Increased Detection of Merkel Cell Polyomavirus in Non-Melanoma Skin Cancer and Its Association with Host Immunogenetic Profile

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates the presence of MCPyV (Merkel cell polyomavirus) in different skin lesions and its association with cytokine gene polymorphisms. However, in order for the study to contribute more to researchers in this field, the following points should be clarified.

1. Images related to the type of cancer addressed in the study should be provided. Visual results of the probabilistic distribution of the medical dataset should also be provided. Such steps will provide more information about the classification process.
2. Formulas for the metrics considered in the statistical analysis should be provided. In this context, the study should also provide qualitative results obtained by considering these formulas.
3. New research in literature in this field, especially in the last 2 years, should be included in the study.

Author Response

Subject: Submission of the revised manuscript (ID: dermato-3597075) and response to reviewers

Dear Editors,

Thank you for your letter and for the valuable feedback from the reviewers on our manuscript, "Increased Detection of Merkel Cell Polyomavirus in Non-Melanoma Skin Cancer and its Association with Host Immunogenetic Profile" (ID: dermato-3597075).

We are grateful for the thorough and constructive comments. We have carefully revised the manuscript to address all the points raised, and we believe the paper has been significantly strengthened as a result. We have also addressed the editorial requests, including the reduction of the similarity index and the addition of the Ethics Committee approval date. All changes in the manuscript have been highlighted using the "Track Changes" feature for easy identification.

Below, we provide a point-by-point response to each of the reviewers' comments.

Reviewer 1

Comment 1.1: Images related to the type of cancer addressed in the study should be provided. Visual results of the probabilistic distribution of the medical dataset should also be provided. Such steps will provide more information about the classification process.

Response: We thank the reviewer for this valuable suggestion. To improve the manuscript's clarity, we have now included:

• A new Figure 1, which displays representative histopathological images of the main cancer types analyzed in our study (Basal Cell Carcinoma and Squamous Cell Carcinoma).
• A new Figure 2, which visually presents the distribution of the different types of skin lesions included in our patient cohort. We believe these additions provide a clearer and more informative overview of our dataset.

Comment 1.2: Formulas for the metrics considered in the statistical analysis should be provided. In this context, the study should also provide qualitative results obtained by considering these formulas.

Response: We appreciate the reviewer's focus on methodological rigor. To ensure transparency, our manuscript's Methods section clearly details our statistical approach, including the specific tests used (Pearson’s chi-square, Fisher’s exact test, McNemar test, and Kappa coefficient), the software employed (IBM SPSS Statistics, version 29), and our defined threshold for significance (p < 0.05). However, we maintain that including the underlying mathematical formulas for these standard tests is generally outside the scope of the journal's format.

That said, we found the reviewer's point about providing clearer qualitative results to be very constructive. Following this suggestion, we have revised the manuscript to better explain the practical meaning of our statistical findings. For example, when discussing the concordance analysis between the lesion and the surgical margin, we now explicitly state the qualitative interpretation of the obtained Kappa value. The text in the Results section (3.2) was modified to include the following sentence: 'According to the widely accepted criteria for interpreting Kappa values, this represents only a 'fair' level of agreement, reinforcing that the presence of MCPyV in the lesion is not a strong predictor of its presence in the surrounding tissue.'

We believe this approach fully addresses the reviewer's concern, making our results more transparent and their interpretation more robust

 

Comment 1.3: New research in literature in this field, especially in the last 2 years, should be included in the study.

Response: We agree completely with the reviewer. We have conducted a thorough literature search and have updated the Introduction and Discussion sections to include and discuss relevant findings published within the last two years. These additions are marked in the text and the new citations have been added to the References section.

We are confident that these revisions have substantially improved the manuscript. We once again thank the reviewers and the editorial team for their time and effort in evaluating our work.

Sincerely,

Prof. Rafael Brandão Varella (Corresponding Author) Federal Fluminense University, Niterói, RJ, Brazil

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a thorough analysis of Merkel Cell Polyoma Virus (MCPyV) in non melanoma skin cancer. Here they compare the presence of the virus in basal cell carcinoma, cutaneous squamous cell carcinoma, the perilesional skin and healthy skin. The authors also correlate the  MCPyV detection dependent on solar exposure. In addition, the cytokine gene polymorphisms with regard to IL-10, IFN-gamma, IL-6 and TNF were analyzed by ARMS-PCR.

The concept of the study is clear and the manuscript is well written. However, MCPyV has been analyzed in non melanoma skin cancer before so that the new findings are reduced to the immunogenetic profile, which is still interesting data for better understanding the role of MCPyV in NMSC but stays descriptive.

There are some points that I recommend to adress to improve the manuscript:

• While the manuscript mixes BCC an cSCC the number of cSCC is quite low. Thus I would recommend to focus on BCC or explicitly and differ between these tumors and also please underline that numbers are very low for cSCC.
• Do the authors also analyze the immunogenetic profile in the perilesional skin? This could give more insight whether the profile correlates with the detection of the virus. Also paired analysis of samples would make sense then.
• The authors mention that they used the tumor location to infer solar exposure. They do not define locations of the tumors for correlation with the sun exposure. This should be added. In addition, a comment on the distribution of MCPyV dependent on the skin area would be helpful. Have there been differences reported? This is important to answer the question whether the MCPyV evidence is due to sun exposure or the location.
• My understanding is that there have been multiple tumors analyzed from one patient. Can you comment whether they were all the same in MCPyV detection or could you find tumors with and without MCPyV in the same patient? Please comment on that.
• Analysis of cytokines on the protein level in the tumor and surroundings would increase the impact of the manuscript.
• Table 3: Why is the order GG, AA, GA and not GG, GA, AA. Suggestion: Depict the data in a graph. That will be easier to be perceived by the reader.

Author Response

Subject: Submission of the revised manuscript (ID: dermato-3597075) and response to reviewers

Dear Editors,

Thank you for your letter and for the valuable feedback from the reviewers on our manuscript, "Increased Detection of Merkel Cell Polyomavirus in Non-Melanoma Skin Cancer and its Association with Host Immunogenetic Profile" (ID: dermato-3597075).

We are grateful for the thorough and constructive comments. We have carefully revised the manuscript to address all the points raised, and we believe the paper has been significantly strengthened as a result. We have also addressed the editorial requests, including the reduction of the similarity index and the addition of the Ethics Committee approval date. All changes in the manuscript have been highlighted using the "Track Changes" feature for easy identification.

Below, we provide a point-by-point response to each of the reviewers' comments.

Reviewer 2

Comment 2.1: While the manuscript mixes BCC an cSCC the number of cSCC is quite low. Thus I would recommend to focus on BCC or explicitly and differ between these tumors and also please underline that numbers are very low for cSCC.

Response: This is an excellent point. We thank the reviewer for highlighting this limitation. We have revised the manuscript to address this as follows:

• In the Results section, we now explicitly state the low number of cSCC samples (n=7) compared to BCC samples (n=56).
• In the Discussion section, we have added a paragraph acknowledging that the small sample size for cSCC limits our ability to draw strong conclusions for this specific tumor type and that our findings regarding NMSC are predominantly driven by the BCC cases.

Comment 2.2: Do the authors also analyze the immunogenetic profile in the perilesional skin? This could give more insight whether the profile correlates with the detection of the virus. Also paired analysis of samples would make sense then.

Response: We thank the reviewer for this question, which allows us to clarify our methodology. The immunogenetic profiling (cytokine gene polymorphisms) was performed once for each patient to determine their constitutional genotype. This profile is inherent to the individual and, therefore, is the same across all tissues sampled (lesional, perilesional, and healthy skin). Our study was designed to correlate this single, systemic genetic profile with the presence or absence of MCPyV in these different tissue microenvironments. We have revised the Materials and Methods section to make this aspect of our study design clearer.

Comment 2.3: The authors mention that they used the tumor location to infer solar exposure. They do not define locations of the tumors for correlation with the sun exposure. This should be added. In addition, a comment on the distribution of MCPyV dependent on the skin area would be helpful. Have there been differences reported?

Response: We agree this information is essential for reproducibility. We have amended the Materials and Methods section to clearly define the criteria used to classify tumor locations into "high," "moderate," and "low" sun exposure categories, based on the framework from our cited reference [11]. Furthermore, we have added a brief discussion in the Discussion section regarding reported differences in MCPyV prevalence by anatomical site, citing relevant literature to address whether detection is linked more to sun exposure or to specific skin areas.

Comment 2.4: My understanding is that there have been multiple tumors analyzed from one patient. Can you comment whether they were all the same in MCPyV detection or could you find tumors with and without MCPyV in the same patient? Please comment on that.

Response: We thank the reviewer for this insightful question regarding the consistency of MCPyV detection within a single patient. Our primary analysis on this topic focused on comparing paired samples from the same individual. As detailed in our Results, we found a significant intra-patient discordance between the lesion and the adjacent surgical margin (p=0.009), with a low Kappa coefficient (κ=0.305) confirming that it was common to find the virus in a tumor but not in the nearby tissue.

To further address the reviewer's specific point about MCPyV dsicrepancies, we had already observed that this principle of intra-patient discordance also held true, where individuals could present both MCPyV-positive and MCPyV-negative lesions.

To better highlight these multi-level findings, we have revised the Discussion section to explicitly frame them as evidence for the localized nature of viral persistence, both between adjacent tissues and between different tumors in the same individual.

 

Comment 2.5: Analysis of cytokines on the protein level in the tumor and surroundings would increase the impact of the manuscript.

Response: This is an excellent suggestion. We agree that analyzing cytokine expression at the protein level (e.g., via IHC or ELISA) would provide valuable functional insight and significantly strengthen our conclusions. However, this was beyond the scope of our current study, which was designed to investigate the association with the host's genetic predisposition. We have now included this point in the Discussion as a key direction for future research.

Comment 2.6: Table 3: Why is the order GG, AA, GA and not GG, GA, AA. Suggestion: Depict the data in a graph. That will be easier to be perceived by the reader.

Response: We thank the reviewer for these helpful suggestions regarding the presentation of our data. We agree that the original order of genotypes in Table 3 could be confusing and that a graph is a more effective way to present these findings.

Embracing this excellent suggestion, we have completely replaced Table 3 with a new, multi-panel bar graph (new Figure 3). This new figure visually represents the percentage of MCPyV-positive and -negative samples for each genotype, with the genotypes now presented in a logical order, making the associations easier for the reader to perceive. We believe this change significantly improves the clarity and impact of our results.

We are confident that these revisions have substantially improved the manuscript. We once again thank the reviewers and the editorial team for their time and effort in evaluating our work.

Sincerely,

Prof. Rafael Brandão Varella (Corresponding Author) Federal Fluminense University, Niterói, RJ, Brazil

Reviewer 3 Report

Comments and Suggestions for Authors

Journal: Dermato (ISSN 2673-6179)

Manuscript ID: dermato-3597075

Type: Article

Title: Increased Detection of Merkel Cell Polyomavirus in Non-Melanoma Skin Cancer and its Association with Host Immunogenetic Profile

 

I appreciate the effort invested in conducting this research. The paper is well written, with a thorough description of the methodology and results; however, I consider it important to clarify the extent to which this research could be applicable to the patients’ management.

I suggest rephrasing the pathology categories; for example, the 'non-malignant lesions' group includes 76 premalignant lesions.

What are the clinical implications of the obtained results, namely: this study provides new evidence that MCPyV is more frequently detected in non-melanoma skin cancers (NMSC) and non-malignant lesions than in surgical margins or healthy skin and that its presence in lesions does not strongly predict its presence in surrounding tissues, suggesting that the lesional microenvironment may favor viral persistence?

 

Author Response

Subject: Submission of the revised manuscript (ID: dermato-3597075) and response to reviewers

Dear Editors,

Thank you for your letter and for the valuable feedback from the reviewers on our manuscript, "Increased Detection of Merkel Cell Polyomavirus in Non-Melanoma Skin Cancer and its Association with Host Immunogenetic Profile" (ID: dermato-3597075).

We are grateful for the thorough and constructive comments. We have carefully revised the manuscript to address all the points raised, and we believe the paper has been significantly strengthened as a result. We have also addressed the editorial requests, including the reduction of the similarity index and the addition of the Ethics Committee approval date. All changes in the manuscript have been highlighted using the "Track Changes" feature for easy identification.

Below, we provide a point-by-point response to each of the reviewers' comments.

Reviewer 3

Comment 3.1 & 3.3: I consider it important to clarify the extent to which this research could be applicable to the patients’ management. (...) What are the clinical implications of the obtained results?

Response: We thank the reviewer for emphasizing the importance of translational relevance. We have added a new paragraph at the end of the Discussion section specifically dedicated to the potential clinical implications of our findings. In this section, we discuss how the association between specific immunogenetic profiles (IL-10 and IL-6 genotypes) and MCPyV detection could, in the future, serve as a potential biomarker for identifying patients at higher risk. We also discuss how our finding—that viral presence is highly specific to the lesional microenvironment—underscores that any successful therapeutic strategy, whether systemic like a therapeutic vaccine or locally administered, must effectively target the unique conditions within the lesion that allow for viral persistence.

Comment 3.2: I suggest rephrasing the pathology categories; for example, the 'non-malignant lesions' group includes premalignant lesions.

Response: We thank the reviewer for this important comment on terminological accuracy. We agree that the term 'non-malignant lesions' was imprecise for a group containing premalignant conditions. To address this, we have revised the Methods section (2.1) to explicitly state the composition of this comparative group, which includes premalignant, benign, and inflammatory conditions. Furthermore, we have adopted the more accurate term 'non-cancerous lesions' (NCL) as a shorthand to refer to this mixed group throughout the manuscript, ensuring clarity in the text, tables, and figures. We believe this approach fully resolves the imprecision while maintaining readability.

We are confident that these revisions have substantially improved the manuscript. We once again thank the reviewers and the editorial team for their time and effort in evaluating our work.

Sincerely,

Prof. Rafael Brandão Varella (Corresponding Author) Federal Fluminense University, Niterói, RJ, Brazil

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

thank you for addressing the suggestions made by the reviewers. Your data are interesting and ready for publication and demand further work on the protein level.