Metabolomics of primary cutaneous melanoma and matched adjacent extratumoral microenvironment

Nicholas J. Taylor; Irina Gaynanova; Steven A. Eschrich; Eric A. Welsh; Timothy J. Garrett; Chris Beecher; Ritin Sharma; John M. Koomen; Keiran S. M. Smalley; Jane L. Messina; Peter A. Kanetsky

doi:10.1371/journal.pone.0240849

Peer Review History

Original SubmissionApril 6, 2020
15 Jun 2020 Decision Letter - Ch Ratnasekhar, Editor PONE-D-20-09783 Metabolomics of primary cutaneous melanoma and matched adjacent extratumoral microenvironment PLOS ONE Dear Dr. Nicholas Taylor, 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. Please submit your revised manuscript by Jul 30 2020 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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Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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: Yes Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 Reviewer #3: 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 Reviewer #3: No ****** 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: Metabolomics-based strategies have become an integral part of present clinical research studies and has been instrumental in cancer precision medicine. This manuscript entitling ‘‘Metabolomics of primary cutaneous melanoma and matched adjacent extratumoral microenvironment’’ by Taylor et al, is an interesting study and authors have collected a unique dataset using an ultra-high-performance liquid chromatography. Overall, this manuscript is well written. However, in my opinion the article has some minor concerns regarding data analyses, and I feel this dataset has not been utilized to its full extent. Furthermore, I would prefer more in-depth functional analyses of the data. 1. Authors did not show any functional analysis of the data. For this, authors can utilize PROFILE clustering to order the metabolites based on their mutual proximity in the metabolic network. This analysis will provide useful insights in the interpretation of metabolomic changes in primary and metastatic melanoma and EM. 2. Authors should create a metabolic map based on the differential expression of metabolites in the primary tumor and EM by using a molecular interaction software like Cytoscape. This metabolic map can be used for predicting predominant metabolic processes in melanoma and will help in building strategies for therapeutic targeting of metabolic pathways in cancer cells. Reviewer #2: The current manuscript describing the LC/MS base metabolomics analysis of melanoma and its matched adjacent extratumoral microenvironment tackles an especially interesting subject. The work done is substantial well designed and executed whereas the statistical treatment of the results was in my opinion accurate and also well designed. The IROA technology used has also led to increased confidence concerning the identification of features. Finally, the authors explained their findings convincingly, rendering the work o significant merit for the readers. A couple of minor points should be taken care of. line 157 please specify if the external calibration refers to mass line 174 using pooled QC samples is a frequently used procedure in metabolomics analyses. Did the authors consider to employ this methodology in order to control the intra batch variation as well? Please comment Table 1 Does the second digit in retention time hold a meaning? Please comment Please also add the statistics of multivariate analyses i.e. at least the R2 and Q2, to indicate their statistical significance. Reviewer #3: Comments Article no. Ms. Ref. No. PONE-D-20-09783, Research Article entitled ‘Metabolomics of primary cutaneous melanoma and matched adjacent extratumoral microenvironment’. In the present study, authors have analysed the metabolomics of primary cutaneous melanoma and matched adjacent extratumoral microenvironment using ultra-high-performance liquid chromatography-mass spectrometry. There are few novel findings and may also hold promising translational relevance to develop the novel biomarker for melanoma patients. However, I do notice many important concerns that should be addressed to further enhance the manuscript quality: Comment 1: First of all, authors need to provide high resolution images for all figures as it’s very hard to figure out anything out of any heatmaps. Comment 2: Authors really need to explain their results in different subheadings and need to cite figure numbers in text rather than in headings. Comment 3: It’s better to provide the box plot or bar diagram of potential differentially altered metabolites with their respective p-values in addition to heatmap. Comment 4: Authors need to analyse and correlate the altered level of their identified metabolites with other pathophysiological characteristics such as TNM staging and survival if possible. Comment 5: Authors need to provide demographic and clinical-pathological details of all the clinical bio specimens collected in their study. ****** 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: Yes: Manish Charan Reviewer #2: No Reviewer #3: 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. Attachments Attachment Submitted filename: plosone.docx https://doi.org/10.1371/journal.pone.0240849.r001
Revision 1
14 Sep 2020 Author Response Reviewer #1 1) “Authors did not show any functional analysis of the data. For this, authors can utilize PROFILE clustering to order the metabolites based on their mutual proximity in the metabolic network. This analysis will provide useful insights in the interpretation of metabolomic changes in primary and metastatic melanoma and EM.” “Authors should create a metabolic map based on the differential expression of metabolites in the primary tumor and EM by using a molecular interaction software like Cytoscape. This metabolic map can be used for predicting predominant metabolic processes in melanoma and will help in building strategies for therapeutic targeting of metabolic pathways in cancer cells.” We have given these suggestions careful consideration. Our original functional analysis was a pathway-based approach via Metaboanalyst and Mummichog focused on mapping compounds to known KEGG pathways in human beings. Our intention was, and remains, to highlight potentially meaningful differences in biological pathways between tissue types. We retain this approach as our primary analysis in our revised manuscript. However, in order to fully address the Reviewer’s comments, and to further explore the functional characteristics of the metabolite differences between tissue types, we performed a metabolite set enrichment analysis (MSEA) within Metaboanalyst 4.0 as a secondary analysis. Again, we considered metabolites with statistically significantly higher abundance (or lower abundance) in primary melanoma (or metastatic melanoma) compared to EM. For each condition, the MSEA of pathway-associated metabolite sets—based on the small molecule pathway database (SMPDB—smpdb.ca)—was considered. A network of metabolite-metabolite interactions was plotted for each condition, with nodes representing metabolites and edges indicating a relationship (from STITCH—an interaction network database for small molecules and proteins) between metabolites. STITCH incorporates several databases (including KEGG) when producing the “interactome” of metabolites (e.g. Protein Data Bank, Reactome, NCI-Nature Pathway Interaction Database, DrugBank, and MATADOR) We highlighted specific KEGG pathways within the network for comparison to our main analysis. We have made appropriate additions to the Methods and Results sections describing these secondary analyses, and we have highlighted the relevant text in the revised manuscript. We also present the results of these exploratory analyses in supplemental material (Supplemental Figures 8-11), and add some discussion as highlighted in the manuscript. Reviewer #2 1) “line 157 please specify if the external calibration refers to mass.” External calibration refers to both mass calibration and mass resolution calibration; we have added this clarification to the methods section of the revised manuscript. 2) “line 174 using pooled QC samples is a frequently used procedure in metabolomics analyses. Did the authors consider to employ this methodology in order to control the intra batch variation as well? Please comment.” We did not use pooled QC samples in this study. The melanoma tissue samples are a scarce resource (especially the primary melanoma samples) and were generally low in mass; the decision was made to preserve tissue for multiple omics studies and other assays rather than using a pool for batch-to-batch quality control. We used a pooled red cross plasma sample to assure instrument performance in both retention time and mass accuracy. Our process of detecting and mitigating inter-batch bias is presented in detail in the manuscript from lines 175-182, as well as Figure 1. 3) “Table 1 Does the second digit in retention time hold a meaning? Please comment.” We thank the reviewer for pointing out this oversight. We have removed the unnecessary digits from all reports of retention times in the manuscript. 4) “Please also add the statistics of multivariate analyses i.e. at least the R2 and Q2, to indicate their statistical significance.” The Authors are confused by the Reviewer’s comment; we did not perform multivariate analyses of these high dimensional data. Reviewer #3 1) “First of all, authors need to provide high resolution images for all figures as it’s very hard to figure out anything out of any heatmaps.” We previously (and again) provided the journal with high resolution images conforming to PLoS ONE guidelines and requirements. Please note that images/figures contained in the manuscript pdf file appear in lower resolution; however, the url links contained within the pdf file allow for images/figures to be viewed externally at full resolution. 2) “Authors really need to explain their results in different subheadings and need to cite figure numbers in text rather than in headings.” We have added subheadings to the results section per the Reviewer’s suggestion. The formatting of figure citations in the text of the manuscript was made to adhere to PLoS ONE requirements. 3) “It’s better to provide the box plot or bar diagram of potential differentially altered metabolites with their respective p-values in addition to heatmap.” We now provide bar diagrams illustrating a) significantly differentially abundant metabolites between primary melanoma and paired EM tissues which we highlight as being identified and named in both positive and negative ion modes, as well as two identified and named metabolites that are differentially abundant between primary melanoma and paired EM in separate ion modes (Supplemental Figure 3)—from results section lines 237-239; and, b) all significantly differentially abundant metabolites between primary melanoma and paired EM (Supplemental Figure 4). Further, we now include box plots illustrating differentially abundant metabolites between EM and unmatched metastatic melanoma tissues. Supplemental Figure 5 illustrates results described in the Metastatic melanoma vs. EM section in lines 255-261; Supplemental Figure 6 illustrates all significantly differentially abundant metabolites between EM and unmatched metastatic melanoma. 4) “Authors need to analyse and correlate the altered level of their identified metabolites with other pathophysiological characteristics such as TNM staging and survival if possible.” We appreciate the Reviewer’s suggestion, and we have previously considered this approach. We decided against including results from such analyses in the manuscript for important reasons: 1) Due to the scarcity of primary melanoma tissue, our study sample essentially represents a convenience sample. This limits our ability to select optimal tissue from those patients who developed multiple primary melanomas; moreover, we were limited in obtaining data indicating whether or not a patient had multiple primary melanomas. This limitation hinders our ability to make reasonable inferences from survival analyses; 2) Our sample size is relatively small in comparison to the number of metabolites identified, which makes external validation highly variable and power low. Nevertheless, we convey results from 3 different analyses of the data with respect to survival below. 1) We performed a Cox proportional hazard model on the raw difference between tumor and EM (i.e. tumor minus EM) and the fold difference (i.e. tumor divided by EM) for each of the 824 statistically significant metabolites identified. The resulting p-values are all non-significant after adjustment for FDR, indicating they are either not important for survival (unlikely) or we have insufficient power to detect their effects (likely). 2) We binned 33 subjects into two clusters based on the profiles of the 824 statistically significant metabolites identified and compared the resulting Kaplan-Meier survival curves. There is no significant survival separation between the two clusters (again, likely due to insufficient power). 3) We performed a cross-validation with penalized Cox regression model on the 824 differentially abundant metabolites (meaning ridge regression so that all metabolites are used in the prediction). The 33 samples were split into 10 folds (100 splits total, each has 10 folds); the best model was fit on 9 folds and the risk evaluated on the remaining one. The measure of model prediction is Harrel’s concordance (https://statisticaloddsandends.wordpress.com/2019/10/26/what-is-harrells-c-index/), with values of 0.5 equating to a completely random guess and 1.0 being maximum prediction. Results from this approach were highly variable across 100 splits and range from 0.3 to 0.8. On average, the best model with 824 metabolites on EM tissue gave better prediction than the best model with 824 metabolites on tumor tissue (concordance means 0.58 vs. 0.52), though both means were similar and close to 0.5, indicating metabolite abundances in EM and primary tissues are not predictive of survival in our dataset (or likely we have insufficient power to detect their effects). Lastly, the Reviewer suggested analyses of associations between metabolites and pathophysiologic characteristics. Unfortunately, we were unable to reliably perform these analyses due to the limited data we had on these factors. 5) “Authors need to provide demographic and clinical-pathological details of all the clinical bio specimens collected in their study.” We have added a new Table 1, which now includes descriptive summaries of sex, vital status, age at diagnosis, histology, TNM staging, anatomic site of primary, and anatomic site of metastasis. Attachments Attachment Submitted filename: (Response to Reviewers)(9-7-20)(PLoS One).docx https://doi.org/10.1371/journal.pone.0240849.r002
5 Oct 2020 Decision Letter - Ch Ratnasekhar, Editor Metabolomics of primary cutaneous melanoma and matched adjacent extratumoral microenvironment PONE-D-20-09783R1 Dear Dr. Nicholas, 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, Ch Ratnasekhar, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0240849.r003
Formally Accepted
16 Oct 2020 Acceptance Letter - Ch Ratnasekhar, Editor PONE-D-20-09783R1 Metabolomics of primary cutaneous melanoma and matched adjacent extratumoral microenvironment Dear Dr. Taylor: 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. Ch Ratnasekhar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0240849.r004

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