Prognostic Value of P63 Expression in Muscle-Invasive Bladder Cancer and Association with Molecular Subtypes—Preliminary Report

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Interesting results

1. The main question is the possible role of p63 as a predictive marker in invasive bladder cancer.

2. Although is a limited research of only one marker, the topic is completely related in the field without any significant gap.

3. In his aproach, the manuscript is well formed. I would like to have more patients included and their follow up.

4.The conclusions are consistent with the evidence and the discussion.

5. The references seem to be appropriate

6. In my point of view ,I would like to have 2-3 photos more of the immunohistochemistry markers.

Author Response

We thank the Reviewer for His/Her meaningful comments.

[4] We added the following sentence (lines 307-310): “We studied patients for whom follow-up data was available and selected them based on the inclusion and exclusion criteria mentioned above. To overcome this limitation, and achieve more significant results, we are planning to expand our cohort in a future study”

[6] Additionally, we have included photographs of the immunohistochemical markers as suggested by the Reviewer.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled: “Prognostic Value of P63 Expression in Muscle-Invasive Bladder Cancer and Association with Molecular Subtypes. Preliminary report” submitted to the journal Current Issues in Molecular Biology with manuscript ID: cimb-2889792 explores the prognostic role of P63 in a limited cohort of 65 patients with muscle-invasive bladder cancer (MIBCs), by assessing of P63, CK5/6, CK20 and GATA3 protein expression by IHC staining.

The study failed to demonstrate an association between P63 expression and disease stage or nodal metastases, as well as between P63 status and CSS or OS, but a trend towards worse OS in P63-positive tumours was noted.

Although the study fits the journal's scope and is well-written I found it too preliminary to be accepted for publication. The main issues include limited sample size and methodological limitations that prevent definitive conclusions.

Reliance solely on the IHC of a narrow panel of markers is insufficient for a comprehensive understanding of molecular subtypes of MIBCs. Other methods are crucial for confirming expression level.

There are no samples of the histologically normal bladder, used as controls. There is no result of a sample with a cut-off of 150 of the H-score.  

The limited sample size probably leads to the low power of the study to demonstrate an association between P63 expression and disease stage or nodal metastases, as well as between P63 status and CSS or OS.

 

Implementing a follow-up study to track dynamic changes could strengthen conclusions and identify potential prognostic markers.

Author Response

We thank the Reviewer for His/Her meaningful comments. We acknowledge the limitations of our study (see lines 306-313) and the fact that we achieved only preliminary findings (see line 316). We decided to use IHC as the only technique in our study due to its wide availability in routine practice. Since we use standardized protocols, and p63 is a reliable marker, we did not need to use negative/positive controls.

Reviewer 3 Report

Comments and Suggestions for Authors

In recent years, scattered reports have examined the association between p63 expression status and disease prognosis in bladder urothelial carcinoma. I recognize that the present study is one of them. This is not highly novel, but I think it is important data. The main question addressed by the research is to evaluate the prognostic value of P63, which is one of the transcription factors, in patients with MIBC.

There have been several studies that evaluate the significance of P63 in the prognosis of bladder cancer. But the results of them are confusing. One potential reason for such divergent findings across previous studies may be due to the fact they included tumors of different Stages and Grades as well as to large variability in analytical parameters, specifically different antibodies and staining protocols, different cut-offs of stained cells (10%, 50%, 80%), combined evaluation of staining intensity and percentage of positive cells. To minimize such potentially confusing factors, the present study chose to analyze a homogeneous cohort of MIBCs treated with RC only. Next, since there is no current standardized scoring system for P63, they used the H-score, a type of immunoreactivity score. Moreover, they used two double-antibody panels as surrogate markers to stratify MIBCs according to current molecular subtyping systems.

Comparing the past studies, the present study is well designed one.

The conclusions of this study is consistent and this reviewer agrees with them.

The references are appropriate.

Author Response

We thank the Reviewer for His/Her meaningful comments.

Reviewer 4 Report

Comments and Suggestions for Authors

This article presents an interesting investigation into the prognostic value of the transcription factor P63 in muscle-invasive bladder cancer (MIBC) post-radical cystectomy. The authors reveal that while P63-negative tumors displayed adverse pathological features such as specific patterns of muscularis propria invasion and variant histology, P63 status alone did not predict Cancer-specific Survival (CSS) and Overall Survival (OS). However, P63 may play a role in improving molecular subtyping of MIBC.

To enhance the manuscript, I offer the following recommendations:

Specific Comments:

1. Lines 73-76: Kindly add the number of excluded patients due to the stated exclusion criteria to provide better context for the final count of 65 patients included in the study. Moreover, clearly state the rationale behind excluding patients who received neoadjuvant or adjuvant chemotherapy and those with evidence of distant metastasis.
2. Lines 78-85: Ensure clarity by specifying whether these variables were retrospectively gathered or recorded during initial treatment. Furthermore, indicate if missing values were encountered and what strategies were implemented to handle them.
3. Line 101: Precisely articulate the rationale behind choosing an H-score of 150 as an optimization cut-off for marker positivity or provide a reference supporting that decision.
4. Line 157: Begin the discussion by restating the main objective of the study and summarizing the key findings. This will provide readers with a clear overview of the investigation and its outcomes.

 

General Comments:

The manuscript is worthy of publication, but certain methodological issues must be addressed. The title and abstract are appropriate, and the level of English is satisfactory.

 

Commendably, the authors have openly acknowledged the limitations of their study, demonstrating maturity and a nuanced understanding of their research's scope and potential impact.

 

In conclusion, the study offers valuable insights into the role of P63 in MIBC. Implementing the suggested modifications will greatly enhance the manuscript and facilitate a smoother reading experience.

 

Comments on the Quality of English Language

The level of English in the manuscript is generally acceptable, with occasional room for improvement in sentence construction and word choice for enhanced clarity.

Author Response

We thank the Reviewer for His/Her meaningful comments.

Reviewer 5 Report

Comments and Suggestions for Authors

The study is well conducted and interesting. However, several errors need to be addressed before it may be accepted for publication:

1. In abstract the background section should state about the clinical problem that the paper addresses. Next you can add the info about the aim of the study.

2. In line 27 there is information about the Algorithm #1 but the first-glance reader of the abstract is not familiar with that specific aspect of your paper. 

3. In M&M section please clarify which exact antibodies were used by providing the manufacturer and some kind of identification number. 

4. I think that reference column in table 3 should be in the end of the table. 

5. I think that the discussion section about the algorithms should be heavily revised. First you should clarify why you developed these two particular analysis schemes and then you can compare the outcomes of using both of them. In the end you can compare to the other papers which also use similar approaches. 

Author Response

We re-wrote the section according to the Reviewer’s suggestion (see lines 277-281)

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors significantly improved their manuscript and answered most of my previous concerns. Although the study is preliminary it is clearly stated by the authors and hopefully, the results will be confirmed in expanding cohort in a future study.

Reviewer 4 Report

Comments and Suggestions for Authors

Upon careful consideration and assessment of the revisions made by the authors in response to our earlier feedback, I am pleased to report that they have adequately addressed each point raised in the review process. Consequently, I believe that the final manuscript is worthy of publication.

Comments on the Quality of English Language

The quality of English is appropriate, exhibiting adequate vocabulary selection, and effective sentence construction