PONE-D-25-03475Epigenetics Assessments of Alcohol Consumption Predict Mortality in Smokers at Risk for Lung Cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening TrialPLOS ONE

Dear Dr. Philibert,

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PLOS ONE

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Thank you for stating the following in the Acknowledgments Section of your manuscript: [This work was supported by 1R44CA285136 (Philibert and Dawes, MPI).   This work is dedicated to the memory of Dr. Meg Gerrard of the University of Connecticut whose work to prevent the effects of alcohol and smoking on risk for cancer have had a profound and lasting impact on the investigative team and the field in general. ]We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. 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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: No

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

Reviewer #1: Yes

Reviewer #2: No

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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: Yes

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: Thank you for the opportunity to read this interesting and well written manuscript that explored the association of epigenetic biomarkers of heavy alcohol drinking and smoking, with all-cause mortality. Findings indicated that Alcohol T Score was significantly related to all-cause mortality in smokers who developed and did not develop lung cancer. DNA methylation at cg05575921 did not have significant association with mortality. The author conclude that heavy alcohol consumption is a significant risk factor of all-cause mortality in smokers. The present study has several strengths including advanced modeling, long follow-up time for death, using participants from case-control study. Despite these strengths, there are several areas that limit my enthusiasm for the study. Below describe the major and minor weaknesses:

Key Weaknesses

1. In the results, you mentioned estimated survival split with AST median, but in the methods, you did not clearly state this analysis. Please add more details for it in the method.

2. Alcohol consumption had different burden on women and men due to their difference on body size, muscle mass, body fat and hormone level. Can you add sex-stratified analysis to further investigate the effect of heavy alcohol drinking on all-cause mortality?

3. Figure3 shows that ATS has a strong correlation with cg05575921. Please provide a more detailed explanation of how the adjusted R-squared value and p-value were calculated. Additionally, clarify the method used to generate the red line in the figure. Furthermore, Figure 3 appears to depict a negative relationship between ATS and cg05575921. Please explicitly state the direction of correlation in the text.

4. Can you add a test for interaction between ATS and cg05575921 in the cox model?

Additional Minor Weaknesses

1. In the introduction, line 70-72 “In abstinent individuals, ATS is zero-centered metric with a standard deviation of 2.2 that non-linearly increases as a function of increasing chronic alcohol consumption.” needs more clarification. Does this mean that, among non-drinkers, ATS has a mean of zero and a standard deviation of 2.2? Additionally, does the statement imply that if non-drinkers start drinking, then the relationship between alcohol consumption and ATS becomes non-linear?

2. In the Methods, the format of each small part is not consistent. Can you add subtitle for each part to improve consistency and readability? Suggested subtitles include Study sample, DNA methylation, AST value, Outcome, statistical analysis.

3. In the results, line 151 “LC cases were not more likely than controls to be current smokers (45% vs 39%, NS). ” What does “NS” mean? What was the p value to support your statement “LC cases were not more likely than controls to be current smokers”?

4. In the results, line 167-168 “median of 20.7 years (interquartile range; 5460-8521 days).”, can you convert the unit of days into years for consistency and clarity?

5. The sentence in the discussion line 205-207 “In a study using a marker for recent (past 3 weeks) heavy alcohol consumption, referred to a ZSCAN25, we found a 0.56 correlation” requires clarification. Could you specify which two variables the 0.56 correlation refers to? Additionally, explain how this correlation supports the claim that "binge drinking is not perfectly correlated with the ATS."

6. In the discussion, line 208-211 “Therefore, we believe that our findings with respect to mortality reflect the entirety of lifestyle risk biology, including risks from poor diets and lack of exercise, that track with the heavily tobacco use and chronic alcohol consumption. ” could benefit from further clarification. Specifically, how do the findings support the conclusion that risks from poor diet and lack of exercise are reflected? Are there specific references? Expanding on this would strengthen the argument.

7. In the discussion, line 222-224 “effects of alcohol measured through this method are greater than those that can be shown using cg05575921, and that with greater sample size, the effects assessed by cg05575921 will become significant.” What specific outcome is being affected by alcohol or cg05575921? Providing this information will help the reader understand the context and implications of these effects more precisely.

Reviewer #2: The authors investigated the associations among cg05575921 (a DNA methylation marker for smoking prediction), the alcohol methylation T score (ATS), and all-cause mortality in lung cancer patients and controls. The results indicate that the ATS score is associated with longer survival times. While the manuscript addresses an important topic, however clarification of the methods and presentation of results are needed to enhance its impact and clarity. Specific questions and comments regarding the manuscript are as follows:

1. The study focuses on two epigenetic markers for smoking and alcohol consumption and investigates their associations with mortality. Although methylation markers reflect a person’s cumulative lifestyle exposure, it would be more informative to compare these markers with actual lifestyle variables (e.g., smoking pack-years and alcohol consumption levels). While I understand that one of the major motivations of the study is the known unreliability of self-reported data in epidemiologic studies, including these variables and comparing the results from the epigenetic signatures could strengthen the findings.

2. Abstract: In lines 45–47, the phrase “in this small group” needs clarification.

3. Also, why did the authors control the occurrence of lung cancer in the analysis? Did the model for cg05575921 include both lung cancer patients and controls? If so, this approach appears inconsistent with that used for the ATS; please clarify.

4. The dataset comprises lung cancer patients and cancer-free individuals, with significant associations observed in both groups. However, the manuscript refers to “at risk for LC” and lung cancer screening efforts. Could the authors provide more contextual information on this point? If space in the abstract is limited, consider including additional details in the Methods section.

5. What is the rationale for providing sex-stratified descriptive statistics? It may be helpful to include P-values for comparisons of demographic and clinical characteristics in Table 1. In lines 156–159, the authors note differences in methylation values by sex. If such differences exist, why were the main association analyses not stratified by sex?

6. Line 110: the abbreviation “EEMS” is used. Please provide the full term when the abbreviation first appears.

7. Methods: The association was investigated using Cox proportional hazards models; however, the details of the models are not well specified. For instance, did the regression model include cg05575921 and ATS simultaneously? Clarification of the modeling strategy is needed.

8. Covariates: Was information on cancer stage and grade available for lung cancer patients? If so, these variables should be included in the analyses. Additionally, please explain why race/ethnicity was excluded from the model construction.

9. Figures: The point estimates mentioned in the text are not displayed in Figures 3 and 4. I am also uncertain whether Figures 1-3 provide sufficient additional information to warrant their inclusion in the main text. Reorganizing the presentation of the findings (both tables and figures) in the Results section would help readers follow the key messages more clearly.

10. Lines 187–189: the authors draw a conclusion that I find difficult to support based on the results presented. Specifically, the analyses were not conducted exclusively among smokers, and referring to “baseline alcohol consumption” is misleading when it is measured via a proxy marker (i.e., the methylation signature). Please clarify how these conclusions were derived from the results.

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Reviewer #1: No

Reviewer #2: No

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Epigenetic Assessments of Alcohol Consumption Predict Mortality in Smokers at Risk for Lung Cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial

PONE-D-25-03475R1

Dear Dr. Philibert,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Chunyu Liu, PhD

Academic Editor

PLOS ONE

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: All comments have been addressed

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: Yes

Reviewer #2: Yes

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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

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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: I did not have any additional comments. All my previous questions were solved. And the manuscript is well written.

Reviewer #2: (No Response)

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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

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