Dear Dr. Kanahara,

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PLOS ONE

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“N.K. reports honoraria from Otsuka Pharmaceutical Co., Sumitomo Pharma Co., Janssen Pharmaceutical., Meiji Seika Pharma Co., Mitsubishi Tanabe Pharma Co., MSD. Y.Od. reports honoraria from Eisai Co., Otsuka Pharmaceutical Co., Meiji Seika Pharma Co. T.N. reports honoraria from Otsuka Pharmaceutical Co., Viatris Inc., Daiichi Sankyo Co., Sumitomo Pharma Co., Meiji Seika Pharma Co., and Yoshitomi Yakuhin Co. He also received research funding from NTT Precision Medicine, Inc. Y.M., Y. Ot., Y.S., T.I., H.S., K.I., H.K., Y.F., Y.H., and M.I. have no conflicts of interest to declare.”

We note that you received funding from a commercial source: “Otsuka Pharmaceutical Co., Sumitomo Pharma Co., Janssen Pharmaceutical., Meiji Seika Pharma Co., Mitsubishi Tanabe Pharma Co. Otsuka Pharmaceutical Co., Viatris Inc., Daiichi Sankyo Co., Sumitomo Pharma Co., Meiji Seika Pharma Co., and Yoshitomi Yakuhin Co”

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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?

Reviewer #1: No

Reviewer #2: Partly

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

Reviewer #1: No

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

Reviewer #1: Yes

Reviewer #2: No

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

Reviewer #1: No

Reviewer #2: No

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Reviewer #1: The manuscript investigates cortical volumetric alterations in patients with treatment-resistant schizophrenia (TRS), and explores whether structural MRI findings are associated with response to clozapine (CLZ). The study leverages clinically acquired MRI data (n = 104 patients, plus 109 external healthy controls) and FreeSurfer-based cortical parcellation according to the Desikan–Killiany atlas.

Altough the topic is clinically relevant, the study suffers from significant methodological and interpretative limitations that limit the robustness and novelty of its conclusions. The results are largely negative or nominal, and the discussion sometimes overstates their significance.

Overall, in my opinion the manuscript does not meet the miniml publication standards and should be rejected, moved to a lower impact factor journal, or modified as a brief report with preliminary exploratory nature. I attach below a list of my major concerns.

1. The total number of TRS patients treated with CLZ is small (n=20, with only 12 responders and 8 non-responders). This precludes adequate power for voxel- or region-based analyses, especially when correcting for multiple comparisons. Indeed, none of the reported associations survived FDR correction. Therefore, the interpretation of uncorrected results shoud be presented with strong caution or omitted. Moreover, I advise the authors to explicitly acknowledge the exploratory nature of the analyses and avoid causal or mechanistic interpretations

2. MRI scans were obtained from five different scanners and protocols, and the healthy control (HC) group was drawn from an external public dataset acquired on different systems. Although the authors included scanner type as a covariate and used ratio normalization, this ususally does not completely correct for inter-scanner variability. Authors should try to run harmonization methods or discuss this issue as a serious limitation that prevents both generalizability and reliable interpretation of data.

3. No correction for antipsychotic dose or age of onset (and therefore duration of illness) is given, altough these parameters are significantly different between groups and could represent strong confounders for structural brain abnormalities. This precludes any reliable inference on outcomes. And the same attains for the sub-classification in clozapine-responders and non-responders, although the reliability of this analysis is already prevented from the very low number of cases.

4. The operationalization of TRS and the criteria for defining clozapine response/non-response should be described in more detail in the main text (not only in Supplementary Material). The use of clinical global impression (CGI-C) and GAF change over 1 year is reasonable, although not the standard, but the reliability of retrospective classification should be discussed. Moreover, I would avoid to make comparisons between R-CLZ and nR-CLZ patients, given the extremely low sample size in this subclassification and its propensity to false outcomes with this limited power. In my opinion, the section on the comparison between clozapine responders and non-responders should be eliminated as it has poor or no scientific value.

5. The central hypothesis that cortical morphology correlates with the delay before CLZ introduction was not confirmed. This negative finding, which contradicts the stated rationale, should be given greater prominence in the abstract and discussion, rather than being overshadowed by nominal findings on other regions. Moreover, it is not clear on which literature base the authors posited this hypothesis. And in general, the introduction is not useful to capture the rationale of the study or a clear neurobiologically-based hypothesis, other than assuming that there are few previous reports on the issue. And the strong claim on the evaluation of biomarkers related to clozapine therapy should be mitigated, given than only 20 patients of the sample were under clozapine

6. The discussion is in large part speculative, as it repeatedly refers to “involvement” of IFG, ACC, and occipital cortex in CLZ response, yet all findings are at p < 0.05 uncorrected. These findings should be clearly labelled as exploratory and interpreted accordingly, with great caution and no causal inferences

7. The choice to analyze cortical volume ratios rather than absolute values or thickness measures is unconventional and not well justified. The authors should clarify why this approach was preferred and discuss its possible drawbacks.

8. The manuscript would benefit from linguistic editing to improve clarity and conciseness. Several sections are redundant or overly descriptive. Figures and tables are informative but should highlight key results more effectively (e.g., provide effect sizes and FDR-adjusted p-values in the main text).

9. Authors should clarify the time relationship between MRI acquisition and the initiation of clozapine treatment (was the MRI always before CLZ introduction or any other else?). The ethics section should specify the form of consent (written vs. opt-out) for both institutions.

Reviewer #2: This study investigates grey matter volumen correlates to TRS and CPZ repsonse in patients with schizophrenia. The study questions are interesting and clinically relevant, and the authors aim to answer the research questions based on pragmatic solutions on data-quality, sample-size, methodological complexity a.o.. There are however a range of major and minor concerns about the study:

1. It is a major concern that the study and data acquisition was not designet for these hypothesis, but was derived from a pragmatic pooling of data from 7 different scanners with vital differences in acquisition parameters such as Field Strenght, REp. time, Inversion, flip angle etc. (according to Supplem. Table S1). The authors state as imitations that harmonization attempts failed, hence they used the very crude correction for scanner type as covariate. As the major results reported is groupdiffernce between patients and controls, where the MRI data for controls were from a very different public dataset, the results are unfortunately questionable. What could validate the results more would be transparence about an extensive examination of data, including an option of visual inspection of e.g. scatterplots etc. by reader and reviewers. However, as there are more major considerations, such as a lack of power calculation, resulting in a far to small sample (8 non-responsers vs 12 responding CPZ patients when examining groupdifferences) to meaningfully answer the relevant research questions. The authors may - with the caveats from the pooling of data from different scanners - meaningfully investigate differences between controls and patients, but further subgrouping of patients will reduce the degrees of freedom to a point where analyses cannot be evaluated as sound / nor results as robust with this sample size. It is a huge concern with yet another MRI study presenting non-robust results on insufficient data of limited quality. The authors state (line 228-229) that they provide several insights into the relationship between brain structure and responsiveness to CPZ - but this conclusion is not valid. At best, the study may be hypothesis generating due to the pragmatic design and major concerns.

2. Another major concern is the attempt to link MRI GMV data to CPZ response. First, it is very uncertain that any difference has an MRI-signal, as the patients were not antipsychotic naive before start CPZ. Secondly, it is not proper to present this mixture of uncorrected (i.e. "nominal level of significance" as in line 182)and corrected results, particularly with the huge number of correlation analyses - a method known for producing to many false positive results. If the authors - after correction for multiple analyses - find that no GM regions survived control when comparing CPZ responders to nonresponders, that is the result which has to be clearly stated in eg. the abstract. It is not, to the contrary it appears like there is a difference, which is not trustworthy.

3. The use of unusual words and terms, which are a bit uncorrect, and claims which demonstrate lack of clinical understanding (such as line 36 "broader brain regions"; line 153 "MRI-system" (usually we term it scanner / scannersites); line 165-167 are not understandable . do the authors correct for different scanners or not in the analyses?; line 301 "cortex was less damaged"; line 286-287, claiming that hallucinations, negative symptoms and cognitive dysfunction is a "potential core psychopathology of TRS" (these are decisive symptoms for all patients with schizophrenia??).

4. Lack of clarity in reporting the study design, materials and methods is a huge obstacle for transparency in this study. The methods reported in supplements needs to go to the main text, and it would be beneficial with a illustration of the analyses plan, and which data came from which group / subgroup / timepoint, as it is hard to deciphre in the current format. Further, it is unclear in Table 2 that you actually appear to have tested every clinical measure at against coorticsl regions, but in some it is delta (change), but not in others etc.

5. Minor concerns would be the lenght of the discussion, with a lot of redundance, such as line 22-225 repeated in 237-239, and it appears unstructured, with far too much speculation on results which cannot justify such discussions.

A major revision, using the principle of Occams razor, where the authors refrain from underpowered analyses, not overreport results, and instead simplify the study questions and enhance clarity and transparency of the pragmatic and preliminary hypothesis-generating results would result in an article of much higher scientific quality.

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Reviewer #1: Yes: Felice Iasevoli

Reviewer #2: No

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Dear Dr. Kanahara,

Please submit your revised manuscript by Mar 19 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.

• 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,

Kenji Tanigaki, Ph.D., M.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.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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

Reviewer #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: (No Response)

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors have addressed most of my concerns and appropriately toned down their conclusions. While important methodological limitations remain, the manuscript now presents a clearer and more transparent exploratory analysis that is suitable for publication in PLOS ONE.

Reviewer #2: The authors have not adressed my concerns sufficiently. The first major concern reg. data acquisition from a pragmatic pooling of data from 7 different scanners with vital differences in acquisition parameters where authors state that harmonization attempts failed, plus the MRI data for controls were from a very different public dataset. I suggested

" more transparence about an extensive examination of data, including an option of visual inspection of e.g. scatterplots etc. by reader and reviewers", but the only action taken by the authors is to move text from supplementary to main text. I would like a thorough examination of how attempts to harmonize was failing, including the results as shown by eg.scatterplots etc. - if not, the conclusions cannot be regarded as valid.

My second concern "a lack of power calculation, resulting in a far to small sample (8 non-responsers vs 12 responding CPZ

patients when examining groupdifferences) to meaningfully answer the relevant research questions. The authors may - with the caveats from the pooling of data from different scanners - meaningfully investigate differences between controls and patients, but further subgrouping of patients will reduce the degrees of freedom to a point where analyses cannot be evaluated as sound / nor results as robust with this sample size." was solved by authors by moving the subgroup analyses to the supplementary and change some text - but still in abstract authors write predominantly about this subject - in results: "The correlational analysis of regions related to clozapine responsiveness identified the inferior frontal gyrus (IFG), anterior cingulate cortex (ACC), and the occipital cortex at the uncorrected level of significance, but none of these survived t he correction for multiple comparisons" and in Conclusion "These results suggest that the IFG, ACC, and occipital cortex might be responsible for the treatment response to clozapine....". It is very difficult to infer from what could have been a result in case of more power, but here I truly find this to be scientifically unsound, and not just something that be framed differently by small reformulaitons. I cannot recommend accept without thorough rewriting from a much more humble perspective on what is possible to infer by this study design and underpowered samplesize.

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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

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

Neural substrates of treatment-resistant schizophrenia and the response to clozapine A structural MRI study in a clinical setting

PONE-D-25-50031R2

Dear Dr. Kanahara,

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,

Kenji Tanigaki, Ph.D., M.D.

Academic Editor

PLOS One