-->PONE-D-25-56784-->-->A multicenter, randomized, parallel-group confirmatory study protocol to evaluate the efficacy of Soft Protector CPC, a novel oral mucosal protectant, in preventing oral mucositis and alleviating pain in patients with breast cancer-->-->PLOS One

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The reviewers made specific comments please address them, apologies for taking this long with the review process.

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This research is supported by the Japan Agency for Medical Development (AMED) under grant number JP25ck0106033.

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The authors received financial support from the Japan Agency for Medical Research and Development (grant number JP25ck0106033). This study supported by San Medical Co., Ltd., for providing the investigational material (Soft Protector CPC).

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.-->

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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-->3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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-->4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 #1: Yes

Reviewer #2: Yes

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 #1: Yes

Reviewer #2: Yes

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 and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: As the statistical reviewer I will focus on methods and reporting

Major

1) The power calculation is necessarily arbitrary. report as cohen's d for clarity, the effect size. is ICC expected since this is a multi centre trial? is there no chance of drop outs? that is not accounted in the power calculations.

2) there is no mention of the consort statement and its relevance here.

3) i'd advise analyses to follow a regression framework so effect sizes and uncertainty can be easily quantified and reported - moving away a bit from p-values, they shouldn't be the focus.

4) what characteristics will be reported for each arm, on which balanced will be evaluated? what will the authors do if the randomisation has not ensured balanced across all covariates considering the small sample? and will missing data be dealt with (perhaps multiple imputation as a sensitiivty analysis)

5) again going back to expected heterogeniety, or not, across centres, the authors need to consider how this affects the study. in the power calculation but also in the analyses. in a regression framework one would expect a mixed effects model with a random intercept for centre.

6) there are numerous secondary outcomes. analyses for these are likely to be underpowered and authors need to be clear these analyses will be described as exploratory.

7) any interventions (medications) need to be monitored and pehaps used in the regression model if there is no complete balance.

Minor

1) is there a plan for safety to be monitored statistically? this relates to the fact there are no interim analyses planed.

Reviewer #2: GENERAL ASSESSMENT

This manuscript describes a multicenter, randomized, open-label controlled trial protocol evaluating the efficacy of the Soft Protector CPC for the prevention or alleviation of chemotherapy-induced oral mucositis in patients with breast cancer. The topic is clinically relevant, and the randomized design and inclusion of both clinician-assessed and patient-reported outcomes are strengths. However, the protocol raises substantial methodological concerns related to the definition of the primary endpoint, its alignment with the two study periods, and the justification of the sample size and statistical analysis, all of which affect the interpretability and confirmatory strength of the study.

MAJOR COMMENTS:

1. Primary endpoint definition and clinical relevance

The primary endpoint is defined as “the proportion of patients achieving prevention of the onset or alleviation of pain during the comparative period” and is planned to be analyzed as a binary outcome using a Pearson chi-square test. This definition raises important concerns. It combines two conceptually distinct phenomena: prevention of oral mucositis onset, which applies to the entire study population, and alleviation of pain, which is relevant only to the subset of patients who develop symptoms. Collapsing these constructs into a single binary outcome obscures clinical interpretation and makes patient classification unclear.

In addition, the protocol does not specify how “alleviation of pain” is operationally defined (e.g., magnitude of change, timing, or applicable patient subset), leaving this component open to subjective interpretation and inconsistent application. These issues are particularly relevant given that oral mucositis in breast cancer chemotherapy is of moderate incidence: published data suggest rates of approximately 20–40%, with severe cases (CTCAE grade ≥3) in fewer than 10% of patients (e.g., Elting et al. 2003 DOI: 10.1002/cncr.11671; Sonis, 2004 DOI: 10.1038/nrc1318; Lalla et al. 2014 DOI: 10.1002/cncr.28592). As many participants may never develop mucositis or pain, inclusion of pain alleviation within the primary endpoint risks inflating apparent success without clearly reflecting a preventive effect. The primary endpoint should therefore be redefined with clear operational criteria and aligned with the preventive nature of the study population.

2. Relationship between endpoints and study periods

The protocol distinguishes between a randomized “comparative period” and a subsequent “continuous period” during which all participants receive Soft Protector CPC. While this design may be ethically motivated, its analytical implications are not sufficiently clarified. The comparative period is the only phase that allows a randomized comparison and must therefore underpin all primary efficacy conclusions. Outcomes assessed during the continuous period, when no control group remains, cannot support causal inference and should be considered descriptive or exploratory.

However, the protocol does not clearly restrict the primary endpoint to the comparative period nor explicitly define the continuous period as exploratory, creating a risk of overinterpretation of non-randomized results. This ambiguity is further compounded by the unclear endpoint definitions, particularly if symptom alleviation is emphasized beyond the randomized comparison. The authors should clearly delineate the analytical roles of the two study periods and explicitly limit confirmatory conclusions to the comparative period.

3. Statistical analysis inconsistencies

The sample size calculation is based on the primary endpoint described above and on a Pearson chi-square test comparing proportions between groups. The validity of this approach depends on a clearly defined binary outcome with a well-specified underlying probability structure. Given the composite and ambiguously defined nature of the primary endpoint, it is unclear how the assumed response rates used for the power calculation were derived or justified. In particular, the protocol appears to assume a relatively high success rate in the intervention group (>50%), which is difficult to reconcile with the moderate incidence of chemotherapy-induced oral mucositis in breast cancer patients unless the endpoint definition is overly broad.

Moreover, the chi-square–based power calculation implicitly assumes a uniform binary outcome assessed over a comparable observation window for all participants. This assumption is questionable in light of the heterogeneous occurrence of mucositis, the mixture of preventive and symptom-based components within the endpoint, and the variability in chemotherapy cycle length across patients. Consequently, the sample size justification lacks a transparent clinical and statistical foundation.

The protocol further specifies Pearson’s chi-square test as the sole inferential method for evaluating the primary endpoint while simultaneously stating an intention to demonstrate superiority of the intervention. The use of superiority language implies a directional hypothesis, which is conceptually inconsistent with a non-directional testing framework unless explicitly justified. Additionally, given the anticipated event rates and the composite nature of the endpoint, some contingency table cells may have small expected counts, potentially violating chi-square test assumptions. The authors should therefore clarify the intended hypothesis structure, ensure consistency between hypothesis, statistical test, and power calculation, and provide a clear justification for the chosen analytical approach.

MINOR COMMENTS:

1. Different CTCAE versions (v3.0 and v5.0) are referenced; a single version should be specified and consistently applied.

2. The procedures for screening, enrollment, and allocation concealment are insufficiently described and should be clarified.

3. The standard of oral care provided to the control group should be more precisely defined to ensure consistency across centers.

4. The duration of participation per patient and the definition of study completion should be explicitly stated.

CONCLUSION

This protocol addresses an important clinical question and has the potential to generate useful evidence. However, substantial clarification is required regarding the definition of the primary endpoint, its relationship to the study periods, and the justification of the statistical analysis and sample size. Addressing these issues will significantly strengthen the methodological rigor and interpretability of the trial.

Reviewer #3: All detailed comments are provided in the attached PDF document of the submitted manuscript.

The main concern relates to the description of all interventions planned in this study. Currently, the descriptions are vague and insufficiently detailed, and they should be more clearly and comprehensively reported. In particular, the protocol should explicitly describe how the tested device will be applied in the clinical setting, in accordance with the TIDieR checklist, to ensure transparency, reproducibility, and scientific rigor.

**********

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Reviewer #1: No

Reviewer #2: Yes: Darko Kero

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-25-56784 Review MR.pdf

A multicenter, randomized, parallel-group confirmatory study protocol to evaluate the efficacy of Soft Protector CPC, a novel oral mucosal protectant, in preventing oral mucositis and alleviating pain in patients with breast cancer

PONE-D-25-56784R1

Dear Dr. Omori,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Yu Uneno

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.-->

Reviewer #1: Yes

**********

-->2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.-->

Reviewer #1: Yes

**********

-->3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.-->

Reviewer #1: Yes

**********

-->4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 #1: Yes

**********

-->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 #1: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: I am satisfied with all the authors' responses and the resulting changes to the paper. I have nothing else to add.

**********

-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

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