PONE-D-22-00869Central nervous system hemangioblastomas in von Hippel-Lindau disease: total growth rate and risk of developing new lesions not associated with circulating VEGF levelsPLOS ONE

Dear Dr. Sundblom,

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.

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Reviewers' comments:

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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: Partly

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: Hemangioblastomas are known to secrete vascular endothelial growth factor (VEGF), suggesting a potential role of VEGF as a biomarker for tumor growth. The authors looked plasma VEGF samples from 24 patients with von Hippel-Lindau disease were

analyzed by solid-phase proximity ligation assay (PLA). Levels were monitored over time together with numeric and volumetric CNS tumor burden, and compared to plasma VEGF levels in healthy controls. They found no difference not surprisingly. The VEGF level may be different locally or in CSF and major changes are unlikely systematically this negative study verifies this suggestion. Hopefully they can look at CSF level in the future and over various time periods although this may be difficult to have patients consent to. I would like the authors to clarify the tumor volume change in patient #4 in Figure #1. Add the volumes with SD over time year 1,2,3, and 4 and also indicate the volumes for female and male with st deviation as well.

Reviewer #2: The authors have presented evidence for the lack of association between circulating VEGF levels and CNS tumor burden in VHL patients. The authors developed a solid phase PLA to measure VEGF165a. They then measured the serum VEGF levels in VHL patients (clinical criteria) and normal volunteers. They report no difference in serum VEGF levels with VHL disease or with CNS tumor burden.

As with other publications that have reported on paracrine/autocrine actions for pro-angiogenic factors in VHL (PMID: 27748427), the authors report no detectable difference with VHL disease or with increasing tumor burden. The manuscript is well written and covers the prior literature well. However, I have some comments for the authors:

1. The authors do not discuss the rationale for developing a brand new assay to measure circulating VEGF165a levels. Can they explain why off-the-shelf ELISA assays were not used? How was the solid phase PLA validated? What were the positive and negative controls used?

2. Another major issue is the single time-point measurement for VEGF in this study. The study design will inherently fail to detect changes with time/intervention. There is at least some evidence from animal studies that while VEGF levels are not different with/without VHL-/- tumors, interventions can lead to decreased VEGF levels (PMID: 30497198). This finding is like the clinical data (senaxamib study) cited by the authors. Can the authors explain the rationale for a single time point measurement?

3. Did the authors use ellipsoid volume or volumetric segmentation to arrive at tumor volumes?

4. The authors need to correct the text when citing the range of tumor numbers and tumor volumes in Results. Table 2 suggests that the lowest tumor volume is 2,46 mm3 while in the main text, the authors cite 0 mm3.

5. The authors make incorrect statements regarding the lack of information about increase in tumor growth in VHL in Discussion. Information about saltatory growth and the unpredictability has been well described in the prior literature with much larger cohorts.

6. In Figure 1, the authors need to mark the time-point for each patient when the serum VEGF sampling was done.

7. In Figure 2, individual values need to be shown (rather than a summary box-whisker plot).

Reviewer #3: Sundblom et al aimed to study the relations between circulating VEGF levels and HB development/progression in patients with VHL.

The idea is generally sound, but highly simplistic, as the increase in VEGF levels is cellular, and depends on the full loss of VHL in the tumors. Hence, the chances to find such an association is very low, to begin with. Moreover, the sample size is extremely small (although dealing with rare diseases, there are much larger cohorts and multi-center collaboration would be expected on such topics).

The paper could have been written better, both in terms of clinical accuracy and structure. The methods are too detailed in regard to the lab techniques, while the MRI interpretation is quite poor. This is especially important in VHL, where the T2 reflects the cystic portion of the tumors and is not described at all. While this may be explained by the aim to correlate with VEGF, the cystic part, together with the edema, might be at least as important as the solid part. Thus the clinical utility of VEGF must be tested vs. these components as well.

The diagnostic criteria for VHL include multiple hemangioblastomas (this might be the 1st one, in fact), and it is surprising that this criterion of all is not mentioned in the introduction...

The genetic data should be written more clearly, with both cDNA variant detail and protein-levels changes (VHL c.499G>C, p.R167W, for example).

The results should be thoroughly revised, written in a more fluent order, with results supporting the text, and not in isolated short phrases (for example line 232).

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-22-00869R1Central nervous system hemangioblastomas in von Hippel-Lindau disease: total growth rate and risk of developing new lesions not associated with circulating VEGF levelsPLOS ONE

Dear Dr. Sundblom,

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 submit your revised manuscript by Dec 21 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'.
• 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Prashant Chittiboina

Guest Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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

Reviewer #3: (No Response)

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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

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: 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

Reviewer #3: Yes

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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

Reviewer #3: 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: They have addressed my concerns. They add the requested information and the manuscript should be considered for publication.

Reviewer #3: The manuscript writing improved significantly, although a few minor issues remained.

My main criticism for the study is yet in its core hypothesis – most of the VEGF in the circulation derive from peripheral blood cells, and the chances to see hemangioblastoma-derived VEGF in a meaningful concentration is extremely low. To prove that, even comparing patients with various VHL-related, VEGF-dependent tumors, to controls, shown no difference.

In my view, the authors might want to discuss this point in the discussion.

In addition, please revise the variants descriptions in Table 1 – the 167 codons is described as nc499 in VHL011 and nc712 in VHL012.

Minor

Editing, typos – therefor -> therefore, missing full stop in the abstract, changing fonts in the abstract.

Genetic analysis – VHL gene should be written in capital letters and in Italics.

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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

Reviewer #3: No

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[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.

Central nervous system hemangioblastomas in von Hippel-Lindau disease: total growth rate and risk of developing new lesions not associated with circulating VEGF levels

PONE-D-22-00869R2

Dear Dr. Sundblom,

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.

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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,

Prashant Chittiboina

Guest Editor

PLOS ONE