Dear Dr. Moss,

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.

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Mohamed Gouda, MD, PhD

Academic Editor

PLOS ONE

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2. Thank you for stating the following in the Funding Section of your manuscript:

“The studies have been funded by a Rare Gynaecological Cancer Research grant (EVE 0042) from The Eve Appeal gynaecological cancer charity. The funder has not had a role in the design of the study and will not be involved in the conduct, analysis, or reporting of trial.”

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“EM, JS and DD were the recipients of the grant from The Eve Appeal, the gynaecological cancers charity (EVE 0042) https://eveappeal.org.uk/. The University of Leicester is the study sponsor. The research team (EM/JS and DD) are employees of the sponsor and have designed of the study and will conduct, analyse, and report of outcomes.”

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“EM/DD have received research funding from Sarcoma UK for an unrelated sarcoma-focused research study.”

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Additional Editor Comments:

Reviewer #1:

This manuscript describes a study protocol aiming to use circulating tumor DNA (ctDNA), a minimally invasive procedure, for diagnosis and monitoring of uterine sarcoma. I have some comments:

- I suggest revising the word order In the title, so that “diagnosis” precedes “monitoring” fitting the sequence of the abbreviations in the title (“DOORS-D and DOORS-M”) and the study objectives.

- Based on the described proposed cohort of 50 patients, 20 will have uterine sarcomas and the remaining 30 will have uterine fibroids. My concern is that leiomyomas are considerably more common and with these small numbers, the study may be underpowered to detect distinct genetic alterations or provide meaningful comparative data between the two categories. Also, limiting sarcoma cases to 20—most likely leiomyosarcomas—may not adequately represent different histotypes.

- Although there are still limitations and variations in MRI performance reported in the literature, the discussion in the manuscript seems to understate its diagnostic value. A meta-analysis of eight studies involving 2,495 women (2,253 with leiomyomas and 179 with uterine sarcomas) reported a pooled sensitivity of 0.90 and specificity of 0.96 for differentiating sarcomas from fibroids (PMID: 37732472). In addition, there is growing consensus on refining MRI evaluation in uterine masses for risk of leiomyosarcoma (PMID: 36194109).

- I would suggest the authors to provide more details on previous efforts exploring the role of ctDNA in uterine sarcomas.

- It would be helpful to discuss the diagnostic challenges during intraoperative pathology assessment, which are often limited by time constraints and restricted tissue sampling. These factors frequently delay definitive diagnosis until permanent sections are reviewed. The proposed study could significantly help bypassing this dilemma.

Reviewer #2:

I think it will be a useful study to answer your scientific question and proof the hypothesis.

However, none is specified about the technique of ctDNA which is core in this particular work.

Reviewer #3:

The manuscript Study Protocol: Development of a blood test for uterine sarcoma – monitoring and diagnosis studies which aims to explore the role of circulating tumor DNA to diagnose and to monitor uterine sarcomas.

The study is sponsored by the University of Leicester and a grant from the Eve Appeal (EVE 0042) and is approved by the South Central – Berkshire B Research Ethics Committee.

Introduction – well written and organized. The study has a strong rationale and potential impact in the diagnosis and care of patients with uterine sarcomas.

Sample size – aim to recruit 50 patients in each study, per the authors it’s feasible based on high volume at the University Hospitals of Leicester.

Study population/Inclusion criteria – DOORS-D women due for hysterectomy/myomectomy for uterine fibroids. DOORS-M previous diagnosis of uterine sarcoma in the previous 10 years. 18-99 years.

Overall, this is a very well written protocol and well-designed studies that answer an important and clinically relevant question. However, there are some comments that could further addressed by the authors:

Methods sections

- authors will use WES as the method to analyze ctDNA from patients diagnosed with uterine sarcomas and will create a tumor-uninformed (agnostic) liquid biopsy test using genomic/methylation profile from those patients to detect uterine sarcoma signatures in patients presenting with fibroids. The authors could provide additional details in this section regarding their analysis and bioinformatics plan.

- There is also no clear guidance on the “semi-structured qualitative interviews” and how those will be evaluated and included in the study.

- There is a mention the use of AI and without providing additional details on the methods planned for this analysis.

Study Design

– The authors mention the inclusion of additional sites without blood collection. The authors could clarify the reason/criteria to include, and which additional sites are planned to be part of the study. In addition, since there is no blood collection, is there a limit of patients from the planned 50 included that will be enrolled in the “additional sites” vs. the University of Leicester?

Primary and secondary objectives

– the authors describe a very broad definition for primary and secondary objectives, without providing a clear outcome/value to consider the study positive. Based on sample size calculation they expect 40% prevalence of uterine sarcoma, what is the accuracy, sensitivity/specificity and ROC/AUC to consider this test feasible to use in clinical practice?

[Note: HTML markup is below. Please do not edit.]

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?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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??>

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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.

Reviewer #1: This manuscript describes a study protocol aiming to use circulating tumor DNA (ctDNA), a minimally invasive procedure, for diagnosis and monitoring of uterine sarcoma. I have some comments:

- I suggest revising the word order In the title, so that “diagnosis” precedes “monitoring” fitting the sequence of the abbreviations in the title (“DOORS-D and DOORS-M”) and the study objectives.

- Based on the described proposed cohort of 50 patients, 20 will have uterine sarcomas and the remaining 30 will have uterine fibroids. My concern is that leiomyomas are considerably more common and with these small numbers, the study may be underpowered to detect distinct genetic alterations or provide meaningful comparative data between the two categories. Also, limiting sarcoma cases to 20—most likely leiomyosarcomas—may not adequately represent different histotypes.

- Although there are still limitations and variations in MRI performance reported in the literature, the discussion in the manuscript seems to understate its diagnostic value. A meta-analysis of eight studies involving 2,495 women (2,253 with leiomyomas and 179 with uterine sarcomas) reported a pooled sensitivity of 0.90 and specificity of 0.96 for differentiating sarcomas from fibroids (PMID: 37732472). In addition, there is growing consensus on refining MRI evaluation in uterine masses for risk of leiomyosarcoma (PMID: 36194109).

- I would suggest the authors to provide more details on previous efforts exploring the role of ctDNA in uterine sarcomas.

- It would be helpful to discuss the diagnostic challenges during intraoperative pathology assessment, which are often limited by time constraints and restricted tissue sampling. These factors frequently delay definitive diagnosis until permanent sections are reviewed. The proposed study could significantly help bypassing this dilemma.

Reviewer #2: I think it will be a useful study to answer your scientific question and proof the hypothesis.

However, none is specified about the technique of ctDNA which is core in this particular work.

Reviewer #3: The manuscript Study Protocol: Development of a blood test for uterine sarcoma – monitoring and diagnosis studies which aims to explore the role of circulating tumor DNA to diagnose and to monitor uterine sarcomas.

The study is sponsored by the University of Leicester and a grant from the Eve Appeal (EVE 0042) and is approved by the South Central – Berkshire B Research Ethics Committee.

Introduction – well written and organized. The study has a strong rationale and potential impact in the diagnosis and care of patients with uterine sarcomas.

Sample size – aim to recruit 50 patients in each study, per the authors it’s feasible based on high volume at the University Hospitals of Leicester.

Study population/Inclusion criteria – DOORS-D women due for hysterectomy/myomectomy for uterine fibroids. DOORS-M previous diagnosis of uterine sarcoma in the previous 10 years. 18-99 years.

Overall, this is a very well written protocol and well-designed studies that answer an important and clinically relevant question. However, there are some comments that could further addressed by the authors:

Methods sections

- authors will use WES as the method to analyze ctDNA from patients diagnosed with uterine sarcomas and will create a tumor-uninformed (agnostic) liquid biopsy test using genomic/methylation profile from those patients to detect uterine sarcoma signatures in patients presenting with fibroids. The authors could provide additional details in this section regarding their analysis and bioinformatics plan.

- There is also no clear guidance on the “semi-structured qualitative interviews” and how those will be evaluated and included in the study.

- There is a mention the use of AI and without providing additional details on the methods planned for this analysis.

Study Design

– The authors mention the inclusion of additional sites without blood collection. The authors could clarify the reason/criteria to include, and which additional sites are planned to be part of the study. In addition, since there is no blood collection, is there a limit of patients from the planned 50 included that will be enrolled in the “additional sites” vs. the University of Leicester?

Primary and secondary objectives

– the authors describe a very broad definition for primary and secondary objectives, without providing a clear outcome/value to consider the study positive. Based on sample size calculation they expect 40% prevalence of uterine sarcoma, what is the accuracy, sensitivity/specificity and ROC/AUC to consider this test feasible to use in clinical practice?

**********

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Reviewer #1: No

Reviewer #2: Yes: Oriol Mirallas

Reviewer #3: Yes: Eduardo Edelman Saul

**********

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Study Protocol: Development of a blOOd test for uteRine Sarcoma – Monitoring and Diagnosis (DOORS-D and DOORS-M) studies

PONE-D-25-49314R1

Dear Dr. Moss,

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,

Mohamed Gouda, MD, PhD

Academic Editor

PLOS One