Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas

Michael E. Berens; Anup Sood; Jill S. Barnholtz-Sloan; John F. Graf; Sanghee Cho; Seungchan Kim; Jeffrey Kiefer; Sara A. Byron; Rebecca F. Halperin; Sara Nasser; Jonathan Adkins; Lori Cuyugan; Karen Devine; Quinn Ostrom; Marta Couce; Leo Wolansky; Elizabeth McDonough; Shannon Schyberg; Sean Dinn; Andrew E. Sloan; Michael Prados; Joanna J. Phillips; Sarah J. Nelson; Winnie S. Liang; Yousef Al-Kofahi; Mirabela Rusu; Maria I. Zavodszky; Fiona Ginty

doi:10.1371/journal.pone.0219724

Peer Review History

Original SubmissionJune 27, 2019
20 Aug 2019 Decision Letter - Joseph Najbauer, Editor Bélatelep, Hungary August 19, 2019 PONE-D-19-18177 Tumor cell phenotype and heterogeneity differences in IDH1 mutant vs wild-type gliomas PLOS ONE Dear Dr. Berens, 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 by the Reviewer, listed below. We would appreciate receiving your revised manuscript by Oct 04 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 include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: 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 ****** 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: Based on my review, these results have not been published elsewhere (except Biorxiv preprint). In this study, the authors have used multiple platforms to investigate phenotype and heterogeneity differences in IDH1 mutant vs wild-type gliomas. Some interesting findings are presented and the study represents an essential contribution to the field. The approach for developing multiscale and multi parametric data to study differences in glioma subtypes and the way to assemble the analysis is the convincing and novel part of the study. The manuscript contains large number of figures and tables demanding a more thorough presentation and conformity. This will make it easier to keep a track on the story. There are some gaps in the development of the arguments and some of the details/discussion/explanations were difficult to find. Following are the points to work on and need to be answered. 1) Experimental statistics are not performed to a technical standard and are not described in sufficient detail. There is not much information about the statistical analysis in the whole manuscript and the figures legends do not have any details about the statistics performed such as tests, p value (not everywhere), significance and the correction (if used). (for example figure 1, 6 and S6A and S11). The author should provide a section in materials and method for detailed statistical analysis and software used. 2) The authors should provide the gene/protein name of the lollipop bars in Figure 3B and 3E (where the other genes are unchanged or slightly changed) for transparency. 3) In the Figure 5A and B, where author is comparing the cell dive biomarker intensity with bulk mRNA expression, there is high contradiction in the FASN and GSK3b expression. The author should also explain or discuss about the difference in expression values coming from cell dive and mRNA seq data for FASN and GSK3B. Western blot should be performed to verify the difference in the expression level of FASN and GSK3B in IDHwt and IDHmt tumors. 4) Figure 3A(h) where FASN overlaid on DAPI, DAPI staining does not look convincing (surprisingly, not all the cells were positive for DAPI in 3A(h) while in figure 3D (h) the DAPI staining looks fine). Any explanation? 5) (in the provided excel sheet) It is mentioned that false staining means staining failed or not observed in any of the cores and this were excluded from the analysis. There is a possibility that particular proteins are expressed in that tissue type. Could this be a source of bias? Did authors use positive controls tissue sections in these situations and were lack of staining with antibodies and presence of protein validated? 6) The mRNA seq data analysis could give many other interesting findings. The authors should also show the expression of few genes for CD8, NK cells, GD T cells, microglia/macrophages and MDSC cells in IDHmt and IDHwt, as they are important shares of the brain tumor microenvironment and contribute up to 50% of the total population. 7) The title of the paper should be changed and should mention the use of multiscale workflow/technology in the study. Especially, because major part of this study is describing the method and new way of analysis. 8) The abstract could be more informative on the specific findings of the study. 9) On the line 689 it says weather, it should be whether. ****** 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 [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. Attachments Attachment Submitted filename: PLOS ONE comments.docx https://doi.org/10.1371/journal.pone.0219724.r001
Revision 1
23 Oct 2019 Author Response The critiques of the prior submission, as well as the authors’ responses/edits are noted below. 1) Experimental statistics are not performed to a technical standard and are not described in sufficient detail. There is not much information about the statistical analysis in the whole manuscript and the figures legends do not have any details about the statistics performed such as tests, p value (not everywhere), significance and the correction (if used). (for example figure 1, 6 and S6A and S11). The author should provide a section in materials and method for detailed statistical analysis and software used. >>> Yes, this was an oversight on our part. A new section on statistical analysis methods has been added at the end of the methods section. Here we have included the statistical methods and cited R software for biomarker, cluster, MR feature and heterogeneity comparison between IDHmt vs wt patients and survival analysis. Extensive detail was already provided on the k-means clustering and spatial analysis methods. 2) The authors should provide the gene/protein name of the lollipop bars in Figure 3B and 3E (where the other genes are unchanged or slightly changed) for transparency. >> >Figure 3 has now been updated with all the protein names 3) In the Figure 5A and B, where author is comparing the cell dive biomarker intensity with bulk mRNA expression, there is high contradiction in the FASN and GSK3b expression. The author should also explain or discuss about the difference in expression values coming from cell dive and mRNA seq data for FASN and GSK3B. Western blot should be performed to verify the difference in the expression level of FASN and GSK3B in IDHwt and IDHmt tumors. >>> Despite the differences for FASN, GSK3b and NCad, the concordance between the two technologies is notable for most other markers, particularly given the differences in sample amount and preservation (fixed vs frozen). Otherwise, directional discordance between mRNA and protein for any given gene is not an unanticipated finding, and on an individual gene level, is more discordant in tumor versus cognate normal tissue (Idit Kosti, Nishant Jain, Dvir Aran, Atul J. Butte & Marina Sirota. Cross-tissue Analysis of Gene and Protein Expression in Normal and Cancer Tissues. Scientific Reports volume 6, Article number: 24799 (2016). Underlying reasons for this discordance include variant turnover rate of the protein versus mRNA, translational efficiency due to ribosomal density and occupancy, RNA secondary structure, among others (Correlation of mRNA and Protein in complex biological samples. 2009). Unfortunately it is not possible to conduct additional western analysis due to sample depletion. These points have also now been noted in the revised paper. 4) Figure 3A(h) where FASN overlaid on DAPI, DAPI staining does not look convincing (surprisingly, not all the cells were positive for DAPI in 3A(h) while in figure 3D (h) the DAPI staining looks fine). Any explanation? >>>Since all images in the same group are set to same window/level in ImageJ, DAPI staining in 3A(h) is not readily apparent, however, the relative DAPI intensities between two images are different. Since these are two different patients the differences may be due to fixation conditions or age of the sample. 5) (in the provided excel sheet) It is mentioned that false staining means staining failed or not observed in any of the cores and this were excluded from the analysis. There is a possibility that particular proteins are expressed in that tissue type. Could this be a source of bias? Did authors use positive controls tissue sections in these situations and were lack of staining with antibodies and presence of protein validated? >>>Yes, a control TMA containing both positive and negative controls used for antibody validations were included in the multiplexing study to confirm antibody performance (described in the methods section, lines 197 and 201). Additionally, “Control cores were included on all slides for glioma, prostate, melanoma, lung, breast cancer (two per cancer type) to verify antibody performance.” (line 183-184). Her2 was not expected to stain positive, nor cytokeratin and many of the other markers with negative staining were immune related, suggesting perhaps an immunosuppressive environment. 6) The mRNA seq data analysis could give many other interesting findings. The authors should also show the expression of few genes for CD8, NK cells, GD T cells, microglia/macrophages and MDSC cells in IDHmt and IDHwt, as they are important shares of the brain tumor microenvironment and contribute up to 50% of the total population. >>> We agree the RNA Seq data could provide other findings. Our focus in this paper was to investigate the RNA and protein expression profiles and metrics at a pathway/hallmark level and compare those with MR features. However, immune evasion hallmark was not found to be a significantly correlated with MR features for either this cohort, nor in an earlier TCGA analysis of RNA and MR data from the Cancer Imaging Archive. Data will be made available for further analysis. 7) The title of the paper should be changed and should mention the use of multiscale workflow/technology in the study. Especially, because major part of this study is describing the method and new way of analysis. >>> Appreciate the helpful suggestion, we have changed the title to: Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas 8) The abstract could be more informative on the specific findings of the study. >>> The information content of the abstract now contains additional specific findings of the study 9) On the line 689 it says weather, it should be whether. >> Changed Attachments Attachment Submitted filename: Response to Critique.docx https://doi.org/10.1371/journal.pone.0219724.r002
13 Nov 2019 Decision Letter - Joseph Najbauer, Editor Pécs, Hungary November 12, 2019 Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas PONE-D-19-18177R1 Dear Dr. Berens, We are pleased to inform you that your manuscript (R1 version) 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, Joseph Najbauer, Ph.D. Academic Editor PLOS ONE --------------------------------------------------------------------------------------------------- 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy 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 ****** 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 ****** 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: (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 https://doi.org/10.1371/journal.pone.0219724.r003
Formally Accepted
12 Dec 2019 Acceptance Letter - Joseph Najbauer, Editor PONE-D-19-18177R1 Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas Dear Dr. Berens: 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 Dr. Joseph Najbauer Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0219724.r004

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