Evaluating immune response and metabolic related biomarkers pre-allogenic hematopoietic stem cell transplant in acute myeloid leukemia

Sharareh Siamakpour-Reihani; Felicia Cao; Jing Lyu; Yi Ren; Andrew B. Nixon; Jichun Xie; Amy T. Bush; Mark D. Starr; James R. Bain; Michael J. Muehlbauer; Olga Ilkayeva; Virginia Byers Kraus; Janet L. Huebner; Nelson J. Chao; Anthony D. Sung

doi:10.1371/journal.pone.0268963

Peer Review History

Original SubmissionDecember 14, 2021
10 Jan 2022 Decision Letter - Francesco Bertolini, Editor PONE-D-21-39481Evaluating immune response and metabolic related biomarkers pre-allogenic hematopoietic stem cell transplant in acute myeloid leukemiaPLOS ONE Dear Dr. Sung, 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 by both Reviewers, experts in the field. Please submit your revised manuscript by Feb 24 2022 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. 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Thank you for stating the following in the Acknowledgments Section of your manuscript: "This research was in part supported, in part, by the American Society of Hematology (ASH) Scholar Award and the NIH/National Institute on Aging 1R21AG066388-01 award" 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. Currently, your Funding Statement reads as follows: "YES - Specify the role(s) played A.D.S.: American Society of Hematology (ASH) https://www.hematology.org/ Scholar Award and the NIH/National Institute on Aging 1R21AG066388-01 award. 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Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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 ******** 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: Summary: In the present study, the authors evaluated immune/inflammatory responses and metabolic biomarkers associated with overall survival (OS) in acute myeloid leukemia (AML) after hematopoietic stem cell transplantation (HCT). Using plasma from 34 AML patients after HCT, they analyzed 65 blood-based metabolomics markers and 61 immune/inflammatory related biomarkers, comparing long-term OS (1 year) with short-term OS (3 years), or comparing younger (30 years) with older (55 years) patients. They found several markers that were elevated or reduced in older versus younger patients, as well as many immune/inflammatory markers associated with outcomes such as graft-versus-host disease (GVHD), infection, or relapse. For metabolic markers, they found higher levels of medium- and long-chain acylcarnitines in older patients with GVHD. Further studies would be required to assess the use of these biomarkers in disease prognosis and therapy. Comments for improvement are listed below: Comments: 1. Overall survival (OS) is spelled out twice in the abstract. 2. Abstract Line 32: “This retrospective study was designed with an exploratory and hypothesis generating….” Introduction Line 113-114, “We hypothesized that metabolomics and blood-based biomarkers have the potential to be used as predictive biomarkers for AML patients who receive HCT treatment.” Please clearly state the hypothesis within the abstract. 3. Please spell out all abbreviations upon first usage. For example, MMP-3 is spelled out on line 152, but first used on line 99. 4. Methods: For biomarkers of inflammation and aging, please provide more details regarding how the 54-plex plates were read. What equipment did you use? Were serial dilutions performed? 5. The metabolomics data are presented haphazardly in the Results section. They are hard to follow. Revision is recommended. 6. Discussion: A recent article (PMID: 34722316) regarding BET inhibition in GVHD after HCT should be discussed in relation to IL-23/IL-17. 7. Discussion Lines 341-346 are not necessary and do not have relevance to the current manuscript. 8. Do the authors speculate that increased acylcarnitines in older patients correlates with enhanced oxidative phosphorylation? In what cell type? This would be supported by the lower mean levels of lactate. Please clarify within the text. 9. Figure 1, panel labels are recommended (e.g. A, B, C, D). These should be updated within the text. Please use consistent font and font sizes for all graph labels. 10. Figure legends: Please indicate the meaning of * within the figures. 11. There is a lot of supporting information that is not described or called out within the manuscript text. Reviewer #2: Siamakpour-Reihani et al investigate the expression of a large number of immune/inflammatory and metabolomic biomarkers in plasma samples from patients with acute myeloid leukemia (AML) before hematopoietic stem cell transplantation (HCT) and explore associations with disease outcome (overall survival-OS), young (≤30years) or old age (≥ 60 years) and other clinical outcomes (chronic graft versus host disease-cGVHD, acute graft versus host disease-aGVHD, infection and relapse). They found that several biomarkers were elevated in older patients compared to younger patients, whereas only IL23 was upregulated in younger patients. Three markers (IL13, SAA, CRP) were lower in patients with OS ≥ 3 years, whereas IL-9, Eotaxin-3 was associated with aGVHD, Flt-1 with cGVHD, D-dimer with infection, or IL-17D, bFGF, and Eotaxin-3 with relapse. Higher baseline levels of medium- and long-chain acylcarnitines (AC) were found in older patients, whereas lactate and long-chain AC were associated with aGVHD, and medium-chain AC with cGVHD. These findings are interesting, and the manuscript is well-presented; however, the following issues need attention. 1. Sex and age matched healthy controls are missing. Thus, it is not clear whether the differential expression of immune/inflammatory and metabolomic biomarkers in plasma samples from AML patients owes to disease, age or comorbidities related to elder. 2. The characteristics of the patients in Table 1, should be presented in more detail. All the parameters examined (cGVHD, aGVHD, infection, relapse, deaths, etc), as well as disease comorbidities (such as thrombosis, cardiac disease, etc) must be presented. 3. In this context, it should be excluded that the factors that are differentially expressed in the various subsets of AML patients, do not owe their differential expression to an infection or comorbidity. 4. Metabolomic analysis was performed in retrospectively collected samples. Were the samples collected appropriately for this kind of analysis? The samples were collected at similar period of the day, were the patients fasting? All this information needs to be included in the manuscript (if they were not performed, along with comment #1, they should be discussed). 5. It is not reported why the authors chose these age limits (e.g., why young were considered those under 30 years and not those under 32 or 35 years). It was based on statistical analysis, or it was an arbitrary selection? This must be clearly stated. ******** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
17 Mar 2022 Author Response PONE-D-21-39481 03/04/2022 Rebuttal letter We are greatly appreciative for this careful and detailed review. Please see a point-by-point response to the reviewers’ comments. We have revised the paper accordingly. Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf 2. and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. Response: Written, witnessed informed consent was obtained for sample collection for future research purposes, as well as use of associated clinical data. De-identified samples and de-identified clinical data were accessed under a separate, IRB-approved protocol; because deidentified samples and data were used, and patients already consented to future use of samples for research purposes, the IRB waived the requirement for consent for this use protocol. (IRB #s are: Pro00006268 and Pro00100650).: 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This research was in part supported, in part, by the American Society of Hematology (ASH) Scholar Award and the NIH/National Institute on Aging 1R21AG066388-01 award" 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. Currently, your Funding Statement reads as follows: "YES - Specify the role(s) played A.D.S.: American Society of Hematology (ASH) https://www.hematology.org/ Scholar Award and the NIH/National Institute on Aging 1R21AG066388-01 award. https://reporter.nih.gov/search/5NyHmfz0skuWJC2JAuThRw/project-details/9980757" Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: We have removed funding-related text from the manuscript. In the cover letter we have requested the following funding information (* is a new request): We also would like to update our Funding Statement to include the following information (per your request this section has bene removed from the acknowledge section): 1. American Society of Hematology (ASH), (PI: Anthony D Sung) https://www.hematology.org/ 2. NIH/National Institute on Aging 1R21AG066388-01 award (PI: Anthony D Sung). https://reporter.nih.gov/search/5NyHmfz0skuWJC2JAuThRw/project-details/9980757 3. NIH/National Institute on Aging Duke Pepper Older Americans Independence Center P30 AG028716, (PI: Schmader, Mini #6, PI of Mini: Anthony D Sung) https://reporter.nih.gov/search/KsvVX_8rwUKk6CXVf2k0vg/project-details/9971412 4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. "Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Response: The minimal data set underlying the results described in our manuscript have been uploaded as supplement/ supporting document. We have also added this statement to the cover letter: “This revised manuscript includes the revisions and edits requested by reviewers. In addition, for the re-submission of our revised manuscript, we have uploaded our study’s minimal underlying data set as Supporting Information files.” 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author ________________________________________ Reviewer #1: Comments: 1. Overall survival (OS) is spelled out twice in the abstract. Response: This has been corrected 2. Abstract Line 32: “This retrospective study was designed with an exploratory and hypothesis generating….” Introduction Line 113-114, “We hypothesized that metabolomics and blood-based biomarkers have the potential to be used as predictive biomarkers for AML patients who receive HCT treatment.” Please clearly state the hypothesis within the abstract. Response: Revisions have been added 3. Please spell out all abbreviations upon first usage. For example, MMP-3 is spelled out on line 152, but first used on line 99. Response: Revisions have been added 4. Methods: For biomarkers of inflammation and aging, please provide more details regarding how the 54-plex plates were read. What equipment did you use? Were serial dilutions performed? Response: Revisions have been added to the methods sub- section for “Biomarkers of inflammation and aging” 5. The metabolomics data are presented haphazardly in the Results section. They are hard to follow. Revision is recommended. Response: The section has been revised in results section. 6. Discussion: A recent article (PMID: 34722316) regarding BET inhibition in GVHD after HCT should be discussed in relation to IL-23/IL-17. Response: This has been added to the discussion section 7. Discussion Lines 341-346 are not necessary and do not have relevance to the current manuscript. Response: Section removed 8. Do the authors speculate that increased acylcarnitines in older patients correlates with enhanced oxidative phosphorylation? In what cell type? This would be supported by the lower mean levels of lactate. Please clarify within the text. Response: We show no significant difference with age for lactate, so would not appear to support an association with the increase in long/median-chain AC with age. Our data do however show a significant decrease in lactate with aGVHD, as they also do decreases in various ACs. This could potentially signify a pathway emphasis towards Krebs (and oxidative phosphorylation) and away from lactate or fatty acid synthesis, or alternatively a drive in the opposite direction through pyruvate towards gluconeogenesis. In order to have the data and conclusion, we would need a more intensive study to support such claims (this would be out of the scope for this paper). A paragraph also has been added with references to the discussion related to this comment. 9. Figure 1, panel labels are recommended (e.g. A, B, C, D). These should be updated within the text. Please use consistent font and font sizes for all graph labels. Response: The figure has been revised and updated within the text 10. Figure legends: Please indicate the meaning of * within the figures. Response: Please note there is no * sign in the figures. The only * signs are for table 2 with the explanation of: “* are separate ELISAs not part of the 54-plex. ** Higher baseline levels in younger patients”. However, in the figures there are dots/points (which might have been mistaken for ), so we have added the following to Fig 1 legend: data is presented as a boxplot. Boxes represent 25th (Q1) and 75th (Q3) percentiles; Horizontal lines indicate the medians; Upper whiskers indicate maximum; Lower whiskers indicate minimum. Points indicate any observations outside the whiskers.” 11. There is a lot of supporting information that is not described or called out within the manuscript text. Response: We have added more data and supporting information, so we hope we have responded to the comment to an extent (since the comment is general, we did not know if adding specific information was intended). Reviewer #2: Siamakpour-Reihani et al investigate the expression of a large number of immune/inflammatory and metabolomic biomarkers in plasma samples from patients with acute myeloid leukemia (AML) before hematopoietic stem cell transplantation (HCT) and explore associations with disease outcome (overall survival-OS), young (≤30years) or old age (≥ 60 years) and other clinical outcomes (chronic graft versus host disease-cGVHD, acute graft versus host disease-aGVHD, infection and relapse). They found that several biomarkers were elevated in older patients compared to younger patients, whereas only IL23 was upregulated in younger patients. Three markers (IL13, SAA, CRP) were lower in patients with OS ≥ 3 years, whereas IL-9, Eotaxin-3 was associated with aGVHD, Flt-1 with cGVHD, D-dimer with infection, or IL-17D, bFGF, and Eotaxin-3 with relapse. Higher baseline levels of medium- and long-chain acylcarnitines (AC) were found in older patients, whereas lactate and long-chain AC were associated with aGVHD, and medium-chain AC with cGVHD. These findings are interesting, and the manuscript is well-presented; however, the following issues need attention. 1. Sex and age matched healthy controls are missing. Thus, it is not clear whether the differential expression of immune/inflammatory and metabolomic biomarkers in plasma samples from AML patients owes to disease, age or comorbidities related to elder. Response: lack of matched healthy controls is a limitation of our study, we have add this to our “limitation section of the manuscript”. In this retrospective study the comparison is only made between the blood based biomarkers in different groups of AML patients based on age and survival. Also, we did not have access to matched healthy control samples for this retrospective study. 2. The characteristics of the patients in Table 1, should be presented in more detail. All the parameters examined (cGVHD, aGVHD, infection, relapse, deaths, etc), as well as disease comorbidities (such as thrombosis, cardiac disease, etc) must be presented. 3. In this context, it should be excluded that the factors that are differentially expressed in the various subsets of AML patients, do not owe their differential expression to an infection or comorbidity. Response (#2): Table 1 have been updated to present the parameters and conditions examined. Response (#3): Based on the updated Table 1, we have not seen balance issue between age groups in our outcomes. The p-values are not significant for differences between groups for infection or other comorbidities we studied. The only two significant p-values for difference between the two groups in Table 1 are: for the age (which was by design), and for Conditioning (which is expected since the conditioning for younger HCT patients will be different 4. Metabolomic analysis was performed in retrospectively collected samples. Were the samples collected appropriately for this kind of analysis? The samples were collected at similar period of the day, were the patients fasting? All this information needs to be included in the manuscript (if they were not performed, along with comment #1, they should be discussed). Response: All samples were appropriate for the metabolomics assays as the DPMI core is highly specialized in these studies. The blood samples have been collected as part of the ABMT Biorepository protocol and we used them retrospectively. However, the samples are not collected at the same time of the day. As part of our IRB approved protocol the biorepository samples have been collected during routine appointments pre and post HCT at routine lab visits. Thus, they are not all collected at the same time. The patients are not required to be fasting unless requested by the physician. 5. It is not reported why the authors chose these age limits (e.g., why young were considered those under 30 years and not those under 32 or 35 years). It was based on statistical analysis, or it was an arbitrary selection? This must be clearly stated. Response: The information was added to the methods section: The age cut-offs were arbitrary selection and mostly based on what we had samples for. Though there is support that AML in patients 55years behaves differently than AML in patients 55 years (Appelbaum, Gundacker et al. 2006). Appelbaum, F. R., H. Gundacker, D. R. Head, M. L. Slovak, C. L. Willman, J. E. Godwin, J. E. Anderson and S. H. Petersdorf (2006). "Age and acute myeloid leukemia." Blood 107(9): 3481-3485. Attachments Attachment Submitted filename: Response to Reviewers-Reihani- Sung etal -03-08-2022.docx* https://doi.org/10.1371/journal.pone.0268963.r002
3 Apr 2022 Decision Letter - Francesco Bertolini, Editor PONE-D-21-39481R1Evaluating immune response and metabolic related biomarkers pre-allogenic hematopoietic stem cell transplant in acute myeloid leukemiaPLOS ONE Dear Dr. Sung, 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 by Reviewer #1. Please submit your revised manuscript by May 18 2022 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 rebuttal 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-emailutm_source=authorlettersutm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Francesco Bertolini, MD, PhD Academic Editor PLOS ONE Journal Requirements: 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. [Note: HTML markup is below. Please do not edit.] 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 ******** 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: No 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: Yes 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: Summary: In the present study, the authors evaluated immune/inflammatory responses and metabolic biomarkers associated with overall survival (OS) in acute myeloid leukemia (AML) after hematopoietic stem cell transplantation (HCT). Using plasma from 34 AML patients after HCT, they analyzed 65 blood-based metabolomics markers and 61 immune/inflammatory related biomarkers, comparing long-term OS (1 year) with short-term OS (3 years), or comparing younger (30 years) with older (55 years) patients. They found several markers that were elevated or reduced in older versus younger patients, as well as many immune/inflammatory markers associated with outcomes such as graft-versus-host disease (GVHD), infection, or relapse. For metabolic markers, they found higher levels of medium- and long-chain acylcarnitines in older patients with GVHD. Further studies would be required to assess the use of these biomarkers in disease prognosis and therapy. Comments for improvement are listed below: Comments: 1. Please provide statistics within each panel of every figure. 2. Supplemental data are still not adequately called out within the text. Reviewer #2: The authors addressed all issues and the manuscript is appropriate for publication. The only correction that is needed in the new version is the deletion of the phrase in lines 350-351 because it is a repetition of the next phrase. ****** 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.] 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.0268963.r003
Revision 2
12 Apr 2022 Author Response 04-11-2022 Rebuttal letter: Thank you for your time and comments. We are greatly appreciative for this second review on the revisions previously made. Please see the response to the additional comments and minor changes requested (blue color font). We have revised the paper accordingly. PONE-D-21-39481R1 Evaluating immune response and metabolic related biomarkers pre-allogenic hematopoietic stem cell transplant in acute myeloid leukemia PLOS ONE Please include the following items when submitting your revised manuscript: • A rebuttal 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'. Response: This had been done • 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'. Response: This had been done • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. • Response: This had been done 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 ________________________________________ 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: No 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: Yes 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: Summary: In the present study, the authors evaluated immune/inflammatory responses and metabolic biomarkers associated with overall survival (OS) in acute myeloid leukemia (AML) after hematopoietic stem cell transplantation (HCT). Using plasma from 34 AML patients after HCT, they analyzed 65 blood-based metabolomics markers and 61 immune/inflammatory related biomarkers, comparing long-term OS (1 year) with short-term OS (3 years), or comparing younger (30 years) with older (55 years) patients. They found several markers that were elevated or reduced in older versus younger patients, as well as many immune/inflammatory markers associated with outcomes such as graft-versus-host disease (GVHD), infection, or relapse. For metabolic markers, they found higher levels of medium- and long-chain acylcarnitines in older patients with GVHD. Further studies would be required to assess the use of these biomarkers in disease prognosis and therapy. Comments for improvement are listed below: Comments: 1. Please provide statistics within each panel of every figure. Response: Thank you for your suggestion. We have added information requested to the figures. Figures 1-3 have been revised. Some additional statistical data has also been added to the figure legends and the main text in the revised manuscript. 2. Supplemental data are still not adequately called out within the text. Response: Information has been added to the manuscript (Lanes 198 and 281) Reviewer #2: The authors addressed all issues and the manuscript is appropriate for publication. The only correction that is needed in the new version is the deletion of the phrase in lines 350-351 because it is a repetition of the next phrase. Response: The phrase has been deleted / revised Attachments Attachment Submitted filename: Response to Reviewers_ Reihani_Sung et al 04-11-22.docx https://doi.org/10.1371/journal.pone.0268963.r004
12 May 2022 Decision Letter - Francesco Bertolini, Editor Evaluating immune response and metabolic related biomarkers pre-allogenic hematopoietic stem cell transplant in acute myeloid leukemia PONE-D-21-39481R2 Dear Dr. Sung, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Francesco Bertolini, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): 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 ******** 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: Yes 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: No ****** 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: The authors have adequately addressed my concerns, specifically regarding statistical analysis and references the supplemental materials throughout the text. Reviewer #2: (No Response) ****** 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 https://doi.org/10.1371/journal.pone.0268963.r005
Formally Accepted
1 Jun 2022 Acceptance Letter - Francesco Bertolini, Editor PONE-D-21-39481R2 Evaluating immune response and metabolic related biomarkers pre-allogenic hematopoietic stem cell transplant in acute myeloid leukemia Dear Dr. Sung: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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. If we can help with anything else, please email us at plosone@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. Francesco Bertolini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0268963.r006

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