Diagnostic Performance and Clinical Utility of the Uromonitor^® Molecular Urine Assay for Urothelial Carcinoma of the Bladder: A Systematic Review and Diagnostic Accuracy Meta-Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a systematic review of the performance of Uromonitor® versus urine cytology. The authors conclude that Uromonitor® can complement cystoscopy for surveillance, but it is not adequate as a stand-alone surveillance test due to low sensitivity and heterogeneity.

Please add a comparison table of Uromonitor® performance with other commonly used urinary biomarkers and molecular assays (e.g., UroVysion® FISH, NMP22, BTA, and other contemporary molecular assays). Please comment on the comparative performance of Uromonitor® relative to these assays.

In the Discussion, the authors should discuss potential approaches to improve Uromonitor® performance by expanding the mutation panel and/or integrating additional biomarker classes (e.g., methylation or transcriptomic markers) to better capture tumor heterogeneity.

Author Response

Dear Prof. Dr. Qiang Lv,

Dear Dr. Xiao Yang,

Dear Prof. Dr. Kjaer (Editor-in-Chief),

Dear Reviewer,

We would like to express our sincere gratitude to the Editors and all three reviewers for their careful evaluation of our manuscript and for their thoughtful and constructive comments. We highly appreciate the time and expertise invested in the review process. The reviewers’ suggestions have been invaluable in improving the clarity, contextualization, and clinical relevance of our systematic review.

We have addressed all comments point by point below. All revisions are explicitly indicated, with precise references to the revised sections in the manuscript and the Supplementary Materials.

 

Reviewer 1

We thank the reviewer for the careful assessment of our work and for raising important points regarding contextualization of Uromonitor® within the broader landscape of urinary biomarkers.

Comment 1

Please add a comparison table of Uromonitor® performance with other commonly used urinary biomarkers and molecular assays and comment on the comparative performance of Uromonitor® relative to these assays.

Response:
We sincerely thank the reviewer for this important and highly relevant suggestion. We agree that placing the diagnostic performance of Uromonitor® into the broader context of urinary bladder diagnostic tests is essential for appropriate interpretation.

In response, we have expanded the Discussion to explicitly introduce a structured conceptual framework for urinary bladder diagnostic tests. We now clarify that contemporary urinary bladder diagnostic tests can broadly be categorized into protein-based, cell-based, RNA-based, and DNA-based assays. Within this framework, we emphasize that current European guidelines for surveillance primarily address urine cytology and molecular multiplex urine marker tests.

To provide readers with a comprehensive and transparent overview without disrupting the narrative flow of the Discussion, we have added a new Supplementary Table that closely follows the structure and scope of the corresponding table in the 2025 EAU Guidelines. This table summarizes the reported diagnostic performance of molecular multiplex urinary marker assays in the surveillance setting, including DNA-based assays such as Uromonitor®, while explicitly acknowledging differences in study design, patient populations, and reference standards, as well as the lack of direct head-to-head comparisons.

Manuscript changes:

– The Discussion section has been expanded to introduce a clear classification of urinary bladder diagnostic tests and to contextualize molecular multiplex urine marker assays within current guideline recommendations.

– A new Supplementary Table has been added summarizing the reported diagnostic performance of molecular multiplex urinary markers in the surveillance setting, modeled after the EAU guideline framework.

 

Comment 2

The authors should discuss potential approaches to improve Uromonitor® performance by expanding the mutation panel and/or integrating additional biomarker classes.

Response:
We thank the reviewer for this thoughtful and forward-looking comment. We have revised the Discussion to more explicitly address potential biological and technical strategies to improve assay performance.

Specifically, we now discuss tumor heterogeneity, variability in tumor DNA shedding, and pre-analytical and analytical factors that influence test sensitivity. We further emphasize that while expansion of mutation panels or integration of additional biomarker classes may increase sensitivity, such approaches may also compromise specificity and positive predictive value. We therefore highlight technical optimization strategies, including improved DNA extraction and digital PCR-based approaches, as rational avenues for future development.

Manuscript changes:

– An additional paragraph has been added to the Discussion addressing biological heterogeneity, technical optimization, and future development strategies for molecular urine assays.

Reviewer 2 Report

Comments and Suggestions for Authors

1-I recommend to add Uromonitor to keywords.

2-The main concern is high heterogeneity between studies , so it couldn't apply effectively in daily practice.

3-The difference in sensitivity for Uromonitor from conventional urine cytology is modest , regarding the probable cost ,I recommend to discuss about cost in discussion part.

4-It is better that authors compare these finding with other urinary- based biomarkers in discussion ,not only with conventional urine cytology.< !--/data/user/0/com.samsung.android.app.notes/files/clipdata/clipdata_bodytext_260101_092107_342.sdocx-->

Author Response

Dear Prof. Dr. Qiang Lv,

Dear Dr. Xiao Yang,

Dear Prof. Dr. Kjaer (Editor-in-Chief),

Dear Reviewer,

We would like to express our sincere gratitude to the Editors and all three reviewers for their careful evaluation of our manuscript and for their thoughtful and constructive comments. We highly appreciate the time and expertise invested in the review process. The reviewers’ suggestions have been invaluable in improving the clarity, contextualization, and clinical relevance of our systematic review.

We have addressed all comments point by point below. All revisions are explicitly indicated, with precise references to the revised sections in the manuscript and the Supplementary Materials.

 

Reviewer 2

We thank the reviewer for the constructive feedback and for highlighting several important aspects related to presentation, heterogeneity, and clinical applicability.

Comment 1

I recommend adding Uromonitor to the keywords.

Response:
We thank the reviewer for this suggestion and have revised the keyword list accordingly.

Manuscript changes:

– “Uromonitor®” has been added to the list of keywords.

 

Comment 2

The main concern is high heterogeneity between studies, limiting applicability in daily practice.

Response:
We fully agree with the reviewer. Study heterogeneity represents a major limitation of the current evidence base and directly impacts clinical applicability. This aspect is now more explicitly emphasized in the Limitations section and in the interpretation of our findings. Importantly, heterogeneity was a key determinant of the low certainty of evidence ratings in our GRADE assessment.

Manuscript changes:

– The Limitations section has been revised to more explicitly link heterogeneity to restricted generalizability.

– The GRADE rationale in the Supplementary Materials has been clarified accordingly.

 

Comment 3

The difference in sensitivity compared with urine cytology is modest; discussion of cost is recommended.

Response:
We thank the reviewer for raising this important point. We have expanded the Discussion to include a qualitative consideration of cost and resource implications. We emphasize that costs of urine cytology and molecular assays vary substantially across healthcare systems and reimbursement frameworks, precluding meaningful quantitative economic comparison within the scope of this review. Cost considerations are therefore framed in terms of potential impact on clinical pathways and the need for future context-specific cost-effectiveness studies.

Manuscript changes:

– A qualitative discussion of cost considerations has been added to the Discussion section.

 

Comment 4

Comparison with other urinary biomarkers should be included, not only urine cytology.

Response:
We appreciate this comment and have addressed it by expanding the Discussion and adding the new Supplementary Table summarizing molecular multiplex urinary marker assays, while maintaining urine cytology as the reference standard used in the primary studies.

Manuscript changes:

– The Discussion section has been expanded to introduce a clear classification of urinary bladder diagnostic tests and to contextualize molecular multiplex urine marker assays within current guideline recommendations.

– A new Supplementary Table has been added summarizing the reported diagnostic performance of molecular multiplex urinary markers in the surveillance setting, modeled after the EAU guideline framework.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors investigated the potential value of Uromonitor as a urine marker in bladder cancer. They reviewed systematically and selected 8 articles to define the diagnostic efficacy of Uromonitor. The significant value of this article is important; however, there are still several points to be addressed.

My main question is whether current urine-based biomarkers can truly distinguish high-risk disease. While many urinary biomarkers are known to detect the presence of bladder cancer cells, differentiating high-risk features such as tumor grade and carcinoma in situ (CIS) is a separate and more challenging issue. It would therefore strengthen this study if the authors emphasized this aspect more clearly and provided additional analyses and results specifically addressing the ability of the assay to identify high-risk disease.

I am wondering whether you have analyzed Uromonitor results specifically in cases where urine cytology is reported as “undetermined” or “atypical.” If so, it would be helpful to present these data, as they could highlight the added value of the assay in exactly those equivocal situations where cytology is limited. If not, I would recommend discussing whether and how Uromonitor might overcome some of the known limitations of urine cytology, particularly in the context of indeterminate or atypical cytology findings.

I would be interested to know what characteristics were observed in the bladder cancer cases that were not detected by Uromonitor. For example, do these false‑negative cases share specific pathological or molecular features that might explain the negative result? It would also be valuable if the Discussion could address how such limitations might be mitigated when using Uromonitor, for instance, by combining it with other biomarkers, refining the mutation panel, or integrating it with cystoscopy and clinical risk factors to improve detection of these “missed” tumors

 

 

Author Response

Dear Prof. Dr. Qiang Lv,

Dear Dr. Xiao Yang,

Dear Prof. Dr. Kjaer (Editor-in-Chief),

Dear Reviewer,

 

We would like to express our sincere gratitude to the Editors and all three reviewers for their careful evaluation of our manuscript and for their thoughtful and constructive comments. We highly appreciate the time and expertise invested in the review process. The reviewers’ suggestions have been invaluable in improving the clarity, contextualization, and clinical relevance of our systematic review.

We have addressed all comments point by point below. All revisions are explicitly indicated, with precise references to the revised sections in the manuscript and the Supplementary Materials.

 

Reviewer 3

We sincerely thank the reviewer for the insightful and clinically nuanced comments, which helped to further sharpen the interpretation of our findings.

Comment 1

Can urine-based biomarkers distinguish high-risk disease, including tumor grade and CIS?

Response:
We thank the reviewer for this important question. We have clarified in the Discussion that urine-based biomarkers are primarily designed to detect tumor-associated molecular alterations, whereas discrimination of high-risk features such as tumor grade and carcinoma in situ represents a distinct and more complex challenge. Subgroup analyses stratified by stage and grade were limited to what was reported in the primary studies and are summarized in Supplementary Table 1. Further stratification is currently not supported by the available published data.

Manuscript changes:

– The Discussion has been revised to clearly distinguish tumor detection from risk stratification.
– Cross-references to Supplementary Table 1 have been strengthened.

 

Comment 2

Were Uromonitor results analyzed in cases with atypical or undetermined urine cytology?

Response:
We thank the reviewer for this insightful comment. All included studies applying urine cytology used dichotomized reporting according to the Paris System, classifying atypical urothelial cells as negative in binary analyses. Separate analyses for atypical or indeterminate cytology were therefore not consistently available across studies. We have clarified this methodological aspect and expanded the Discussion to address the potential role of molecular assays in equivocal cytology scenarios.

Manuscript changes:

– Clarification added to the Methods section regarding cytology dichotomization.
– Expanded Discussion on potential value in equivocal cytology settings.

 

Comment 3

What characterizes false-negative Uromonitor cases and how might these limitations be mitigated?

Response:
We thank the reviewer for this important comment. Detailed characterization of false-negative cases was only available in a subset of studies and could not be aggregated quantitatively. However, available subgroup analyses demonstrate reduced detection rates in muscle-invasive and high-grade tumors, supporting our conclusion that Uromonitor® should not be used as a stand-alone test. We have expanded the Discussion to address potential biological and technical explanations and to outline mitigation strategies, including combination with cystoscopy and clinical risk stratification.

Manuscript changes:

– Expanded Discussion addressing false-negative patterns and mitigation strategies.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors revised the manuscript based on the reviewer's requests.