Dear Dr. Hobson,

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Kind regards,

Yazhou He

Academic Editor

PLOS ONE

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2. Thank you for stating the following financial disclosure:

“LMH is supported in part by grant MR/N0137941/1 for the GW4 BIOMED MRC DTP, awarded to the Universities of Bath, Bristol, Cardiff and Exeter from the Medical Research Council (MRC)/UKRI

RMM is a National Institute for Health Research Senior Investigator (NIHR202411). RMM, LJG and PCH are supported by a Cancer Research UK 25 (C18281/A29019) programme grant (the Integrative Cancer Epidemiology Programme). RMM is also supported by the NIHR Bristol Biomedical Research Centre which is funded by the NIHR (BRC-1215-20011) and is a partnership between University Hospitals Bristol and Weston NHS Foundation Trust and the University of Bristol. Department of Health and Social Care disclaimer: The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

SERB was supported by an NIHR Advanced Fellowship (NIHR 301666) whilst undertaking this work. Additional support was provided by the Higgins family.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

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“RMM, LJG and PCH have received funding from Cancer Research UK. LMH receives funding from the GW4 BioMed2 MRC DTP.”

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Additional Editor Comments:

This study presents a protocol of a potentially important contribution to cancer prognosis research.  

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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?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: 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??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: 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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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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: Very interesting and overall fascinating work.

While the protocol provides a solid framework for Mendelian Randomization (MR) analysis, I recommend expanding the discussion in regards to horizontal pleiotropy as a potential methodological limitation. Pleiotropy, particularly when genetic instruments influence the outcome via pathways other than the exposure of interest, constitutes a major threat to the validity of causal inference in MR.

To strengthen the translational relevance and external validity of the colorectal cancer (CRC)-specific findings, I recommend that the authors consider incorporating a standalone Europe population based analysis before performing the analysis with the Asia Colorectal Cancer Consortium (ACCC).

Reviewer #2: This is a well-structured and timely protocol that proposes a novel application of polygenic risk scores (PRS) to identify circulating protein biomarkers associated with cancer development. The study is based on a solid theoretical framework, incorporates advanced analytical strategies, and is grounded on high-quality datasets such as UK Biobank and GWAS consortia.

The authors clearly articulate the rationale for using PRS as a proxy for genetic liability and justify the methodological choices with appropriate references. The distinction between forward and reverse causality is well addressed through the planned use of bidirectional Mendelian randomization, colocalization, and clustering approaches.

The methodology is technically robust, with transparent criteria for inclusion/exclusion of participants, detailed statistical procedures, and clear descriptions of software and tools to be used. The strategy to handle potential biases, including population stratification, horizontal pleiotropy, and survivorship bias, is particularly commendable.

The data management and sharing plan aligns with best practices in open science, and the authors demonstrate commitment to reproducibility through open code and future data availability. The language of the manuscript is precise, clear, and appropriate for a scientific audience.

In summary, this is a strong and comprehensive study protocol with the potential to generate valuable insights into cancer-related proteomics. I recommend its acceptance without revisions.

Reviewer #3: Greetings to the editor and the author. To be honest, the study protocol was very well planned. It took into account the scientific methods used and the rules of the PLOS ONE journal. When it comes to research ethics, the author must give the journal copies of the ethical approvals they got, in line with the timeframe of the research.

I would like to thank the editors and authors for their hard work and for the way they organised the scientific work.

Reviewer #4: I uploaded my review as an attachment.

I have no concerns or additional comments for the author on the research or publication ethics

Reviewer #5: This manuscript presents a study protocol to identify circulating protein biomarkers for early detection of colorectal and lung cancer using UK Biobank genetic data. Polygenic risk scores (PRS), derived through PRS-CS and PRS-CSx methods, are used to assess genetic liability to cancer and its association with 2,023 plasma proteins measured via the Olink platform. Bidirectional Mendelian Randomization (MR) distinguishes between causal, reverse, and non-causal relationships. Supporting analyses, including colocalization and time-to-diagnosis assessments, aim to refine causal inference and characterize protein changes relative to cancer onset. The study ultimately aims to improve early detection and risk prediction.

Strengths

-The study tackles an important and timely issue.

-The use of PRS-CS and PRS-CSx is a thoughtful and advanced methodological choice.

-The analysis plan is thorough, using multiple approaches like bidirectional Mendelian randomization and colocalization to strengthen causal inference.

-The study’s aims are clearly explained, with a solid rationale and potential for clinical benefit.

To further strengthen the manuscript, consider the following minor revisions;

Weaknesses

-The manuscript does not mention collider bias, which is a potential issue given the selection of prevalent cancer cases and volunteers from UK Biobank.

-The protocol does not assess whether subgroup analyses (e.g., by cancer subtype or smoking status) will be sufficiently powered to yield reliable results.

-There is no mention of using internal validation within UK Biobank or applying regularisation techniques to reduce overfitting, which is important given the high-dimensional proteomic data

-Writing Clarity:

The sentence “advantages of measuring protein within the blood are the reduced volume…” is grammatically awkward. Rephrasing it to “an advantage of measuring proteins in blood is the small volume required” would improve clarity.

• The sentence “five-year survival rates fall considerably…” is hard to follow. It should be rewritten with clearer structure and full sentence form for precision.

• Noun–verb mismatches, “advantages of measuring protein within the blood … are…”).

• The explanation sentence of Mendelian Randomisation and reverse Mendelian Randomisation can be more clarified.

Reviewer #6: Comments to Authors:

The authors present a study protocol that will use human genetic datasets to identify circulating proteins that may be causal, and thus used as early screening biomarkers, in the development of colorectal and lung cancer. There are many strengths to the proposed protocol, including cost and ease to implement in a clinical setting. Outcomes will include the identification or protein biomarkers associated with PRS for a given cancer, and protein changes may be more meaningful compared to gene changes when discussing functional biological processes. My specific comments are below:

Major Comments:

- The authors include an aim for the analyses, but not a clear aim for the study as a whole. The “overall aim of these analyses is to identify protein changes that are the causal consequence of genetic liability to cancer”. The hypothesis is that “protein level changes resulting from cancer development can be identified via an individual’s PRS for the disease, representing their genetic liability to developing that cancer”. The aim does not seem to match the hypothesis. Inferring the purpose of the study from the hypothesis suggests that the aim is to develop a protocol to determine if the genetic liability to cancer, identified using an individual’s PRS, is associated with measurable changes in circulating (blood plasma or serum) proteins. Then, the identified protein changes can be used to identify specific proteins that may be causal (forward or reverse) in the development of cancer. Please clarify.

- The text should be specific when referring to ‘disease’ and ‘cancer’. While the analyses may be relevant for different diseases and cancer types, the aims and hypothesis should be specific to the study – colorectal cancer and lung cancer.

- A workflow figure would be helpful in clarifying the protocol discussed in the methods section of the text. For example, summary statistics will be used from GWAS studies, then weights will be generated via PRS-CS, the O-link will be performed (on biobanked samples or will subjects be recruited and samples collected – not clear) …and so on.

- Include legends that describe what each figure is showing, annotate symbols, lines, colors, abbreviations etc.

Minor Comments:

- Indicate which panel is being discussed when describing the figures in the text. For example, figure 1 has three panels (A, B, C). When mentioning fig 1 in the text, it is not clear when authors are referring to a specific panel.

- Differentiate between PRS-CS and PRS-CSx. First line under methods and analysis section suggests that PRS-CS and PRS-CSx are synonymous acronyms.

- The text states that “forward and reverse Mendelian randomization sensitivity analyses, as well as colocalization analyses, will be performed….”. In the Strengths of the Study section, the authors state that a strength of the study is that the proposed method will increase the power compared to conventional MR. As currently written, it appears that the proposed method is ‘better’ than MR, but the proposed method still uses MR? Please clarify if MR will just be used to statistically show that the proposed method has more power compared to conventional MR.

- The text describes the assumptions shown figure 2, please make sure to include the description of IV1 (not annotated in figure), IV2, and IV3 either in the figure legend or a short description in the figure itself.

- A figure depicting what is discussed in the ‘study design’ section would be extremely helpful in clarifying the main points of the analyses. For example, depicting the predicted outcome for an association in the forward direction (protein is causal for the disease) or if association is found in the reverse direction (may suggest genetic liability to cancer is causing protein level change). Having simplified figures for these complex analyses will aid in communicating the science to broader populations.

- Please include additional details for performing data harmonization. For example, is this performed to ensure the same alleles are associated with the same effects across datasets and that all are coded in the same manner?

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes:  Érika Carvalho de Aquino

Reviewer #3: Yes:  Amran Ibrahim

Reviewer #4: No

Reviewer #5: No

Reviewer #6: No

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<p>A protocol for using human genetic data to identify circulating protein level changes that are the causal consequence of cancer processes.

PONE-D-24-41052R1

Dear Dr. Hobson,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Yazhou He

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?

Reviewer #5: Yes

Reviewer #6: 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 #5: Yes

Reviewer #6: Yes

**********

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

Reviewer #5: Yes

Reviewer #6: 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

Reviewer #5: Yes

Reviewer #6: Yes

**********

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

Reviewer #5: Yes

Reviewer #6: 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 #5: All major and minor reviewer comments have been adequately addressed. Fix the study design typo “willl be defined” to “will be defined.”

Reviewer #6: Thank you for addressing each of the comments. The addition of figure three strengthens the manuscript.

**********

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 #5: No

Reviewer #6: No

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