-->PONE-D-25-50692-->-->Exploring Hypoxia-Related Genes as Prognostic Indicators in Lung Adenocarcinoma-->-->PLOS One

Dear Dr. Rocha,

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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We look forward to receiving your revised manuscript.

Kind regards,

Hongtao Bi, Ph.D.

Academic Editor

PLOS One

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7. 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.

Additional Editor Comments:

This study has a solid research foundation and potential value, but there are the following issues that require systematic revision and improvement:

1.Numerous studies on the association between hypoxia-related genes and LUAD prognosis have been reported in the field. Although this study has identified a novel candidate gene combination, it has not fully compared the differences and advantages with existing research. It is necessary to supplement the discussion on the functional associations and uniqueness of DSG2, EIF6, and EXO1 with previously reported prognostic genes. Additionally, combined with recent advances in the field (e.g., the association between hypoxia and immune checkpoint inhibitor response), the mechanistic exploration of gene functions should be deepened to avoid merely staying at the level of expression correlation.

2.Lack of independent dataset validation: A core requirement of PLOS ONE for bioinformatics research is result extrapolation. The current study only relies on two datasets for screening and lacks validation of the prognostic value of DSG2, EIF6, and EXO1 in a third-party independent cohort. This part of the analysis needs to be supplemented to enhance the credibility of the conclusions.

3.The rationality of the number of HRDEGs is questionable: The 3101 HRDEGs identified far exceed the conventional scale of similar studies in the field, and there is a lack of direct biological validation. It is necessary to supplement the strict quality control criteria for gene screening or explain the biological rationality of the large-scale HRDEGs.

4.Separate the "Results" and "Discussion" sections, deepen the comparative analysis with existing studies in the field, and strengthen the discussion on innovation.

5.Standardize the figure and table formats (supplement the flowchart of the research process, optimize the visualization effect of Fig. 6, and correct label abbreviations), and adjust the reference format in accordance with the journal's requirements.

6.Improve the methodological details, and supplement the statistical analysis parameters and complete database links.

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. 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 #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?

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 #1: Yes

Reviewer #2: Yes

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-->4. 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: No

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: GENERAL COMMENTS

The authors have designed the study effectively and employed an appropriate discourse for the methodology, statistical analyses, and experimental work. The results are presented with sufficient detail and clarity, and the statistical analysis is appropriate for the given context. The language throughout the manuscript is precise and suitable for scientific publication.

However, the manuscript would benefit from improved flow, with careful attention to avoiding repetition, and ensuring the relevance of statements. The narrative should be simplified while maintaining a level appropriate for general scientific discourse. In addition, figure legends should be concise and should not reiterate results already described in the main text.

Specific comments are provided below.

SPECIFIC COMMENTS

1. Adding line numbers would facilitate clearer reviewer feedback on specific areas of concern as per the guidelines of the journal too.

2. Abstract: The abstract is professionally written.

3. Introduction

i. The introduction begins with the heading “Lund Adenocarcinoma and Hypoxia” and extends to mention non-small cell lung cancer (NSCLC). However, the relevance and significance of NSCLC to the study are not explained. This should be clarified and addressed.

ii. If subheadings are to be included in the introduction, which is generally not needed—the author should ensure consistent formatting throughout the section.

iii. Appropriate references should be incorporated in relevant places, for example, at the end of the third paragraph.

iv. The final paragraph of the introduction is more suitable for the methodology section and should be moved accordingly.

4. Methodology

i. Data downloading and Processing: The methodology section should be written with consistency. For example, web links to all databases should be provided. While TCGA is a well-known resource within the field, it is advisable to include a direct link to the TCGA LUAD dataset for accessibility. Additionally, sufficient and appropriate details should be provided across all subsections.

ii. References should be included for the methodology, as the approach adopted is a well-established method for identifying prognostic factors in cancers.

iii. Differentially Expressed Gene Identification: Repetitions in sample details should be removed (e.g., lines 1–4 in this section).

iv. Other sections of the manuscript should also be reviewed to address issues of language clarity and redundancy.

v. A separate subsection for statistical analysis should be added within the methodology. This will provide a clearer structure and make the analysis plan easier to follow.

5. Results and Discussion

i. All figure legends must be appropriate, concise and should not reiterate results already described in the main text.

ii. The flowchart of the analyzing process for full study should be added as the figure.

iii. All figure labels should be self-explanatory. For example, in Fig. 1A, “TCGA-LUAD” should be used in the volcano plot. Instead of the abbreviated term “TCGA up,” the complete phrase “TCGA-LUAD upregulated” should be written. Similar adjustments should be made for Fig. 1B, as appropriate. Fig. 1A and 1B titles should be correctly defined as well.

iv. HRDEGs are associated with functional enrichment section should be supplemented with proper references.

v. Figure 9 legend does not belong to it completely. Revise the legend.

vi. The discussion added in the article provides limited linkage between the results and previous studies. At present, it primarily restates the findings of this work without sufficient contextualization. It is recommended that the authors strengthen the discussion by justifying the results with supportive evidence from relevant literature where applicable.

6. Conclusion: It is proper from the work done.

7. References

The references provided are not formatted as per the journal guidelines.

Reviewer #2: The authors analysed two publicly available datasets from TCGA and an expression study performed in lung adenocarcinomas and normal tissues. Using different algorithms, the authors aim to select hypoxia-related genes and compare them with differentially expressed genes in the two datasets: upregulated and downregulated. As expected, selected genes play different roles in cellular processes. Using top-associated hypoxia genes, survival analysis was performed to demonstrate their utility as biomarkers. The manuscript brings an interesting piece of data to enhance the usage of hypoxia genes in tumor progression monitoring; however, some conclusions fall short.

Major comments

1. The expression profiles performed by RNA-seq TCGA samples and array technologies PMID: 20878980 highlight the need to validate the selected genes in an independent lung adenocarcinoma dataset, which will strengthen the study's reliability.

2. The analysis revealed 3101 hypoxia-related differentially expressed genes (HRDEGs), a number that appears higher and is challenging to associate with hypoxia in the absence of direct biological validation.

3. In figure 1, the lower overlap between GSE18842 and HRG compared to TCGA and HRG is expected. Please explain why the overlap between TCGA and HRG is underestimated, considering array technology's limitations compared to RNA-seq. Also, clarify the differences between the gene lists of 2330 and 2301 to support robust data analysis.

4. It remains crucial to emphasize that biological validation of hypoxia-induced genes is essential to confirm the underlying assumptions and enhance the study's credibility.

5. Separating the results from the discussion will help readers better contextualize the findings and appreciate the study's contribution within the field.

6. By grouping the genes by function/similar expression level as the authors did for “hubs”, is it possible to observe the same prediction power for survivals using the hubs normalized values for gene expression as for single genes, i.e., EXO1, EIF6, or RAD51 in figure 8? And classify the hubs following the obtained results.

7. It is difficult to extract any information from Figure 6; it would be helpful to move the supplemental figures to the main text.

Minor comment

Formatting the manuscript to improve the quality of the main figures is greatly appreciated.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Review report for manuscript ID PONE-D-25-50692.pdf

-->PONE-D-25-50692R1-->-->Exploring Hypoxia-Related Genes as Prognostic Indicators in Lung Adenocarcinoma-->-->PLOS One

Dear Dr. Rocha,

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  Mar 28 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.

Please include the following items when submitting your revised manuscript:-->

• A 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,

Hongtao Bi, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

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.

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.

Additional Editor Comments:

Please ask the authors to carefully revise the manuscript according to the reviewer's comments.

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: (No Response)

Reviewer #2: 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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

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.-->

Reviewer #1: No

Reviewer #2: 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

Reviewer #2: Yes

**********

-->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 #1: GENERAL COMMENTS

I appreciate the authors for their comprehensive responses to the comments raised in the first round of review. The revisions demonstrate a clear effort to strengthen the manuscript, and manuscript has benefited from the revisions. The changes have substantially improved the overall readability of the text and arguments are better supported with contextualization of the findings in the discussion. Most of the concerns have been addressed satisfactorily, albeit with some minor changes remain, which should be incorporated to further refine the manuscript and ensure its readiness for acceptance by PILOS ONE.

SPECIFIC COMMENTS

1. Introduction: The author has satisfactorily addressed the concerns regarding the introduction by improving the flow, eliminating redundancy and adding appropriate references. However, minor concerns persist with subheadings and typographical errors, notably at line 69 of page 5, which require correction.

2. Methodology: This section has improved through the addition of appropriate data sources, proper acknowledgment of methodological references, removal of redundancy, and inclusion of a statistical section. However, several concerns remain and should be addressed.

i. Differentially expressed gene identification: The text followed in line127-128 describes HRDEGs identification too. It is recommended to be added in the main heading as “Identification of Differentially expressed gene (DEGs) and Hypoxia Related Differentially expressed gene (HRDEFs)”, to facilitate the reader.

ii. Page 9 line 133: The main heading reads as “Curation of hypoxia related genes and identification of hypoxia related differentially expressed genes (HRDEGs)”. However, the following text from line135-152 does not describe curation of the HRDEGs. Hence, the section heading should be restated as appropriate. Further, lines 137-144 should be with the correct names of the gene sets which are not readable in the current form.

iii. Statistical Section: I acknowledge the authors’ addition of this section, which improves the manuscript. However, the section remains incomplete. The statistical tests used for the survival analysis of HRDEGs should be specified here, consistent with the reporting provided for other analyses.

3. Line 206: “Results and Discussion” This section is detailing only on results hence, “discussion” should be removed from the section heading.

4. Table 1: The values in the table should be expressed in proper scientific notation, with exponents presented as superscripts (e.g., 9.12 X 10-6) rather than in “E” format. This will ensure consistency with standard scientific reporting practices

5. Figures

i. Figure 1: I appreciate for adding the methodology workflow chart as figure 1.

ii. Figure 2: The y axis labels in the volcanic plot should be expressed in a scientifically consistent manner (i.e. subscripts for log values like log10 instead of log10). In addition, the figure legend appropriately explains the abbreviations and acronyms used; however, please also include an explanation for HRDEGs to ensure clarity for readers.

iii. Figure 3 and 6: The figures have duplication of labels as “KEGG pathways”

iv. Figure 4 and 5: Legends for Figures 4 and 5 lack consistency. Figure 4 is too brief, and Figure 5 repeats results. Please revise both to maintain a uniform style.

v. Figure 7: The primary PPI figure presents a densely interconnected network with limited interpretability. In its current form, it resembles a hairball visualization lacking functional annotation, modular clustering, or confidence-based filtering. To enhance clarity and utility, the figure should either be refined to highlight biologically relevant subnetworks or relegated to supplementary materials.

vi. Figure 8: 8A is PPI for hub shows nodes in different colors but without a legend clarifying which represents up- or downregulated genes. Standard conventions (e.g., red for up, green for down) should be used with clear labeling. Edges are uniformly styled, providing no information on interaction strength or confidence; varying edge thickness or color would improve interpretability.

vii. Figure 13: No figure was found.

6. Supplementary Tables: The supplementary Excel file includes two additional sheets (“Sheet 3” and “Sheet 4”) that currently lack identification labels. To enhance clarity and strengthen the support for the findings, I recommend adding appropriate labels or titles to these sheets. Furthermore, the supplementary data for the revalidation is also deemed to be added.

Reviewer #2: I have no further comments; all the questions were properly addressed. The title Exploring Hypoxia-Related Genes as Prognostic Indicators in Lung Adenocarcinoma is more representative of the author's findings

**********

-->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

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.

-->

-->PONE-D-25-50692R2-->-->Exploring Hypoxia-Related Genes as Prognostic Indicators in Lung Adenocarcinoma-->-->PLOS One

Dear Dr.  Rocha,

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 Apr 24 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.

Please include the following items when submitting your revised manuscript:-->

• A 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,

Hongtao Bi, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

1. 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.

2. 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.

Additional Editor Comments:

Upon further careful verification, I have found that this manuscript has critical flaws, including a complete lack of academic discussion of the already cited literature, a total failure to cite or discuss core relevant studies in the field, and an absence of clear clarification of the research necessity and the knowledge gaps in the field. Based on a subsequent in-depth review of the manuscript, I hold the view that the deficiencies in the literature discussion do not directly negate the novelty of the research itself, but they will render the claimed novelty unsubstantiated and expose the manuscript to a high risk of rejection on the grounds of being classified as a "derivative and duplicative study".

For the above reasons, to meet the publication requirements of PLOS ONE, the authors must conduct comprehensive and substantive revisions to the manuscript addressing the aforementioned deficiencies. The core revision contents are divided into the following four modules:

1.Introduction Section: Reconstruct the research background, systematically sort out the progress in the field, and clarify the necessity of the study

Supplement a systematic review of existing studies in the field of hypoxia-related prognostic biomarkers for LUAD, with a focus on incorporating and introducing the main contents and core findings of the three key studies: Li et al. (2022), Xiong et al. (2022), and the study published in the Malawi Medical Journal (2024).

Accurately point out the limitations of existing studies and clarify the knowledge gaps that this study intends to fill, including:

(1) Most previous studies adopted a narrow set of hypoxia signature genes, resulting in insufficient representativeness of the screened biomarkers for the hypoxic tumor microenvironment;

(2) Most of the reported hypoxia-related prognostic signatures for LUAD lack validation in independent external cohorts, casting doubt on their clinical applicability;

(3) Previous studies mostly focused on multi-gene combined models, with insufficient exploration of the independent prognostic value and biological mechanisms of the core genes.

Based on the above limitations, clearly elaborate the scientific design, core scientific questions, and research significance of this study, and explicitly demonstrate that "this study is not a duplication of existing work, but a supplement and optimization of research in the field".

2.Discussion Section: Supplement in-depth comparative analysis with relevant studies and clarify the innovative increments of this study

Add a dedicated subsection to compare the similarities and differences between this study and the three core relevant studies, with a focus on highlighting the innovations of this work:

(1) Comparison with the dual hypoxia-immunity study by Li et al. (2022): Clarify that this study focuses purely on hypoxia-related genes, the three biomarkers screened have no overlap with the seven immune-hypoxia genes identified in that study, and the signature has been validated in an independent cohort, thus providing a brand-new biomarker panel for the prognostic assessment of the hypoxic microenvironment in LUAD.

(2) Comparison with the study by Xiong et al. (2022): Clarify that this study is not limited to classical hypoxia-related genes but adopts a more comprehensive hypoxia-related gene set. Meanwhile, without introducing additional non-core analyses such as the competing endogenous RNA (ceRNA) network, this study focuses more on the screening of core prognostic biomarkers and the validation of their independent prognostic value, leading to research conclusions with stronger clinical relevance.

(3) Comparison with the lncRNA study published in the Malawi Medical Journal (2024): Clarify that this study focuses on protein-coding mRNAs, fills the gap in the field of hypoxia-related mRNA biomarkers, complements lncRNA biomarkers, and provides potential targets at the protein level for the prognostic assessment of hypoxia in LUAD.

Supplement an objective discussion of the limitations of this study, and simultaneously clarify the follow-up research directions of this work, forming an academic echo with existing research in the field.

3.References Section: Improve citation specifications and supplement the missing core literature

(1) Formally include the study by Xiong et al. (2022) published in Scientific Reports and the 2024 study from the Malawi Medical Journal in the references, with one-to-one correspondence to the discussion content in the text.

(2) For the study by Li et al. (2022), add standardized citation annotations at the corresponding positions in the text to ensure that citations match the discussion, and eliminate the academic non-standard behavior of "citation without substantive discussion".

(3) Check the format of all references in the full text to ensure compliance with the reference citation specifications of PLOS ONE.

4. Supplement the demonstration of the biological rationality of the core biomarkers to strengthen the scientific value of the study

Combined with existing literature, supplement the discussion on the biological association between the three genes (DSG2, EIF6, and EXO1) and tumor hypoxia as well as LUAD progression, and clarify the biological rationality of these three genes as hypoxia-related prognostic biomarkers. This will further distinguish this study from previous pure bioinformatics screening studies, strengthen the scientific connotation of the research, and completely avoid the risk of being classified as a "derivative and duplicative study".

Based on the above assessment, it is recommended that the editorial office grant the authors a 14-day revision period, requiring the authors to complete the supplements and improvements strictly in accordance with the above contents.

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-->PONE-D-25-50692R3-->-->Exploring Hypoxia-Related Genes as Prognostic Indicators in Lung Adenocarcinoma-->-->PLOS One

Dear Dr. Rocha,

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 May 15 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.

Please include the following items when submitting your revised manuscript:-->

• A 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'.

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We look forward to receiving your revised manuscript.

Kind regards,

Hongtao Bi, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

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.

Additional Editor Comments:

Thank you for submitting the revised version of your manuscript to PLOS ONE. I have carefully evaluated the revised manuscript, the accompanying response letter, and the changes made in relation to the previous editorial comments. The revision has addressed some points in part, including the addition of several relevant references, expansion of the discussion of the three highlighted genes, and inclusion of an external validation cohort. However, the manuscript does not yet adequately or fully address the major issues raised in the previous round, and substantial revision is still required before the manuscript can be considered further.

Below I summarize the main issues that must be addressed in a revised submission.

1. Introduction remains insufficiently revised

A central request of the previous decision letter was to reconstruct the research background and clearly position the study within the existing literature on hypoxia-related prognostic biomarkers in LUAD, with particular emphasis on the following three studies: Li et al. (2022), Xiong et al. (2022), and the Malawi Medical Journal study (2024).

Although these references have now been added, the Introduction still does not provide a sufficiently systematic, explicit, and comparative review of these studies. In particular, the manuscript should do more than cite them collectively. It should clearly describe, for each study: the main study design, the type of biomarkers evaluated, the key findings, the main limitations, and how the present study differs from and extends prior work.

The current Introduction only offers broad statements about prior studies and does not yet satisfactorily establish the specific knowledge gap that this manuscript is intended to fill.

Please revise the Introduction to:

explicitly summarize the three key prior studies;

clearly state the limitations of prior work, including:

(1)the use of relatively narrow hypoxia-related gene sets,

(2)the lack of independent external validation in many prior LUAD hypoxia prognostic studies,

(3)the predominant focus on multi-gene signatures without sufficient emphasis on the independent prognostic value and biological interpretation of core genes;

clearly articulate the scientific rationale, core research question, and significance of the present study;

explicitly explain why this study should be regarded as a supplement and refinement of the field rather than a duplication of previous work.

2. Discussion does not yet provide the requested dedicated comparison with prior studies

The previous editorial comments specifically requested a dedicated subsection in the Discussion comparing the present study with the three core related studies. This remains insufficiently addressed.

At present, the Discussion includes general claims regarding novelty, but it does not provide the requested structured comparison. A revised Discussion should include a clearly labeled subsection that directly compares your findings with those of:

(1) Li et al. (2022)

Please clarify:

that Li et al. examined a hypoxia-immune signature rather than a purely hypoxia-focused gene set;

whether the three genes identified in your study overlap or do not overlap with the genes identified in that study;

how the external validation in your study strengthens clinical relevance;

in what way your panel contributes a distinct prognostic framework for LUAD.

(2) Xiong et al. (2022)

Please clarify:

how your study differs in the composition and breadth of the hypoxia-related gene set;

how your study differs methodologically from a framework that also incorporated ceRNA network analysis;

why your focus on identifying core prognostic genes and evaluating their independent prognostic value may provide stronger translational relevance.

(3) Malawi Medical Journal study (2024)

Please clarify:

that this prior study focused on hypoxia-related lncRNAs,

whereas your work focuses on protein-coding mRNAs;

how your findings complement rather than replicate lncRNA-based prognostic work;

why protein-coding candidates may be of particular interest from a biomarker and mechanistic standpoint.

This comparison must be explicit and substantive. General statements of novelty are not sufficient.

3. Biological rationale for DSG2, EIF6, and EXO1 requires further strengthening

The revised manuscript includes some additional discussion of DSG2, EIF6, and EXO1, which is appreciated. However, the biological rationale still requires clearer and more rigorous development.

In particular, the manuscript should better justify why these genes should be considered hypoxia-related prognostic biomarkers in LUAD, rather than simply genes associated with cancer progression more broadly. Please strengthen this section by:

clarifying the evidence linking each gene specifically to hypoxia, hypoxia-associated pathways, or cellular adaptation to hypoxic stress;

discussing, where possible, evidence relevant specifically to LUAD or lung cancer biology;

integrating this discussion more directly with your own enrichment results and the hypoxia-centered framework of the study.

At present, the discussion still reads in part as a general cancer-biology summary rather than a focused biological justification for these genes in the context of LUAD hypoxia biology.

4. Clarification is needed regarding the meaning of “independent prognostic factors”

The manuscript states that DSG2, EIF6, and EXO1 are independent prognostic factors, based on multivariate Cox regression. However, from the current Methods and Results, it is not sufficiently clear which covariates were included in the multivariate model.

Please clarify:

whether the multivariate Cox model included only gene-expression variables, or also included relevant clinical covariates such as age, sex, stage, smoking status, and/or other available clinicopathologic parameters;

if clinical covariates were included, please report them explicitly in the Methods and Results;

if clinical covariates were not included, please revise the language accordingly and avoid overstating the conclusion as “independent prognostic factors” in the clinical sense.

This point is important for the interpretation of the manuscript and must be corrected.

5. Limitations and future directions remain underdeveloped

Although a short limitations paragraph has been added, it remains too brief for a study of this type.

Please expand the limitations section to address issues such as:

reliance on public retrospective datasets,

cross-platform heterogeneity between datasets,

possible batch effects and annotation differences,

limitations of database-derived hypoxia gene curation,

lack of protein-level validation,

lack of experimental validation in LUAD models or patient samples,

limitations of validation through an online platform rather than a fully reproduced external analytic pipeline.

The future directions should also be more clearly linked to these limitations.

6. Reference section and citation quality require further attention

Although additional key references were added, the current reference list still appears to contain formatting and completeness issues. Several entries appear incomplete or not fully standardized.

Please carefully review all references to ensure full compliance with PLOS ONE formatting requirements. In addition:

ensure that all newly added references are discussed substantively in the main text and not only cited in passing;

check all software/database citations for completeness and consistency;

correct any incomplete or malformed reference entries.

Please note that simply stating that the manuscript follows “Vancouver” style is not sufficient; the references must match the journal’s required style and be internally consistent.

7. Language, clarity, and presentation require substantial improvement

The manuscript still contains a number of issues affecting readability and professionalism, including:

grammatical errors,

awkward phrasing,

typographical errors,

duplicated or repetitive wording,

inconsistent terminology,

occasional imprecise statements.

Examples include inconsistent use of abbreviations and terminology, unclear sentence structure in several sections, and residual editing artifacts.

Please ensure that the entire manuscript undergoes careful English-language editing and thorough proofreading before resubmission.

8. Response letter must be substantially improved

The current response letter is too general and does not allow efficient editorial assessment of how each point has been addressed. Statements such as “we have tried our best” or “we have added this as requested” are not sufficient.

For the next revision, please provide a detailed point-by-point response in which each prior comment is followed by:

(1)your response,

(2)the exact changes made,

(3)and the page and line numbers where the changes can be found.

If you disagree with any point, please explain your reasoning clearly and respectfully.

9. Data availability and reporting clarity

The Data Availability Statement is improved, but please ensure that the manuscript text clearly reports:

all dataset accession numbers,

where each dataset was used in the workflow,

and whether all relevant processed outputs are contained in the manuscript and/or Supporting Information.

Please also ensure consistency between the submission form, cover letter, manuscript text, and supplementary materials.

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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.]

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.

Exploring Hypoxia-Related Genes as Prognostic Indicators in Lung Adenocarcinoma

PONE-D-25-50692R4

Dear Dr. Rocha,

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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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,

Hongtao Bi, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

After a complete assessment of the authors' final revised manuscript, point-by-point response letter, all supplementary materials, and original figure/table files, the authors have fully and thoroughly addressed all core issues raised in the previous editorial review. All revisions for scientific rigor and the journal's formatting requirements have been completed, and the manuscript now complies with the publication guidelines and academic standards of PLOS ONE.

Reviewers' comments: