CCNB2 and AURKA overexpression may cause atypical mitosis in Japanese cortisol-producing adrenocortical carcinoma with TP53 somatic variant

Akira Ikeya; Mitsuko Nakashima; Miho Yamashita; Keisuke Kakizawa; Yuta Okawa; Hirotomo Saitsu; Shigekazu Sasaki; Hironobu Sasano; Takafumi Suda; Yutaka Oki

doi:10.1371/journal.pone.0231665

Peer Review History

Original SubmissionJanuary 14, 2020
25 Feb 2020 Decision Letter - Sumitra Deb, Editor PONE-D-20-01265 CCNB2 and AURKA overexpression may cause atypical mitosis in Japanese cortisol-producing adrenocortical carcinoma with TP53 somatic variant PLOS ONE Dear Dr. Ikeya, 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 Apr 10 2020 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 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: 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 ****** 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: This is a nice study in which the authors have investigated molecular markers in adrenocortical markers that are of diagnostic and prognostic significance. Table-1, 2, 3: some columns are missing in the current version. Please upload again. Line 104: The authors should explain the rationale for interrogating only the selected genes in this study. Line 293-299: I did not find supporting evidence in literature to attribute pathogenicity to PRKAR1A(c.545C>A:p.T182K) variant. In silico tools in by themselves are not sufficient to attribute pathogenicity to a variant. The reasons listed by the authors to consider this variant as pathogenic are insufficient and in the absence of functional evidence, this variant would best be considered as a “variant of uncertain significance”. The reference to the variant in Clinvar is for PRKAR1A:c.545C>T and not c.545C>A as reported here. The authors come to similar conclusion in the discussion, and hence it is not reasonable to list it as a pathogenic variant. Also since ZNRF3 is an uncommon variant, the authors should consider discussing the clinical significance and role in pathogenicity in more detail. Line 331: atypical mitotic figures were positive in all of the cases with TP53 somatic variants and Ki-67 index ≥ 10%. I would need to see the complete Table-2 for verification of this. ****** 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. 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Revision 1
27 Mar 2020 Author Response Response to Reviewer #1: Comment: Q1. Table-1, 2, 3: some columns are missing in the current version. Please upload again. A1. I apologize for not being able to show the whole table in the original manuscript. In accordance with Reviewer #1 comment, the font size has been adjusted for Tables 1 and 3 so that all columns are visible in the revised manuscript. Table 2 has been changed to be shown as Figure 1 in the revised manuscript. Q2. Line 104: The authors should explain the rationale for interrogating only the selected genes in this study. A2. We sincerely accept comments made by Reviewer #1. In accordance with Reviewer #1 comment, we have added Line 106-119 in the revised manuscript explaining the reason why we selected the 12 genes analyzed as candidate genes this study. One of our objectives of this study was to investigate the factors for poor prognosis of adrenocortical carcinoma (ACC) and to consider the potential for treatment. First, we selected ten candidate genes of TP53-RB1 and Wnt pathways, to which TP53 and CTNNB1 belong. TP53 and CTNNB1 somatic variant was reported as a poor prognostic factor in the previous report. We speculated that somatic variants of genes involved in these two genes may also contribute to poor prognosis. Because these candidates ten genes variants were detailed in a previous paper, they were also suitable for comparative studies between Japanese and other races. We further selected PRKAR1A, since it is associated with cortisol production, which is a factor of poor prognosis. We also selected TERF2, which may be associated with disease progression due to shortened telomere length, among ACC driver genes. Q3. Line 293-299: I did not find supporting evidence in literature to attribute pathogenicity to PRKAR1A c.545C>A (p.T182K) variant. In silico tools in by themselves are not sufficient to attribute pathogenicity to a variant. The reasons listed by the authors to consider this variant as pathogenic are insufficient and in the absence of functional evidence, this variant would best be considered as a “variant of uncertain significance”. The reference to the variant in Clinvar is for PRKAR1A:c.545C>T and not c.545C>A as reported here. The authors come to similar conclusion in the discussion, and hence it is not reasonable to list it as a pathogenic variant. A3. We sincerely accept comments made by Reviewer #1.　As suggested, since PRKAR1A:c.545C>T and c.545C>A are not the same, the text describing references to variants of Clinvar has been deleted. Also, we have changed Table S5 because we agree it is correct to classify the PRKAR1A:c.545C>A (p.T182K) variant as "variant of uncertain significance". Regarding to the variant of the PRKAR1A:c.545C>A (p.T182K), it is a de novo somatic variant, a very rare variant with no registration such as gnomAD, and its pathogenic significance is predicted by in silico prediction. The pathogenicity of some missense variants of the cAMP binding domain A, such as c.547G>T (p.A183Y), c.638C>A (p.213D), and c.438A>T (p.R146S) have been demonstrated in vitro, as shown in the new reference 41 and 42. The in silico prediction of those variants, which was proven to be pathogenetic in vitro, were ”Probably damaging” or ”Benign”. The PRKAR1A:c.545C>A (p.T182K) variant is likely to affect cAMP binding domain A and which will affect the enzyme function of PRKAR1A through conformational changes. It is presumed that the change enhances PKA activity and affects tumor formations. The PRKAR1A gene is one of the ACC driver genes, and variants in this gene are presumed to be clinically significant, especially in ACC. Based on the above, we have identified the PRKAR1A:c.545C>A (p.T182K) variant in this study as a potential variant of carcinogenesis. We have added the above content to LINE 559-575 and references 41 and 42 as a new reference in the revised manuscript. Certainly, as pointed out by Reviewer #1, the effect on the actual carcinogenesis will need to be evaluated for the functional studies of the variant. We are aiming to demonstrate the actual functional evaluation of the PRKAR1A:c.545C>A (p.T182K) variant in the future. Q4. Also since ZNRF3 is an uncommon variant, the authors should consider discussing the clinical significance and role in pathogenicity in more detail. A4. In accordance with Reviewer #1 comment, we have added a detailed discussion of the clinical significance and role in the pathogenicity of ZNRF3: c.433C>T (p.R145X) variant at LINE 576-588 in the revised manuscript. The ZNRF3:c.433C>T (p.R145X) variant was a nonsense variant of the transmembrane domain that has been reported to have a high potential for affecting pathogenicity in previous reports. It is speculated that this variant may upregulate the Wnt pathways when variant position are considered. This variant is a very rare variant that is not registered in gnomAD and its pathogenic significance was predicted by in silico prediction. Q5. Line 331: atypical mitotic figures were positive in all of the cases with TP53 somatic variants and Ki-67 index ≥ 10%. I would need to see the complete Table-2 for verification of this. A5. As indicated, in the revised manuscript, we changed Table2 to Figure1 so that you can check the contents of Table2 that Reviewer #1 pointed out. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0231665.r002
30 Mar 2020 Decision Letter - Sumitra Deb, Editor CCNB2 and AURKA overexpression may cause atypical mitosis in Japanese cortisol-producing adrenocortical carcinoma with TP53 somatic variant PONE-D-20-01265R1 Dear Dr. Yamashita, 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, Sumitra Deb, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0231665.r003
Formally Accepted
1 Apr 2020 Acceptance Letter - Sumitra Deb, Editor PONE-D-20-01265R1 CCNB2 and AURKA overexpression may cause atypical mitosis in Japanese cortisol-producing adrenocortical carcinoma with TP53 somatic variant Dear Dr. Yamashita: 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. Sumitra Deb Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0231665.r004

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