Metabolic analysis of amino acids and vitamin B6 pathways in lymphoma survivors with cancer related chronic fatigue

Alexander Fosså; Knut Halvor Smeland; Øystein Fluge; Karl Johan Tronstad; Jon Håvard Loge; Øivind Midttun; Per Magne Ueland; Cecilie Essholt Kiserud

doi:10.1371/journal.pone.0227384

Peer Review History

Original SubmissionJuly 28, 2019
30 Oct 2019 Decision Letter - Gilles J. Guillemin, Editor PONE-D-19-21227 Metabolic analysis of amino acids and vitamin B6 pathways in lymphoma survivors with cancer related chronic fatigue PLOS ONE Dear Dr Fossa, 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. We would appreciate receiving your revised manuscript by Dec 14 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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? 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: No ****** 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: No ****** 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: This is a well written research article with a clear experimental approach and well-presented results. The research area is indeed an interesting area with minimal data available. Here are two comments/questions for the authors: 1. As IFN-y and TNF-alpha are shown to be potent inducers of the pathway, do the authors have data on these cytokines in patient’s blood? It will definitely be interesting to see if there is any correlation between these cytokines and activation of this pathway. 2. Does the author have data on the melatonin/serotonin pathway? This is so as tryptophan is the substrate for both the melatonin and kynurenine pathway. Reviewer #2: In this paper, the authors report decreased levels of circulating tryptophan in chronically fatigued versus non fatigued cancer patients, associated with changes in metabolites of B6 and trending changes in neopterin. Despite its cross-sectional nature the study is a priori interesting as it includes many more patients than what is usually the case in clinical studies of cancer-related fatigue. Unfortunately the study does not hold to its premises because of a number of major weaknesses. Although the reported study is presented as exploratory it fails to adjust for multiplicity to account for the multiple statistical tests carried out in the same patients, which therefore gives limited credibility to the results that are reported. This is made even more problematic by the lack of any post hoc power analysis. Concerning the significance of the observed changes, there are several issues that are not considered including nutritional status and possibility of activation of TDO rather than IDO to account for the observed differences in TRP and KYN. Note that figures 1 to 3 show the existence of outliers that probably drive the differences between fatigued and non-fatigued individuals. Apparently the authors did not attempt to assess whether their results remain identical if the outliers are excluded. In discussion of the trending results the authors omit to consider the possibility of reverse causality, i.e., "stress"-related fatigue causing low grade inflammation (or HPA axis activation) which itself would be responsible for activation of the kynurenine pathway. ****** 6. 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0227384.r001
Revision 1
10 Dec 2019 Author Response Response to review comments to the author Reviewer #1: This is a well written research article with a clear experimental approach and well-presented results. The research area is indeed an interesting area with minimal data available. Response: Thank you. We appreciate the recognition of our work. Here are two comments/questions for the authors: 1. As IFN-y and TNF-alpha are shown to be potent inducers of the pathway, do the authors have data on these cytokines in patient’s blood? It will definitely be interesting to see if there is any correlation between these cytokines and activation of this pathway. Response: We have analyzed TNF alfa, IL-1RA and IL-1Beta in this cohort and reported that previously (Smeland KB et al, 2019, reference 22). Neither TNF alpha nor IL-1 Beta are correlated with the presence of CF, and these cytokines are not considered in this paper. Only IL-1R alpha is correlated with fatigue. Both PAr index, Kyn/Trp ratio and neopterin are significantly correlated with the level of IL-1RA in this cohort. We have added these data in Table 1 and a separate paragraph in the result section. 2. Does the author have data on the melatonin/serotonin pathway? This is so as tryptophan is the substrate for both the melatonin and kynurenine pathway. Response: Unfortunately, we don’t have these data. As for IFN-γ, we agree that this would be interesting. Reviewer #2: In this paper, the authors report decreased levels of circulating tryptophan in chronically fatigued versus non fatigued cancer patients, associated with changes in metabolites of B6 and trending changes in neopterin. Despite its cross-sectional nature the study is a priori interesting as it includes many more patients than what is usually the case in clinical studies of cancer-related fatigue. Unfortunately the study does not hold to its premises because of a number of major weaknesses. 1. Although the reported study is presented as exploratory it fails to adjust for multiplicity to account for the multiple statistical tests carried out in the same patients, which therefore gives limited credibility to the results that are reported. This is made even more problematic by the lack of any post hoc power analysis. Response: In deed, we view our analyses as exploratory and in need of validation it other cohorts of cancer survivors with CF. This is clearly stated in the manuscript. The design of the study was to test whether we could find similar changes in metabolites in CF as in CFS/ME patients previously reported by our group. We did not find these patterns of amino acid changes, despite a reasonably large number of patients with or without CF. As such; the study is negative and clearly stated as such in results and discussion. These differences to CFS/ME are however in themselves important as they may point to different disease mechanisms. The only finding that is confirmed when comparing across studies CFS/ME patients and cancer survivors with CF are the lower values for tryptophan. The analyses of the kynurenine pathway and the vitamin B6 metabolites are conducted in an effort to explain these differences, i.e. they are explanatory. As they all point in similar directions, i.e. implicate low grade inflammation in the pathogenesis of CF, we believe the data deserve to be presented at such awaiting formal validation in the future. When it comes to post hoc power analyses, we believe that stating mean differences, standard deviations and p-values of appropriate (parametric or non-parametric tests) allow for assessment of possible type II errors, that is the risk that there are differences that would be statically significant in larger data sets. Post-hoc power analyses are in our view alternative ways to present the same information. For a broader discussion of the value of post-hoc power analyses our statistician has referred to the following web-site which we as clinician-scientists found useful to remind us of this fact. http://daniellakens.blogspot.com/2014/12/observed-power-and-what-to-do-if-your.html 2. Concerning the significance of the observed changes, there are several issues that are not considered including nutritional status and possibility of activation of TDO rather than IDO to account for the observed differences in TRP and KYN. Response: We have done a number of analyses to look at nutritional status and vitamin supplements and find that these do not influence the results. This is clearly stated in the manuscript as it is. The origin of the proposed increased metabolism though this pathway is not known. It could be IDO or TDO, a hepatic enzyme differently regulated than IDO 1 and 2. We have added sentences in the discussion to discussion to incorporate the role of TDO in view of recent findings and clarifying reviews by experts in the field. 3. Note that figures 1 to 3 show the existence of outliers that probably drive the differences between fatigued and non-fatigued individuals. Apparently the authors did not attempt to assess whether their results remain identical if the outliers are excluded. Response: We do not fully agree that there are obvious outliers in most of the analyses done. We have however repeated the analyses presented for Trp, alpha-ketoglutarate, Kyn/Trp ratio, Neopterin and PAr index related to presence of absence of CF, and related to measurable IL-6 levels after excluding potential outliers by the interquartile range rule. This excludes up to 2 % of the patients from the analyses, depending on which univariate test we are looking at. The results remain stable, that is Trp is significantly reduced and both alpha ketoglutarate and PAr index both significantly increased in patients with CF compared to non-fatigued survivors. For neopterin and Kyn/Trp ration the values remain borderline with p-values of 0.06-0.12. The association with measurable IL-6 as presented in Figure 4 remains highly significant for all comparisons despite omitting the most extreme values at both ends. These data are not presented. 4. In discussion of the trending results the authors omit to consider the possibility of reverse causality, i.e., "stress"-related fatigue causing low grade inflammation (or HPA axis activation) which itself would be responsible for activation of the kynurenine pathway. Response: We agree that this could be the case and we have added a paragraph about reverse causality in the discussion, also including a potential role of cortisol induced TDO activity. Kind regards, Alexander Fosså Attachments Attachment Submitted filename: Response to reviewers PLOS one.docx https://doi.org/10.1371/journal.pone.0227384.r002
18 Dec 2019 Decision Letter - Gilles J. Guillemin, Editor Metabolic analysis of amino acids and vitamin B6 pathways in lymphoma survivors with cancer related chronic fatigue PONE-D-19-21227R1 Dear Dr. Fossa, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Gilles J. Guillemin Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0227384.r003
Formally Accepted
26 Dec 2019 Acceptance Letter - Gilles J. Guillemin, Editor PONE-D-19-21227R1 Metabolic analysis of amino acids and vitamin B6 pathways in lymphoma survivors with cancer related chronic fatigue Dear Dr. Fosså: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Gilles J. Guillemin Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227384.r004

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