In silico analysis identifies a putative cell-of-origin for BRAF fusion-positive cerebellar pilocytic astrocytoma

Subhi Talal Younes; Betty Herrington

doi:10.1371/journal.pone.0242521

Peer Review History

Original SubmissionJune 6, 2020
29 Jul 2020 Decision Letter - Marta M. Alonso, Editor PONE-D-20-17252 In Silico Analysis Identifies a Putative Cell-of-Origin for BRAF Fusion-Positive Cerebellar Pilocytic Astrocytoma PLOS ONE Dear Mr. Younes, 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. After careful revision of your manuscript although very interesting several issues arise that should be address; First please take into account the comments of the reviewer. SEcond the paper could use further review by a biostatistician, as the methodology used is complex. I would also advice you to ask a further review by a senior colleague that could give further input. This is a recommendation not a must for the revision Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact. ============================== Please submit your revised manuscript by Sep 12 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. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Marta M. Alonso, PhD Academic Editor PLOS ONE 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 and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 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 ****** 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: In this study, Younes investigates the cell or origin for pediatric cerebellar juvenile pilocytic astrocytoma through an in silico approach. This is an ambitious study and carries relevance for the study of pediatric low-grade glioma. However, the study and especially the figures are often confusing and challenging to interpret. The lack of in vitro or in vivo validation of the findings is also a major issue. Additional issues are raised below. Introduction: - Line 73 would say radiation or chemotherapy, since both would rarely be necessary in these tumors. - Would add a section on the known molecular alterations common to cerebellar pilocytic astrocytomas Methods: - Line 87, translocation -- does this refer to BRAF fusions? - Need to define PA-DR at first mention in this section, not in the middle of Results - There needs to be more justification of the use of a mouse atlas. Results - Line 162: Again, would change to BRAF fusion (throughout manuscript) - The legend for Figure 2 is unclear -- what do the yellow dots represent? - Figure 3 is also unclear -- color gradients need to be better defined, and images need to be labeled; screen captures from analysis programs generally are not good manuscript figure panels without editing to clarify and remove some unnecessary portions (e.g. a "Refresh" button) - Wouldn't the lack of transcriptional upregulation of MAPK markers within the developmental cerebellum be because the fusion protein deregulating the pathway in tumors is not present? - Again, Figure 5 is not clear and needs to be better explained in both the figure (via labels) and legend. - These findings could likely be validated on a protein level through IHC/IF in human tumor samples, which would substantially strengthen the conclusions. Discussion - Figure 6: While usually figures should be associated with Results, this may be acceptable if allowed by editors; figure again needs to be better explained, however -- what do the colored arrows represent? - More discussion is needed regarding the use of investigating markers from human tumor samples in a mouse brain atlas and then revalidating with a human atlas -- why not just use the human brain atlas throughout? This may be justifiable in terms of these developmental atlases not being available for humans, but this merits discussion. ****** 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.] 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.0242521.r001
Revision 1
21 Oct 2020 Author Response Response to Reviewers We would like to thank the reviewer and the editor for their thoughtful review and suggestions for improvement. In response, the manuscript has been extensively revised to incorporate the aforementioned reviewer and editor comments. Editor Comments Comment: The paper could use further review by a biostatistician, as the methodology used is complex. Response: The methodology has been reviewed by Dr. Kurt Showmaker, a bioinformatician with expertise in differential gene expression analyses. His comments have been used to revise the description of the methodology used. Comment: I would also advise you to ask a further review by a senior colleague that could give further input. Response: Dr. Betty Herrington, a pediatric neuro-oncologist, has carefully reviewed the manuscript and analyses. Based on her contributions, she has been added as an author on the manuscript. Reviewer Comments Comment: In this study, Younes investigates the cell or origin for pediatric cerebellar juvenile pilocytic astrocytoma through an in silico approach. This is an ambitious study and carries relevance for the study of pediatric low-grade glioma. However, the study and especially the figures are often confusing and challenging to interpret. The lack of in vitro or in vivo validation of the findings is also a major issue. Response: The figures and accompanying text have been extensively revised in order to make their interpretation clearer. In particular, the figure legends have been expanded to more carefully walk the reader through the data presented. Regarding the lack of in vitro or in vivo validation, the discussion has been updated to address these potential shortcomings. Most notably, potential avenues of in vivo validation have been proposed. Comment: Line 37 should say radiation or chemotherapy, since both would rarely be necessary in these tumors. Response: The line in question has been updated to correctly state that radiation or chemotherapy would be used for the treatment of pilocytic astrocytoma. Comment: Would add a section on the known molecular alterations common to cerebellar pilocytic astrocytoma. Response: A paragraph commenting on the known molecular alterations in cerebellar pilocytic astrocytoma has been added to the introduction. Comment: Line 87, “translocation” – does this refer to BRAF fusions? Response: This line did indeed seek to refer to BRAF fusion. The entire manuscript has been updated to properly state, “BRAF fusion.” Comment: Need to define “PA-DR” at first mention in this section, not in the middle of Results. Response: A line defining the designation of PA-DR has been added to the methods section Comment: There needs to be more justification of the use of a mouse atlas. Response: Several lines have been added explaining why we chose to utilize mouse atlases first. Briefly, as the reviewer suggests in a later comment, gene expression atlases of the developing human brain are sparse. On the other hand, mouse developmental atlases are more readily available with more robust data. Additionally, a section has been added to the discussion to more fully justify the use of a mouse gene expression atlas. Comment: Line 162: Again, would change to BRAF fusion (throughout manuscript) Response: All references throughout the manuscript have been changed to read, “BRAF fusion.” Comment: The legend for Figure 2 is unclear – what do the yellow dots represent? Response: The legend for Figure 2 has been revised to explain what is depicted. Comment: Figure 3 is also unclear – color gradients need to be better defined, and images need to be labeled; screen captures from analysis programs generally are not good manuscript figure panels without editing to clarify and remove some unnecessary portions (e.g. a “refresh” button). Response: Figure 3 has been extensively revised. The legend has been expanded and the figures themselves have been annotated to better direct the reader to the key data points. Comment: Wouldn’t the lack of transcriptional upregulation of MAPK markers within the developmental cerebellum be because the fusion protein deregulating the pathway in tumors is not present? Response: The results section in question has been revised to address a comment by the editor. Namely, the editor requested that the comment, “data not shown,” be removed from the manuscript. With the removal of that sentence, the reviewer’s comment has been relieved. Comment: Again, figure 5 is not clear and needs to be better explained in both the figure (via labels) and legend. Response: Figure 5 has been extensively revised and annotated to better convey the key data points. Comment: These findings could likely be validated on a protein level through IHC/IF in human tumor samples, which would substantially strengthen the conclusions. Response: A review of the literature was performed. Protein-level expression data could only be identified for a single PA-DR gene, namely, Pax3, confirming that it is overexpressed in cerebellar pilocytic astrocytoma. Unfortunately, a lack of funding precludes the ability to interrogate other markers. Comment: Figure 6: While usually figures should be associated with Results, this may be acceptable if allowed by editors; figure again needs to be better explained, however -- what do the colored arrows represent? Response: The figure legend has been revised. Comment: More discussion is needed regarding the use of investigating markers from human tumor samples in a mouse brain atlas and then revalidating with a human atlas -- why not just use the human brain atlas throughout? This may be justifiable in terms of these developmental atlases not being available for humans, but this merits discussion. Response: The discussion has been revised to more clearly state our rationale for having used mice atlases. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0242521.r002
3 Nov 2020 Decision Letter - Marta M. Alonso, Editor PONE-D-20-17252R1 In Silico Analysis Identifies a Putative Cell-of-Origin for BRAF Fusion-Positive Cerebellar Pilocytic Astrocytoma PLOS ONE Dear Dr. Younes, 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, address the following minor concerns: Improve the color keys to the color gradients in panels 3C, 3D, and 5A on the figures themselves to assist readers in interpreting these figures; 3C-D could likely also use text and/or arrows on the panels themselves to help with interpretation Please submit your revised manuscript by Dec 18 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. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Marta M. Alonso, PhD Academic Editor PLOS ONE [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: (No Response) ******** 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: The authors have done well in revising the manuscript and have addressed the majority of my concerns. While the figure captions are improved, I would still like to see better keys to the color gradients in panels 3C, 3D, and 5A on the figures themselves to assist readers in interpreting these figures; 3C-D could likely also use text and/or arrows on the panels themselves to help with interpretation. ****** 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 [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.0242521.r003
Revision 2
3 Nov 2020 Author Response Response to Reviewers We would like to thank the reviewer for their thoughtful review and suggestions for improvement. In response, the manuscript has been revised to incorporate the aforementioned reviewer comments. Reviewer Comments Comment: The authors have done well in revising the manuscript and have addressed the majority of my concerns. While the figure captions are improved, I would still like to see better keys to the color gradients in panels 3C, 3D, and 5A on the figures themselves to assist readers in interpreting these figures; 3C-D could likely also use text and/or arrows on the panels themselves to help with interpretation. Response: Figures 3C, 3D, and 5A have each been updated to include a color gradient key. In addition, arrows and text descriptions have been added to figures 3C and 3D to aid the reader in interpreting which direction represents which cell lineage. Attachments Attachment Submitted filename: Response to Reviewers R2.docx https://doi.org/10.1371/journal.pone.0242521.r004
4 Nov 2020 Decision Letter - Marta M. Alonso, Editor In Silico Analysis Identifies a Putative Cell-of-Origin for BRAF Fusion-Positive Cerebellar Pilocytic Astrocytoma PONE-D-20-17252R2 Dear Dr Younes, 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, Marta M. Alonso, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0242521.r005
Formally Accepted
6 Nov 2020 Acceptance Letter - Marta M. Alonso, Editor PONE-D-20-17252R2 *In Silico* Analysis Identifies a Putative Cell-of-Origin for BRAF Fusion-Positive Cerebellar Pilocytic Astrocytoma** Dear Dr. Younes: 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. Marta M. Alonso Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0242521.r006

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