Serum IgG titer findings for Fusobacterium nucleatum associated with clinical outcome following surgery in patients with esophageal squamous cell carcinoma

Toru Yoshikawa; Hiromi Nishi; Manabu Emi; Yoichi Hamai; Yuta Ibuki; Tomoaki Kurokawa; Ryosuke Hirohata; Manato Ohsawa; Nao Kitasaki; Hitoshi Komatsuzawa; Hiroyuki Kawaguchi; Morihito Okada

doi:10.1371/journal.pone.0336219

Peer Review History

Original SubmissionJune 25, 2025
18 Sep 2025 Decision Letter - David Ojcius, Editor Dear Dr. Nishi, 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. Specifically, the reviewer recommends revisions that would improve statistical reporting and methodological rigor. Please submit your revised manuscript by Nov 02 2025 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. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). 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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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: This retrospective cohort study explores the prognostic utility of pre-treatment serum IgG antibody titers against Fusobacterium nucleatum (IgG-Fn) in 305 esophageal squamous cell carcinoma patients undergoing esophagectomy. The central finding—that IgG-Fn positivity associates with reduced survival and diminished response to neoadjuvant therapy—addresses a clinically relevant gap in ESCC biomarker research. While the cohort size is appropriate and statistical methods generally sound, several revisions are necessary to strengthen methodological rigor and contextual interpretation. Specific comments 1.The ELISA methodology requires significant elaboration to ensure reproducibility. Specifically, authors should detail the rationale for selecting the F. nucleatum subspecies beyond citing ATCC 10953, explain how serum dilution ratios were optimized. Clarity regarding sample timing is essential—explicitly confirm all specimens were obtained before any treatment initiation, including neoadjuvant therapy. The partial oral health assessment in just 213 patients introduces possible selection bias that warrants explanation or sensitivity analysis. 2.Statistical reporting needs refinement throughout. All Kaplan-Meier figures must be supplemented with "Number at Risk" tables and annotated with hazard ratios to improve interpretability. Critical inconsistencies between text sections require resolution, such as the abstract's misstatement about IgG-Fn-negative patients showing worse pathology—this directly contradicts Supplementary Table 1 findings where IgG-Fn-positive status correlates with adverse pathological features. 3.Interpretation would benefit from deeper biological contextualization. The stark contrast in IgG-Fn's prognostic significance between Stage I versus advanced-stage patients necessitates mechanistic exploration—is this attributable to statistical power limitations, fundamental biological differences in early disease, or the modifying effect of neoadjuvant therapy exposure? While chemotherapy resistance mechanisms are cited, the discussion fails to integrate how the IgG biomarker itself relates to these pathways—does elevated IgG indicate failed bacterial clearance that enables Fn-mediated treatment resistance? ****** 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 ******** [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.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0336219.r001
Revision 1
17 Oct 2025 Author Response Dear Editor, Thank you for providing us with an opportunity to resubmit our study. Please find our detailed responses to each comment below. Comment 1 The ELISA methodology requires significant elaboration to ensure reproducibility. Specifically, authors should detail the rationale for selecting the F. nucleatum subspecies beyond citing ATCC 10953, explain how serum dilution ratios were optimized. Clarity regarding sample timing is essential—explicitly confirm all specimens were obtained before any treatment initiation, including neoadjuvant therapy. The partial oral health assessment in just 213 patients introduces possible selection bias that warrants explanation or sensitivity analysis. Authors’ response: Thank you for these valuable suggestions. The rationale for selecting ATCC 10953 F. nucleatum subsp. polymorphum) among the four subspecies of Fusobacterium nucleatum for ELISA and steps taken to adopt a 1:3200 dilution is now described in the revised Methods section (lines 366-369, 373-375). In addition, to clarify the timing of blood collection, it is noted that it was performed before the start of any treatment in all cases (Methods, lines 361-362). Based on these excellent comments and suggestions, results of sensitivity analysis regarding the response to partial oral health assessment have been added to the revised manuscript. Similar to the main analysis, the findings obtained with the subset of 213 patients showed that IgG-Fn positivity was consistently associated with poor prognosis (OS: HR=1.68, 95% CI 1.13-2.50; p=0.01) (CSS: HR=1.72, CI 1.10-2.72; p=0.02) (multivariate Cox CSS: HR=2.22, CI 1.40-3.53; p<0.001). These results have been added to the Methods (lines 417-418) and Results (lines 139-152) sections and are also presented in Supplementary Figure 1 and Supplementary Table 1. Comment 2 Statistical reporting needs refinement throughout. All Kaplan-Meier figures must be supplemented with "Number at Risk" tables and annotated with hazard ratios to improve interpretability. Critical inconsistencies between text sections require resolution, such as the abstract's misstatement about IgG-Fn-negative patients showing worse pathology—this directly contradicts Supplementary Table 1 findings where IgG-Fn-positive status correlates with adverse pathological features. Authors’ response: We appreciate these helpful comments. We have added number at risk indicating results of Kaplan-Meier analyses to all the figures and also added hazard ratios to the figure legends (lines 102-133). In addition, the misstatement in the original Abstract has been corrected (line 34). Comment 3 Interpretation would benefit from deeper biological contextualization. The stark contrast in IgG-Fn's prognostic significance between Stage I versus advanced-stage patients necessitates mechanistic exploration—is this attributable to statistical power limitations, fundamental biological differences in early disease, or the modifying effect of neoadjuvant therapy exposure? While chemotherapy resistance mechanisms are cited, the discussion fails to integrate how the IgG biomarker itself relates to these pathways—does elevated IgG indicate failed bacterial clearance that enables Fn-mediated treatment resistance?　 Authors’ response: Thank you for these insightful comments. The following has been added to the Discussion section in the revised version. Notably, the prognostic impact of IgG-Fn was found to vary according to stage. In Stage I patients, the survival rate was high and number of events was low, thus limiting the ability to detect a significant effect. In contrast, in advanced clinical stages, tumor-associated inflammation and immunosuppression were more pronounced. Thus, sustained Fn exposure in such cases may promote immune dysregulation and chemotherapy resistance, leading to enhancement of the prognostic significance of IgG-Fn. (lines 292-298). An elevated anti-Fn IgG level suggests prolonged exposure to Fn or persistence in the host rather than a simple past infection. This may activate cell survival-related pathways such as inflammatory signaling and autophagy, possibly resulting in reduced sensitivity to cytotoxic therapy. (lines 251-254). Attachments Attachment Submitted filename: Response to Reviewers .docx https://doi.org/10.1371/journal.pone.0336219.r002
23 Oct 2025 Decision Letter - David Ojcius, Editor Serum IgG titer findings for Fusobacterium nucleatum associated with clinical outcome following surgery in patients with esophageal squamous cell carcinoma PONE-D-25-26990R1 Dear Dr. Nishi, 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 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, David M. Ojcius Academic Editor PLOS ONE Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The authors have addressed all my concerns, I think this manuscript is suitable for publication in PLOS One. Thank you very much. ****** 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 ******** https://doi.org/10.1371/journal.pone.0336219.r003
Formally Accepted
Acceptance Letter - David Ojcius, Editor PONE-D-25-26990R1 PLOS ONE Dear Dr. Nishi, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. David M. Ojcius Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0336219.r004

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