Dear Dr. Leitges,

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 by Jan 23 2026 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.. 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.. 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.. 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.

• 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 . 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 . 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 . 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,

Amr Ahmed El-Arabey

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

3. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse.

4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

5. Thank you for stating the following financial disclosure:

“Canada Research Chairs: 950-232114”

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.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

6. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

3. 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.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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: Overall, this study provides important insights into Protein Kinase D3 as a potential Pan-Cancer biomarker, and with appropriate revisions, it is expected to make a positive impact in the relevant field.

1. It is recommended to include a flowchart of the study to visually present the research design, data processing, and analysis steps, enhancing the readability and logical clarity of the study.

2. I recommend that the authors include more recent literature in their references. For example, the authors cite ‘Global cancer statistics 2020’. However, currently ‘Global cancer statistics 2022’ has been published.

3. Please provide the full definition of abbreviations the first time they are used, and include the abbreviation in parentheses.

4. Some previous pan-cancer studies have utilized similar bioinformatics and statistical methods (PMID: 39702250; PMID: 37437243; PMID: 36213111). While referencing these studies may reduce the novelty of the work to some extent, I believe it would greatly benefit the readers by providing a more comprehensive understanding of the context of the current study.

Reviewer #2: This manuscript presents a comprehensive pan-cancer analysis of PRKD3 using multi-omics datasets from TCGA, GTEx, CPTAC, cBioPortal, and multiple immune-deconvolution platforms. The study examines PRKD3 expression, genetic and epigenetic alterations, clinicopathological associations, prognostic value, immune infiltration patterns, and potential relevance to immunotherapy. Overall, the work addresses an important gap by providing a broad, data-driven overview of PRKD3 across 33 cancer types and offers useful descriptive insights that may inform future mechanistic studies. However, several methodological, statistical, and interpretational issues need to be addressed to strengthen the rigor and reliability of the conclusions.

Introduction

1. The introduction provides a thorough overview of the kinase family and successfully contextualizes PRKD3 as a biologically relevant but understudied gene in cancer. However, the background is somewhat lengthy and occasionally repetitive. The authors may tighten the narrative and present clearer, more specific hypotheses or objectives to guide the reader. While the need for a pan-cancer investigation is justified, stating explicit research questions or expected outcomes would enhance clarity.

Methods

The study makes use of multiple public resources, which is appropriate for a pan-cancer bioinformatics analysis. However, several methodological gaps reduce the technical rigor:

1.Direct comparison between TCGA tumors and GTEx normal samples is performed without any batch-effect correction. This is a well-known issue and can significantly bias differential expression results and diagnostic ROC analyses.

2. Prognostic analyses do not adjust for essential confounders such as stage, age, tumor purity, and sex. Multivariate Cox regression is required to determine whether PRKD3 is an independent prognostic factor.

3.TIMER, MCP-counter, EPIC, TIDE, and QuanTIseq often produce discordant cell-type estimates. The manuscript reports findings but does not explain how inconsistencies were handled or which algorithmic outputs were prioritized.

4. The manuscript states that FDR correction was applied, but this is not clearly indicated for all analyses. Some figures appear to show uncorrected p-values. Please specify the exact correction method (e.g., Benjamini–Hochberg) and where it was applied.

5. Functional implications of CpG island vs. open sea methylation are not fully discussed. Additionally, methylation–expression correlations should consider CNV as a confounder.

Overall, the methods are broadly appropriate, but these omissions weaken the statistical soundness of the study.

Results

The results are extensive and clearly organized, covering expression, stage correlations, survival, CNV/mutations, DNA methylation, immune infiltration, immune checkpoint associations, and immunotherapy relevance. However, several issues need to be addressed:

1. Several conclusions imply causality (e.g., “PRKD3 modulates immune interactions”), although all findings are purely correlative.

2. AUC values from TCGA vs GTEx may be inflated due to uncorrected batch effects and lack of external validation datasets.

3. TISMO datasets are based on mouse syngeneic models, which differ from human tumors. Their relevance to clinical immunotherapy response should be more carefully qualified.

4. The concept of PRKD3 acting as a “paradox gene” is interesting, but conclusions require analyses that adjust for tumor purity, immune subtypes, or molecular subtypes.

Discussion

The discussion synthesizes findings well and connects them to existing literature. The emphasis on context-dependent roles of PRKD3 is appropriate. However, the discussion is overly long and occasionally repetitive. Certain interpretations go beyond what the data support, especially regarding immune regulation and therapy response. The limitations section should explicitly acknowledge the statistical and methodological issues noted above: lack of batch correction, absence of multivariate survival analysis, immune algorithm discrepancies, constraints of mouse-model immunotherapy data. A more concise and conservative discussion will strengthen the manuscript.

Conclusion

The conclusion effectively summarizes key findings, but it should more clearly reflect the descriptive nature of the study and avoid suggesting causal roles for PRKD3. Emphasizing that the results generate hypotheses rather than definitive mechanistic insights would better align with the analyses performed.

This manuscript provides a valuable and comprehensive description of PRKD3 across cancers. However, several methodological, statistical, and interpretational issues need to be addressed to ensure technical rigor. The study has the potential to make a meaningful contribution once these concerns are resolved.

**********

what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: No

Reviewer #2: No

**********

[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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

<p>Pan-Cancer Landscape of Protein Kinase D3: An Integrative TCGA Multi-Omics Analysis of Clinical, Molecular, and Immunological Roles

PONE-D-25-52894R1

Dear Dr. Leitges,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support....

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Amr Ahmed El-Arabey

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

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

Reviewer #2: Yes

**********

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

Reviewer #2: 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.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 #2: Yes

**********

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

Reviewer #2: Yes

**********

Reviewer #2: The authors have satisfactorily addressed all concerns raised in the previous round of review. The revised manuscript shows clear improvements in methodological rigor, statistical transparency, and interpretative caution.

Key issues have been appropriately resolved, including batch-effect correction for TCGA–GTEx analyses, multivariate Cox regression adjusting for relevant clinical variables, and consistent application of false discovery rate correction. The consensus-based approach used to address discrepancies among immune deconvolution methods is appropriate for pan-cancer immune analyses. Causal language has been revised throughout, and interpretations related to immune regulation and immunotherapy response are now properly qualified.

The study employs suitable statistical methods and sufficiently large public datasets, and the conclusions are well aligned with the data presented without overstatement. No concerns regarding research ethics, data availability, or publication ethics were identified.

**********

what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #2: No

**********