Analysis of residual disease in periocular basal cell carcinoma following hedgehog pathway inhibition: Follow up to the VISORB trial

Shelby P. Unsworth; Christina F. Tingle; Curtis J. Heisel; Emily A. Eton; Christopher A. Andrews; May P. Chan; Scott C. Bresler; Alon Kahana

doi:10.1371/journal.pone.0265212

Peer Review History

Original SubmissionFebruary 26, 2022
1 Sep 2022 Decision Letter - Arianna L. Kim, Editor PONE-D-22-05819 Analysis of Residual Disease in Periocular Basal Cell Carcinoma Following Hedgehog Pathway Inhibition: Follow Up to the VISORB Trial. PLOS ONE Dear Dr. Kahana, 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. Your manuscript describing molecular characterization of residual BCCs following the VISORB trial could be of significant interest to the field, particularly in understanding tumor response to currently available Hh-targeted therapy in patients with advanced BCCs. However, concerns were raised regarding the technical aspects related to the mutational status and burden. Please address Reviewer 2’ concerns about Figs 3 and 4 and include the details about sequencing and filtering criteria and subsequent statistic analysis. Please submit your revised manuscript by Oct 15 2022 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'. 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Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 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: I think the paper is innovative and brings light to procedures and investigations that should be performed also in other advance BCC locations. It was a pleasure to review this paper. The Gli-expression in residual tumor should be performed even in advance BCC localized in other than periocular tissues. It is crucial to understand the biology of these particular tumors and the reason some of these patients do not respond in the area of new treatment and advancing new protocols such as neo adjuvant treatment with HH inhibitors. Please add in the introduction after reference 10 "While vismodegib has a relatively high response rate, unfortunately a subset of these tumors become resistant and recur" [9, 10 ) following reference: Dika E, Scarfì F, Ferracin M, Broseghini E, Marcelli E, Bortolani B, Campione E, Riefolo M, Ricci C, Lambertini M. Basal Cell Carcinoma: A Comprehensive Review. Int J Mol Sci. 2020 Aug 4;21(15):5572. doi: 10.3390/ijms21155572. PMID: 32759706; PMCID: PMC7432343. The results show one of the first studies that describe pathogenic variants of PTCH1, and SMO. The authors further characterized each variant using the VarSome.com database. This kind of multi center studies offers to the literature and most importantly to clinicians a better knowledge and an inspiration for further investigations. Reviewer #2: Unsworth et al. present a study of residual disease in periocular (op) BCC following Hedgehog Pathway Inhibition. The authors revealed that in treated with vismodegib opBCC patients who demonstrated a complete response histologically, excision samples contained micro-tumors. In one case of recurrent tumor a SMO W535L mutation was identified. Sequencing of pretreatment tumor, also revealed a SMO W53L mutation but with a low frequency. Major comment The number of detected mutations is not realistic. The authors can consult other papers on BCC, such as Bonilla et al 2018, NatGen, to assess the average number PTCH1 or SMO mutations per tumor. It seems that listed mutations include technical sequencing artifacts (Fig.3) and the germline variants (Fig 4B). It is also possible from fig 4B that in the post-treatment tumor sample no tumor cells were present. The methods should include details about the sequencing and filtering criteria for the variants. ****** 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: Emi DIKA 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.] 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: PLos one review .docx https://doi.org/10.1371/journal.pone.0265212.r001
Revision 1
17 Oct 2022 Author Response We thank the reviewers for their comments and suggestions. All the suggested references have been added. Reviewer #2 is correct that the average number of PTCH1 or SMO “mutations” expected to be found in a basal cell tumor is low; however, in this manuscript we are not reporting tumor-specific mutations but instead all variants present in the whole FFPE tumor biopsy or post-treatment excision sample. These samples include tumor cells, normal keratinocytes, and surrounding dermal cells, meaning our sequencing results include both germ-line variants and tumor-specific mutations. Because we do not have matched non-tumor control skin for study patients – we are unable to distinguish between germ-line and tumor specific mutations. Instead, we characterized all non-silent PTCH variants with a greater than 0.25 allele frequency and all non-silent SMO variants using VarSome to identify which were known to be pathogenic and thus more likely to be tumor-specific. To make this more apparent in the manuscript, we removed all references to “mutations” which suggest tumor specificity, and instead referred only to “variants of interest” present in the samples. We also clarified that sequencing was performed on “pre-treatment formalin-fixed paraffin-embedded (FFPE) biopsy sections” As the reviewer stated – while it is possible that no tumor cells were present in the post-treatment sample for the patient that recurred, the presence of Gli1+ proliferative cell clusters throughout the excision sample suggest otherwise. Instead, we believe that because the excision samples contain a relatively small number of these residual tumor cells compared to surrounding normal keratinocytes and dermal cells the residual tumor cells were undetectable using our targeted sequencing methods. It is possible that these cells could be isolated using laser micro-dissection followed by targeted sequencing; however, this was outside the scope of our study. This has been further described in paragraph 2 of the discussion. We are grateful for the helpful comments to improve our manuscript. Sincerely, Alon Kahana and co-authors Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0265212.r002
1 Nov 2022 Decision Letter - Arianna L. Kim, Editor Analysis of Residual Disease in Periocular Basal Cell Carcinoma Following Hedgehog Pathway Inhibition: Follow Up to the VISORB Trial. PONE-D-22-05819R1 Dear Dr. Kahana, 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, Arianna L. Kim, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0265212.r003
Formally Accepted
23 Nov 2022 Acceptance Letter - Arianna L. Kim, Editor PONE-D-22-05819R1 Analysis of Residual Disease in Periocular Basal Cell Carcinoma Following Hedgehog Pathway Inhibition: Follow Up to the VISORB Trial. Dear Dr. Kahana: 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. Arianna L. Kim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265212.r004

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