PONE-D-22-26758Could the mitotic count improve personalized prognosis in melanoma patients?

PLOS ONE

Dear Dr. Cozzolino,

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The introduction has to be re-­‐structured, and the rationale for the study should be set clear. The manuscript should also be checked for types and grammars errors.

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting study looking at the prognostic role of Mitoses in melanoma patients. This topic has been discussed in the past years , since it gave indication to SLN biopsy in the last AJCC classification (upstaging the T1a to T1b).

The study is well conducted and the results and discussion sound interesting. There authors made correction with SLNB status but not with the involvement of SLN (micro macro metastasis). Please have a look at Quaglino et al . Surg Oncol. 2011 Dec;20(4):259-64. Is there a particular reason not to look at this association ? Please discuss.

Reviewer #2: In this manuscript the authors examine the prognostic significance of mitotic rate in two cohorts of melanoma patients from the Veneto Cancer Registry. They show a correlation between increasing mitotic rate and various histopathologic factors in melanoma. They also show worse survival with increasing mitotic rate by Kaplan-Meier analysis. They investigate various cutoffs for mitotic rate in the entire cohorts, and in individual T categories. They conclude that mitotic rate does have prognostic significance, and may be used in individualized prognostic assessments of melanoma patients. Overall, the manuscript is useful in its reinforcement of the importance of mitotic rate. Attention to the following points is warranted to strengthen their conclusions, and also to address limitations in the available cohorts that precluded additional observations regarding the prognostic significance of mitotic rate.

1. A critical issue regarding assessment of mitotic rate, given the non-linear shape of its impact on survival, is the identification of optimal cut-points, both in the entire cohort of patients, as well as in specific T categories, as has been previously demonstrated (ref 14). To begin with, this should be discussed in greater detail in the Discussion section. While the authors examine this issue in some analyses, additional analyses are suggested. For example, in their K-M analysis of the entire cohort in Fig. 1, the authors examine different cutpoints for mitotic rate (0, 1, 2, 3-5; and >6). This analysis suggests the following “optimal” grouping or index for mitotic rate with differing survival: 0-2; 3-5; and > 6. It would be both interesting and important to analyze this further, both in univariate and multivariate analyses for the entire cohort. Also, given that the analysis of individual cutpoints only showed significant results in T2 patients, it would be interesting to examine this index in each T category to determine whether it would show a significant prognostic significance.

2. For the multivariate analysis of the entire cohort, it would be important to show the significance of mitotic rate (using the best individual cutpoint or the index mentioned in point 1 above) along with the other factors analyzed, so that its impact can be understood in the context of the other factors available.

3. As indicated above, there are significant limitations to the cohorts available that may preclude additional significant observations regarding the prognostic significance of mitotic rate in this analysis. One limitation is the preponderance of stage I cases (76% of the cohort), which in part accounts for 50% of the cases having a mitotic index of 0. Therefore, the proportion of cases with elevated mitotic rate is low. In addition, this results in smaller sample sizes for T3 and T4 cases, precluding a meaningful assessment of the prognostic impact in these subsets. These issues should be explicitly discussed.

4. What is the median follow up of the two cohorts? This should be explicitly stated, as it represents another major limitation of the study, especially in view of the predominance of stage I lesions, which can present with late relapses and deaths (i.e., beyond five years).

5. Table 1- 46% of patients were male. This is in contrast to most studies, which show a male predominance. Do the authors have any explanation for this finding?

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-26758R1Could the mitotic count improve personalized prognosis in melanoma patients?PLOS ONE

Dear Dr. Cozzolino,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within Apr 26 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Dr H. Boukerche, PhD

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Comments to the Author

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Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The assessed manuscript was a 'Revised Manuscript with Track Changes' in the document PONE-D-22-26758_R1_reviewer. In general, the objective is clear, and the methodology is appropriate.

- In the abstract, the authors should add the time of overall survival evaluated (3-year overall survival) and use the term 'histological subtype' instead of 'type of morphology'.

- In Statistical Methods, make it clear that the considered period for short-term overall survival was 3 years.

- In Tables 1 and 2, it is important to include in the caption what 'Malignant (NOS)' means, once it is not a histological subtype of melanoma.

- In the title of Table 2, there is no need for the phrase “p-values < 0.05 are reported in bold”

- In Table 2, the percentage is not consistently represented (Some values have two numbers after the decimal point, others do not).

- Fig.1 is mentioned in the text, the title appears, but I could not find either the figure or its caption.

- Tables 3 and 4: highlight significant p-values in the adjusted model using bold, not red color.

- In the discussion, it is more appropriate to write "increased mitoses per mm2 are associated with a higher risk of mortality, particularly in T2 patients," instead of "mitoses per mm2 are associated with an increased risk of mortality, particularly in T2 patients."

- In the discussion, it is more appropriate to write "In particular, we observe that patients with a high mitotic rate are more likely to be older, male, have a melanoma lesion with a vertical growth pattern, nodular histological subtype, ulceration, greater tumor thickness, and an advanced stage," instead of "In particular, we observe that patients with a high mitotic rate are more likely to be older, male, have a melanoma lesion with a vertical growth pattern, ulceration, greater tumor thickness, and an advanced stage."

- In the discussion, "... the presence of pre-existing nevi were found to be typical of lesions with sparse mitotic activity," however, there is no information about the absence or presence of pre-existing nevi in the results. Please, insert the data or remove this sentence.

- In the discussion, reference 14 appears in the wrong place (page 72 of the document PONE-D-22-26758_R1_reviewer).

- In the last paragraph of the discussion, it should be T2N0M0, not T2N0.

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #3: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Could the mitotic count improve personalized prognosis in melanoma patients?

PONE-D-22-26758R2

Dear Dr. Claudia Cozzolino,

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