS) for early detection of orthotopic tumors via remote urinalysis is presented. To enable its oral delivery to the colon, HATS is designed to have remarkable resistance to acidity and digestive enzymes in the stomach and small intestine and negligible intestinal absorption. Upon reaction with a cancer biomarker in the colon segment, HATS releases a small fragment of tetrazine that can transverse the intestinal barrier, enter blood circulation, and ultimately undergo renal clearance to urine. Subsequently, the urinary tetrazine fragment is detected by bioorthogonal reaction with trans-cyclooctene-caged resorufin (TCO-Reso) to afford a rapid and specific fluorescence enhancement of TCO-Reso. Such signal readout is correlated with the urinary tetrazine concentration and thus measures the level of cancer biomarkers in the colon. HATS-based optical urinalysis detects orthotopic colon tumors two weeks earlier than clinical serological tests and can be developed to a point-of-care paper test. Thereby, HATS-based urinalysis provides a non-invasive and sensitive approach to cancer screening at low-resource settings., (© 2024 Wiley‐VCH GmbH.)

Background: In view of discordance consisting in different reports, a meta-analysis was conducted to comprehensively evaluate the diagnostic efficacy of exosomal noncoding RNAs (ncRNAs) in blood and urine in the detection of bladder cancer., Methods: Eligible studies were acquired by systematic retrieval through PubMed, Cochrane Library, and Embase. The pooled diagnostic efficacy was appraised by reckoning the area under the summary receiver operating characteristic (SROC) curve. The latent sources of heterogeneity were probed by subgroup analyses and meta-regression. STATA 12.0, Meta-DiSc 1.4, and RevMan 5.3 were applied to carry out all statistical analyses and plots., Results: A total of 46 studies from 15 articles comprising 2622 controls and 3015 bladder cancer patients were included in our meta-analysis. Exosomal ncRNAs in blood and urine represented relatively satisfactory diagnostic efficacy in detecting bladder cancer, with a pooled sensitivity of 0.75, a specificity of 0.79, and an area under the SROC curve (AUC) of 0.84. Exosomal microRNAs (miRNAs) exhibited better diagnostic value with a pooled AUC of 0.91 than that of exosomal long noncoding RNAs (lncRNAs). To some extent, the heterogeneity among studies was induced by exosomal ncRNA types (miRNA or lncRNA), exosomal ncRNA profiling (single- or multiple-ncRNA), sample size, specimen types, and ethnicity., Conclusion: Exosomal ncRNAs in blood and urine may play a vital role in diagnosing bladder cancer as prospective noninvasive biomarkers; nonetheless, their clinical performance needs to be confirmed by further massive proactive researches., (© 2024. The Author(s).)

Background: The gold standard for detecting bladder cancer is cystoscopy with biopsy or transurethral resection confirming histologic diagnosis. URO17® employs a chromogenically labeled monoclonal antibody to keratin 17 (k17), an intermediate filament cytoskeleton molecule associated with bladder, pancreatic, and cervical cancers. Preliminary studies evaluating k17 demonstrated a high sensitivity and specificity for the detection of bladder cancer, supporting the need for further study., Objective: To evaluate the sensitivity and specificity of URO17., Methods: This is a cross-sectional study of participants undergoing urologic procedures between July 6, 2018 and July 17, 2019 at a single institution. Patients undergoing cystectomy, endoscopic bladder and/or upper tract procedure for probable urothelial carcinoma comprised cases; patients undergoing urologic procedures for other reasons comprised the control group (i.e. prostatectomy, nephrectomy, etc.). Voided urine samples were at the time of procedure; a minority of participants underwent multiple resections in the study period, thus, as many as three urine samples were taken from any given participant. Samples were distributed for blinded testing with URO17. Sensitivity and specificity were calculated., Results: In 152 participants and 167 samples, URO17 demonstrated an overall sensitivity of 90% and 92% and a specificity of 88% and 87%, respectively. In 76 participants and 91 samples from patients with suspected urothelial carcinoma, the sensitivity was 90% and 92%, and the specificity was 50% and 54%, respectively. No controls demonstrated a positive URO17 result, and URO17 superseded urine cytology detection of low-grade and high-grade Ta. False positive results were associated with inflamed tissue or urothelial atypia on histology; the large majority had a history of intravesical therapy., Conclusion: Limitations include cross-sectional design and convenience sampling. URO17 may improve sensitivity of urine cytology in the detection of urothelial cancer, though further study is required to refine the application of this biomarker in clinical practice., Competing Interests: Declaration of competing interest SJ is co-founder, President, and COO of KDx diagnostics; NK is co-founder and CEO of KDx diagnostics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Withdrawal: Urinary exosomal tRNA-derived small RNA signatures as a predictive biomarker in lung cancer, published in FEBS OPEN BIO, 10.1002/2211-5463.13716. The above article, published online on 9th October 2023 in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the journal Editor in Chief, Miguel De la Rosa, and Wiley Periodicals LLC. The withdrawal has been agreed following no response from the author to requests to sign the Journal's publishing license/to requests to approve their article proof for publication. https://doi.org/10.1002/2211-5463.13716., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Purpose: Despite many efforts, no reliable urinary marker system has so far shown the potential to substitute cystoscopy. Measuring volatile organic compounds (VOCs) from urine is a promising alternative. VOCs are metabolic products which can be measured from the headspace of urine samples. Previous studies confirmed that the urine of bladder tumor patients has a different VOC profile than healthy controls. In this pilot study, the feasibility of discriminating VOCs from urine of bladder cancer patients from that of healthy control subjects was investigated. Aim of this study was to investigate whether VOC-based diagnosis of bladder cancer from urine samples is feasible using multicapillary column ion mobility spectrometry (MCC/IMS) and to identify potential molecular correlates to the relevant analytes., Methods: Headspace measurements of urine samples of 30 patients with confirmed transitional cell carcinoma (TCC) and 30 healthy controls were performed using MCC/IMS. In the results of the measurements, peaks showing significant differences between both groups were identified and implemented into a decision tree with respect to achieve group separation. Molecular correlates were predicted using a pre-defined dataset., Results: Eight peaks with significantly differing intensity were identified, 5 of which were highly significant. Using a six-step decision tree, MCC/IMS showed a sensitivity of 90% and specificity of 100% in group separation., Conclusion: VOC-based detection of bladder cancer is feasible. MCC/IMS is a suitable method for urine-based diagnosis and should be further validated. The molecular characteristics and metabolic background of the analytes require further workup., (© 2024. The Author(s).)

Background: The recurrence rate of thyroid cancer can be as high as 30%. The purpose of this study was to examine changes of urine exosomal peptide levels after thyroidectomy in patients with thyroid cancer to determine if levels can predict the risk of recurrence., Methods: Patients >20 years old as newly diagnosed with papillary thyroid cancer who had received a thyroidectomy were recruited. Urine samples were collected at 12 months after enrollment to the study, and 1 year later. Urine exosomes containing different peptides were identified and compared., Results: A total of 70 patients were enrolled in the study, and were classified by the interval between surgery and enrollment: 42 patients with < 5 years between surgery and enrollment, 14 patients between 5-10 years, and 14 patients longer than 10 years. No recurrence was observed in any patient during the 2 years after enrollment. No significant differences were found in the levels of serum proteins or urine exosomal peptides between groups, or between intervals. Known risk factors for high-risk thyroid cancer had only a mild correlation with serum protein levels and urine exosomal peptides., Conclusion: Our study revealed the long-term basal fluctuation ranges of serum proteins and urine exosomal peptides in patients with thyroid cancer who underwent thyroidectomy. For high-risk patients after thyroidectomy, concentrations of serum proteins or urine exosomal peptides within the ranges may indicate there is a lower risk of thyroid cancer recurrence during long-term follow-up., Trial Registration: ClinicalTrials.gov: NCT03488134., Competing Interests: All authors have declared that no competing interests exist for this work., (© 2024 Wang et al.)

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).)