Increased therapeutic effect on medullary thyroid cancer using a combination of radiation and tyrosine kinase inhibitors

Viktor Sandblom; Johan Spetz; Emman Shubbar; Mikael Montelius; Ingun Ståhl; John Swanpalmer; Ola Nilsson; Eva Forssell-Aronsson

doi:10.1371/journal.pone.0233720

Peer Review History

Original SubmissionJanuary 14, 2020
1 May 2020 Decision Letter - Donald P. Bottaro, Editor PONE-D-20-00504 Increased therapeutic effect on medullary thyroid cancer using a combination of radiation and tyrosine kinase inhibitors PLOS ONE Dear Mr Sandblom, 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 be sure to address all comments raised by both Reviewers. Specifically regarding points #2 and #3 raised by Reviewer#2, please make your best effort to accommodate these recommendations. If there are circumstances that prevent full compliance with points#2 and #3, please explain how any partial compliance addresses the overall concern of each point, and why complete compliance is not feasible. 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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 noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed: https://doi.org/10.1371/journal.pone.0225260 In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the Methods section. Further consideration is dependent on these concerns being addressed. 3. 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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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: No Reviewer #2: 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 ****** 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: The authors used an orthotopic PDX model to test the efficacy of combining clinically approved TKI therapies (vandetanib or cabozantinib) with external beam radiation therapy (EBRT). They found an additive effect by such combinations. The data were appropriately analyzed and are presented fairly. The authors appropriately discussed that EBRT is different from radiolabelled somatostatin analogues. Because of this, I would remove the mention of radiolabelled somatostatin analogues from the abstract and the introduction sections as they are misleading: I expected that they would use a model of radiolabelled somatostatin analogues. They can replace this with the more broad term "radiotherapy". Because the authors used immunocompromised nude mouse models, the potential immunomodulatory effects of EBRT were not captured. This is important as both vandetanib and cabozantinib target the immunomodulatory VEGF pathway and cabozantinib targets other important immunomodulatory pathways such as Axl. This should be mentioned in the discussion section. Reviewer #2: Comments to Authors: In this manuscript, the authors studied the combination irradiation with tyrosine kinase inhibitors, vandetanib, and cabozantinib in a PDX model of MTC. The authors showed an additive inhibition of tumor growth in combination therapy. Patients with high expression SSTRs may benefit from PRRT in combination with tyrosine kinase inhibitors. Specific comments: 1. What is the level of somatostatin receptors expression in GOT2 tumors? Please provide a reference. 2. The tumors should be stained for a marker of cell proliferation, cell death, or angiogenesis, to provide some insight into the mechanism of an additive effect. 3. Figure 3. Provide quantification for all markers and show images in tumors treated with monotherapy and combination. 4. In the discussion, provide some insight into the mechanism by which this combination inhibits tumor growth. 5. The table 1 is hard to read. Please remove monotherapy and treatment in each row. 6. For immunohistochemistry, provide dilution of primary antibodies and the time of incubations. 7. Line 185: Please Describe the results of the study 8. Line 190: what is a clear effect? 9. Line 229: please revise the sentence for clarity. 10. Line 254: provide a title describing the data 11. Line 360: explain the treatment with fractionated 177Lu-octreotate ****** 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 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 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. https://doi.org/10.1371/journal.pone.0233720.r001
Revision 1
8 May 2020 Author Response 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 Authors’ comment: Postal codes were removed from the affiliations list. Figure captions and tables were moved to directly after the paragraph in which they were first cited. 2. We noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed: https://doi.org/10.1371/journal.pone.0225260 In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the Methods section. Further consideration is dependent on these concerns being addressed. Authors’ comment: Rephrasing was made (outside the Methods section) at lines 70-76, 207-213, 255-260, 290-291, and 295-297. 3. 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. Authors’ comment: The phrase that refers to these data was removed, lines 390-393. This data was not important for the discussion. Editor Comments 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 be sure to address all comments raised by both Reviewers. Specifically regarding points #2 and #3 raised by Reviewer#2, please make your best effort to accommodate these recommendations. If there are circumstances that prevent full compliance with points#2 and #3, please explain how any partial compliance addresses the overall concern of each point, and why complete compliance is not feasible. Review Comments to the Author Reviewer #1 The authors used an orthotopic PDX model to test the efficacy of combining clinically approved TKI therapies (vandetanib or cabozantinib) with external beam radiation therapy (EBRT). They found an additive effect by such combinations. The data were appropriately analyzed and are presented fairly. Comments The authors appropriately discussed that EBRT is different from radiolabelled somatostatin analogues. Because of this, I would remove the mention of radiolabelled somatostatin analogues from the abstract and the introduction sections as they are misleading: I expected that they would use a model of radiolabeled somatostatin analogues. They can replace this with the more broad term "radiotherapy". Authors’ comment: We have now made clarifications in the abstract (lines 23-24) and the introduction (lines 81-84). The intended strategy to target the metastases is to use TKIs in combination with radiolabelled somatostatin analogues, but the animal model limits the possibility to test the hypothesis with 177Lu-octreotate, and external irradiation was needed. It should be noted that the GOT2 model is, as far as we know, the MTC model so far developed with the highest expression of somatostatin receptors (ref [30] in the manuscript: Dalmo J, et al. Oncology Reports. 2012;27(1):174-81). As the reviewer states, we have presented this situation in Discussion. Because the authors used immunocompromised nude mouse models, the potential immunomodulatory effects of EBRT were not captured. This is important as both vandetanib and cabozantinib target the immunomodulatory VEGF pathway and cabozantinib targets other important immunomodulatory pathways such as Axl. This should be mentioned in the discussion section. Authors’ comment: The authors are thankful for this suggestion and have added text about this at the end of the first paragraph of the discussion (lines 316-319). Reviewer #2 In this manuscript, the authors studied the combination irradiation with tyrosine kinase inhibitors, vandetanib, and cabozantinib in a PDX model of MTC. The authors showed an additive inhibition of tumor growth in combination therapy. Patients with high expression SSTRs may benefit from PRRT in combination with tyrosine kinase inhibitors. Comments 1. What is the level of somatostatin receptors expression in GOT2 tumors? Please provide a reference. Authors’ comment: There is no specific data on somatostatin receptor expression of GOT2 tumours. However, the biodistribution of 177Lu-octreotate in GOT2-bearing nude mice has been studied and information regarding tumour-to-blood activity concentration ratio was added (line 347-350). It should be noted that the GOT2 model is, as far as we know, the MTC model so far developed with the highest expression of somatostatin receptors, where a comparison is made with the TT animal model (ref [30] in the manuscript: Dalmo J, et al. Oncology Reports. 2012;27(1):174-81). 2. The tumors should be stained for a marker of cell proliferation, cell death, or angiogenesis, to provide some insight into the mechanism of an additive effect. 3. Figure 3. Provide quantification for all markers and show images in tumors treated with monotherapy and combination. Authors’ comments to #2 and #3: In the present study, the animals were followed for as long time as possible in order to study the long-term effects and determine progression-free survival. Thus, no tumour tissue was collected during the initial phase when the tumour volume was declining, which is the period when the mentioned parameters would be most interesting to study. Instead, the mice were followed until tumour regrowth, when the differences between groups probably would be minimal. Furthermore, the radiation dose was limited in order to not reduce the tumour volume too much and still be able to determine the tumour volume with reasonable accuracy. However, the suggested experiments are interesting, but requires new animal studies to be performed, and would be interesting to include in future work trying to elucidate mechanisms. Such studies would, however, require at least six months to complete, and we would then like to include more molecular methods for an even better understanding of the mechanisms, thus generating data amounts too large to be able to present and discuss in the present paper. 4. In the discussion, provide some insight into the mechanism by which this combination inhibits tumor growth. Authors’ comment: We have now included some more information on this issue of interest (line 316-319, and previously 372-375). As discussed, it is likely that the effect of the combination is not fully demonstrated due to the use of immunocompromised animals (which is necessary when using human tumour tissues). 5. The table 1 is hard to read. Please remove monotherapy and treatment in each row. Authors’ comment: These suggested changes were made, together with some more, to make the table easier to read (Table 1). 6. For immunohistochemistry, provide dilution of primary antibodies and the time of incubations. Authors’ comment: This information was added (line 160-162). 7. Line 185: Please Describe the results of the study Authors’ comment: The meaning of the comment is unclear to us. However, we have interpreted it as that we should use a more descriptive subtitle, and thus included one (lines 194-195). Although not suggested by the reviewer, we have changed the following subtitle in a similar way (lines 242-243). 8. Line 190: what is a clear effect? Authors’ comment: We have used this way of writing at several occasions when we have a statistically significant difference between the groups. The specific data are then presented in the previous or following sentence at such occasions. For clarification we also added a reference to Figure 1 at this line (line 200). 9. Line 229: please revise the sentence for clarity. Authors’ comment: This sentence was revised (line 263-264). 10. Line 254: provide a title describing the data Authors’ comment: The title is now descriptive (line 289). 11. Line 360: explain the treatment with fractionated 177Lu-octreotate Authors’ comment: The word fractionated is not necessary here and has been deleted. The sentence is now clarified (line 402). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0233720.r002
12 May 2020 Decision Letter - Donald P. Bottaro, Editor Increased therapeutic effect on medullary thyroid cancer using a combination of radiation and tyrosine kinase inhibitors PONE-D-20-00504R1 Dear Dr. Sandblom, 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, Donald P. Bottaro Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0233720.r003
Formally Accepted
14 May 2020 Acceptance Letter - Donald P. Bottaro, Editor PONE-D-20-00504R1 Increased therapeutic effect on medullary thyroid cancer using a combination of radiation and tyrosine kinase inhibitors Dear Dr. Sandblom: 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. Donald P. Bottaro Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0233720.r004

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