Epigenetic Bridge Between Oxidative Balance of Koreans and TCGA Pan-Cancer Risk: Sex-Specific DNA Methylation Signatures

Round 1

Reviewer 1 Report

Please add more detail description of methodology used to calculate the OBS. You can cite relevant reference or add explanation why did the authors choose to analyze selected parameters. At the same time, the methos for estimation of dietary intake and lifestyle factors determination should be added.

The visibility of Figures 6 and 7 could be improved.

The conduction of study including Korean population dietary and lifestyle habits could be added as limitation of the study. 

In material and methods section, lines from 71-82 Methods for estimation of dietary intake and lifestyle factors could be added

In material and methods section, lines from 83-120 Reference for calculation of OBS is missing. The authors could add reference or explain how they chose factors that were used for calculation of OBS scores.

Figure 6. The black letters in blue cycles have not good visibility

Figure 7. The used points in different colors could be larger to improve their visibility

Strengths and limitations, lines 440-446, The conduction of the study on Korean population could be added as limitation of the study

 

Author Response

Please add more detail description of methodology used to calculate the OBS. You can cite relevant reference or add explanation why did the authors choose to analyze selected parameters. At the same time, the methos for estimation of dietary intake and lifestyle factors determination should be added.

Response: While citing references related to OBS, we failed to cite two papers that we had most heavily cited. Specifically, all authors printed Table 1 of the paper Moon et al., 2024 and coded it in R during our research. This failure was our mistake, and we have cited Moon et al., 2024 and Lin et al., 2025 where appropriate. We have added relevant information to the beginning of section 2.2.

 

The visibility of Figures 6 and 7 could be improved.

Response: We modified two figures to improve readability.

 

The conduction of study including Korean population dietary and lifestyle habits could be added as limitation of the study.

Response: This is similar to the last point, so I explained it in the last point.

 

 

 

In material and methods section, lines from 71-82 Methods for estimation of dietary intake and lifestyle factors could be added

Response: From 2020, when we began this study, to the present, we have used references related to dietary and lifestyle habits in our research and added related content to the methods [Lines 83-85].

 

In material and methods section, lines from 83-120 Reference for calculation of OBS is missing. The authors could add reference or explain how they chose factors that were used for calculation of OBS scores.

Response: While citing references related to OBS, we failed to cite two papers that we had most heavily cited. Specifically, all authors printed Table 1 of the paper Moon et al., 2024 and coded it in R during our research. This failure was our mistake, and we have cited Moon et al., 2024 and Lin et al., 2025 where appropriate. We have added relevant information to the beginning of section 2.2 [Lines 90-95].

 

Figure 6. The black letters in blue cycles have not good visibility

Response: We changed the color of the node to light and made the text bold.

 

Figure 7. The used points in different colors could be larger to improve their visibility

Response: We identified a critical error in Fig. 7. We corrected the incorrectly displayed portion and increased the size of the dots to improve readability. Furthermore, we added a female figure, instead of the male figure, which was previously presented only. We also reduced the left margin so that the figure would fit on a single line.

 

Strengths and limitations, lines 440-446, The conduction of the study on Korean population could be added as limitation of the study

Response: We agree with the reviewer that our findings, based on the Korean population (KoGES), may have limited generalizability to other ethnic groups. While the KoGES cohort provides a robust and large-scale dataset, ethnic-specific dietary patterns, such as high intake of fermented foods and genetic backgrounds could influence the association between OBS and DNA methylation. We have now added this as a formal limitation in the Discussion section 4.6. and emphasized the need for future cross-ethnic validation.

Reviewer 2 Report

Kang and colleagues investigate DNA methylation in cancer patients in Korea.

Major comments include:

*The title should reflect the manuscript's contents, incl. the country investigated and more.
*The determination of OBS needs to be detailed in the Methods section.
*Fig. 2: the writing alongside both panels is barely legible.

*Fig. 3 is simply too small to be assessed.

*Fig. 5: some of it is too small to assessed properly.

*Fig 6: you know....

*Fig. 7: and once again.....

*Both the Discussion and Conclusions are overly verbose must be focused.

See above

Author Response

Kang and colleagues investigate DNA methylation in cancer patients in Korea.

Major comments include:

*The title should reflect the manuscript's contents, incl. the country investigated and more.

Response: The title reflects all the information you provided, and keywords have been added as follows.

“Epigenetic Bridge between Oxidative Balance of Koreans and TCGA Pan-Cancer Risk: Sex-Specific DNA Methylation Signatures”

*The determination of OBS needs to be detailed in the Methods section.

Response: While citing references related to OBS, we failed to cite two papers that we had most heavily cited. Specifically, all authors printed Table 1 of the paper Moon et al., 2024 and coded it in R during our research. This failure was our mistake, and we have cited Moon et al., 2024 and Lin et al., 2025 where appropriate. We have added relevant information to the beginning of section 2.2 [Lines 90-95].

 

*Fig. 2: the writing alongside both panels is barely legible.

Response: We increased the font size and adjusted the parameters to improve readability.

 

*Fig. 3 is simply too small to be assessed.

Response: We also increased the font size and adjusted the parameters to improve readability. Fig. 3 contains a wealth of information, displaying various cancer types and HyPi scores on a coordinate plane. The colors of individual points were also varied, and the optimal parameters were selected to present the points. Additionally, we placed the figure in a larger size up to the left margin.

 

*Fig. 5: some of it is too small to assessed properly.

Response: We tried to increase the font size properly, but the code modification was difficult. So, we tried to change layout of four figures.

 

*Fig 6: you know....

Response: We changed the color of the node to light and made the text bold.

 

*Fig. 7: and once again.....

Response: We identified a critical error in Fig. 7. We corrected the incorrectly displayed portion and increased the size of the dots to improve readability. Furthermore, we added a female figure, instead of the male figure, which was previously presented only. We also reduced the left margin so that the figure would fit on a single line.

 

*Both the Discussion and Conclusions are overly verbose must be focused.

Response: We have revised the figures overall to improve readability. All figures are available as PDF files, ready for immediate presentation at the highest resolution possible upon publication. We have added relevant statistical descriptions. We have presented the limitations of our research as objectively as possible, focusing on key findings. If you have any suggestions for further revision, please let us know. We will do our best to incorporate them.

Reviewer 3 Report

This study presents a novel and well-designed investigation linking lifestyle-based oxidative balance to pan-cancer risk through sex-specific DNA methylation signatures. The integration of population-based epigenomic data with TCGA cancer datasets and the introduction of the Hybrid Piscore (HyPi) represent clear strengths and add methodological innovation. The identification of distinct male- and female-specific methylation patterns provides important insight into sex-dependent oxidative stress–epigenome interactions. However, several gaps should be addressed to strengthen the manuscript.

 

• The biological interpretation of sex-specific CpG sites remains limited; deeper functional annotation and pathway-level analyses would help clarify their mechanistic relevance.
• OBS is derived from self-reported lifestyle factors, which may introduce measurement bias and residual confounding that are not fully discussed.
• While directional consistency with TCGA data is shown, the study lacks experimental or longitudinal validation to support causality between OBS, methylation changes, and cancer risk.
• The applicability of findings beyond the Korean population requires discussion, as ethnic and environmental differences may affect generalizability.
• Although vitamin C dependent demethylation and one-carbon metabolism are proposed mechanisms, direct evidence linking these pathways to the identified CpGs is limited.
• Authors does not sufficiently describe the statistical methods used to support these interpretations.

Overall, the study is innovative and impactful, but incorporating functional validation, improved mechanistic depth, and discussion of population-specific limitations would substantially enhance its translational relevance and robustness.

 

The study incorporates extensive biological interpretation of the identified DNA methylation signatures and their links to oxidative balance and cancer risk. However, the manuscript does not sufficiently describe the statistical methods used to support these interpretations. Greater clarity regarding the statistical models, multiple-testing corrections, threshold selection, and robustness analyses is needed to ensure the validity and reproducibility of the reported findings. Explicitly linking biological conclusions to well-defined statistical evidence would substantially strengthen the rigor of the study.

Author Response

This study presents a novel and well-designed investigation linking lifestyle-based oxidative balance to pan-cancer risk through sex-specific DNA methylation signatures. The integration of population-based epigenomic data with TCGA cancer datasets and the introduction of the Hybrid Piscore (HyPi) represent clear strengths and add methodological innovation. The identification of distinct male- and female-specific methylation patterns provides important insight into sex-dependent oxidative stress–epigenome interactions. However, several gaps should be addressed to strengthen the manuscript.

 

• The biological interpretation of sex-specific CpG sites remains limited; deeper functional annotation and pathway-level analyses would help clarify their mechanistic relevance.

Response: Our research team has been preparing this study for a long time and has analyzed the results from various perspectives. We have related our findings to our thoughts in sections 4.4, 4.5, and the conclusion. However, some parts were lengthy and exaggerated, so we have summarized them concisely, focusing on key points. Our research team does not analyze biological mechanisms through experiments, but rather presents key insights from bio big data. Therefore, we have revised the discussion to present the most important points as much as possible.

 

• OBS is derived from self-reported lifestyle factors, which may introduce measurement bias and residual confounding that are not fully discussed.

Response: We acknowledge the inherent limitations of using self-reported data for calculating OBS, which may introduce recall bias and measurement error. However, the food frequency questionnaire (FFQ) and lifestyle surveys in KoGES have been previously validated and are widely used in epidemiological research. We have expanded our Discussion to address the potential for residual confounding and the necessity of incorporating objective biomarkers in future studies to mitigate this bias (section 4.6).

 

• While directional consistency with TCGA data is shown, the study lacks experimental or longitudinal validation to support causality between OBS, methylation changes, and cancer risk.

Response: The reviewer makes an important point regarding the cross-sectional nature of our study. Although we observed directional consistency with TCGA data, this does not definitively establish causality. We have clarified that our study is primarily associative and serves as a hypothesis-generating work. We have added a statement in the Discussion regarding the requirement for longitudinal tracking to confirm the causal link between OBS, methylation, and cancer risk (Section 4.6).

 

• The applicability of findings beyond the Korean population requires discussion, as ethnic and environmental differences may affect generalizability.

Response: We agree with the reviewer that our findings, based on the Korean population (KoGES), may have limited generalizability to other ethnic groups. While the KoGES cohort provides a robust and large-scale dataset, ethnic-specific dietary patterns, such as high intake of fermented foods and genetic backgrounds could influence the association between OBS and DNA methylation. We have now added this as a formal limitation in the Discussion section 4.6. and emphasized the need for future cross-ethnic validation.

 

• Although vitamin C dependent demethylation and one-carbon metabolism are proposed mechanisms, direct evidence linking these pathways to the identified CpGs is limited.

Response: We agree that direct experimental evidence linking the identified CpGs to the proposed pathways is currently limited. Our discussion on Vitamin C-dependent demethylation and one-carbon metabolism was intended to provide a plausible biological context based on existing literature. In the revised manuscript, we have largely omitted such content and presented a description focusing on the main points (section 4.5).

 

• Authors does not sufficiently describe the statistical methods used to support these interpretations.

Response: We have added a concise summary of the methodology for generating OBS from KoGES, focusing on key points, as cited in Moon et al., 2024 and Lin et al., 2025, where appropriate. We have added relevant information to the beginning of section 2.2 [Lines 83-85, 90-95].

 

Overall, the study is innovative and impactful, but incorporating functional validation, improved mechanistic depth, and discussion of population-specific limitations would substantially enhance its translational relevance and robustness.

Response: We've revised the figures overall to improve readability and added relevant statistical descriptions. We've presented our research as objectively as possible, focusing on key findings and presenting limitations. If you have any suggestions for further revisions or additions, please let us know. We'll do our best to incorporate them.

 

The study incorporates extensive biological interpretation of the identified DNA methylation signatures and their links to oxidative balance and cancer risk. However, the manuscript does not sufficiently describe the statistical methods used to support these interpretations. Greater clarity regarding the statistical models, multiple-testing corrections, threshold selection, and robustness analyses is needed to ensure the validity and reproducibility of the reported findings. Explicitly linking biological conclusions to well-defined statistical evidence would substantially strengthen the rigor of the study.

Response: The Hybrid Pi calculation method we developed is based on research that has explored the connection between DNA methylation and various indicators since 2020, and is revealed for the first time in this paper. Section 2.4 provides a detailed explanation, and related papers are cited. If you find any areas that require further explanation, please let us know and we will add them.

Round 2

Reviewer 2 Report

See above.

See above.

Author Response

Thank you for your meticulous review. Thanks to your review, the manuscript has become even better. We will provide high-resolution original figures upon publication to ensure perfect quality.

Reviewer 3 Report

Authors have addressed all my comments and i don't have any more suggestions.

 

Authors have addressed all my comments and i don't have any more suggestions.

 

Author Response

Thank you for your meticulous review. Thanks to your review, the manuscript has become even better. We will provide high-resolution original figures upon publication to ensure perfect quality.