PONE-D-22-28908FMRP expression in primary breast tumor cells correlates with recurrence and specific site of metastasisPLOS ONE

Dear Dr. Palombi,

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 Feb 10 2023 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,

James A.L. Brown, PhD, MA(Ed)

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. You indicated that you had ethical approval for your study. Please clarify whether minors (participants under the age of 18 years) were included in this study. If yes, in your Methods section, please ensure you have also stated whether you obtained consent from parents or guardians of the minors included in the study or whether the research ethics committee or IRB specifically waived the need for their consent

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This work was supported by: Telethon GGP20137, PRIN 201789LFKB MIUR to CB.”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This work was supported by Telethon GGP20137 and PRIN 201789LFKB MIUR, to CB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Other Authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Additional Editor Comments :

Please pay particular attention to fully address the comments from Reviewers 1 and 2, which I believe will help significantly improve the clarity and impact of that paper.

[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

Reviewer #2: Yes

Reviewer #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: 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: No

Reviewer #2: No

Reviewer #3: 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: PONE-D-22-28908

FMRP expression in primary breast tumor cells…

PLOS ONE

The authors have described a potentially useful biomarker for the propensity of metastatic spread of breast cancer (BC), based on the levels of nuclear and cytoplasmic FMRP determined through immunocytochemical staining. When further developed, this approach would add to the evaluation of BC for the propensity of metastatic spread, and even the expectation for lymphatic vs non-lymphatic spread to other tissues.

However, there are several concerns with the manuscript as written that temper enthusiasm for the work.

(i) The description of FMRP measurements is confusing. Much of the manuscript appears to be describing FMRP in cancer tissue within the primary tumor in breast tissue; however, it is not clear whether additional measures of FMRP are also made in the metastatic foci. For example, in Table 4, the title is “FMRP expression in metastatic site”. This is not clarified in either the Methods section, nor in the legend to the figure.

(ii) Also with respect to Figure 4, under the header, “metastatic site”; there is a “yes” and “no” which are not defined, for each target organ, but which presumably means that a metastasis is/is not located in that organ. Also, for those cases where there is a “no”, presumably meaning no metastasis at that site – what then is the meaning of the nuclear and cytoplasmic FMRP staining, since the table refers to FMRP expression “in metastasis site”?

(iii) In Figure 2, there is an inverse correlation of FMRP expression in cases vs controls, but elsewhere in the manuscript, it is pointed out that among cases, high FMRP levels correlate with lung and liver metastases, while tissues associated with brain and bone metastasis showed a low FMRP expression. Thus, the red bars may represent the average of two very different behaviors, depending on the site of metastasis.

(iv) From the broader perspective, little attention is given to why FMRP levels are up in some types of BC, and why there may be differential expression in nuclei vs cytoplasm. Because all of the reported work is performed in situ, what steps were undertaken to demonstrate that observed differences are not due to differences in antibody accessibility following fixation from one case to the next?

(v) P16 /l334 – if absence of FMRP is associated with lower rates of cancer, why is metastatic spread associated with lower FMRP? p17 /l339 for Basal subtype – the opposite was found.

This work is not specifically concerned with the mechanistic basis for the various levels of FMRP expression; nonetheless, there should have been some discussion as to why such striking differences exist in patterns of FMRP expression.

Reviewer #2: Caredda E et al.’s manuscript titled: “FMRP expression in primary breast tumor cells correlates with recurrence and specific site of metastasis” analyzed the expression levels of FMRP in the primary and its associated metastatic lesions. Form this data, it was concluded that there was a direct and inverse correlation with FMRP expression and metastasis to the different sites.

Major Concerns:

1) There is a lack of mechanistic and functional data as to why FMRP levels (high and low expression) promote metastasis to different organs. Are there are indication of the role of the organ environment that regulates FMRP levels?

2) Is this differential expression of FMRP also seen in preclinical model of breast cancer metastasis?

3) Needs significant more experiments to suggest that FMRP levels could be used as a prognostic factor for site-specific metastasis.

Additional Concerns:

1) With respect to the latter, at the end of line 50 on page 3, given that 685,000 deaths are ~30% of the 2,261,419 cases of BC, this reviewer was uncertain how the authors arrived at a value of 6.8%. The WHO website, that the author’s provide, indicates that the total cases of all cancers is 10 million, which clarifies the issue since, of course, 685,000 is ~ 6.8% of all cancer deaths. Thus, it would be helpful if the author’s included this information; e.g., adding (red-type) to the sentence: “In 2020, the World Health Organization (WHO) reported that, of the 10 million cancers cases, 2,261,419 were new cases of BC….”.

2) Additionally, a misconception needs to be corrected in line 71 as “a single”, within the sentence’s context, is generally incorrect, given that several reports have provided evidence that clumps of tumor cells or tumor cells within groups of other cells (e.g., macrophages or fibroblasts), as often or more often have been found to seed metastasis relative to single cells. Thus, “single cells” should be deleted, which then means that “leaves” (a few words later) becomes singular.

3) At the end of line 88 there is some confusion as to whether the authors meant to refer to metastasis or tumor progression or tumor aggressiveness or a combination of these rather than tumorigenesis, given that the examples cited in the paragraph don’t refer to tumorigenesis. The sentence (lines 95 – 96) is disconnected from the paragraph that it is associated with as what is stated has not been linked to FMRP expression – please clarify this.

4) In line 328 “primitive” is inappropriate as it has been used in the context of embryological defects in neuroectodermal development that led to tumors. Therefore, delete “primitive” and replace with “nascent primary tumor” or “early primary tumor”. More importantly, throughout the Results section the findings that lower FMRP expression is associated with metastasis is very much ambiguous, due to the wording of several sentences. As such, contrary to the findings that FMRP is a suppressor of metastatic progression/lethality, a reader is left either with a sense that FMRP promotes metastatic progression or is confused as to when it does or doesn’t contribute to poor prognosis. This needs to be corrected; e.g., by: 1) in line 193, immediately in front of “FMRP” insert “low expression of”; 2) in line 216, immediately in front of “FMRP” insert “lower”; 3) emphasize in the legend to Table 2 (or in the text describing Table 2) that Cases have lower FMRP expression; 4) in line 266, immediately before “cytoplasmic” insert “low”; and, 5) in line 289 immediately in front of “We” insert “Although lower expression of FMRP was consistent with increased frequencies of metastasis, within cases of metastasis…”.

5) Adding to the confusion are statements in the Discussion that indicate that FMRP is an oncogene, apparently (referred to in an ambiguous manner) during tumorigenesis and/or primary tumor progression. As such, in these cases some explanations/distinctions between high levels of FMRP and its function in the onset/progression of primary tumor development and low levels of expression FMRP that are associated with poor prognosis need to be made.

6) Authors must add a discussion of the limitations of the small cohort size especially with respect to the very few TNBC samples studied. This was acknowledged in the Results but must be further discussed in more detail in the Discussion.

Reviewer #3: The manuscript (Luca R is well written. The study design is good with stratification into cases and controls. The study while not novel with a similar study being published by Luca R et al in EMBO in 2013, ref 29 is still a good validation and proof of principal study with low levels of FMRP in cases vs control. Additionally the association of FMRP with site- specific metastases is interesting. The role of FMRP in breast cancers especially in the various molecular types is key to understanding the aggressive subtypes of breast cancers (TNBC) which was illustrated by the authors.

The major deficiency in this paper is the lack of good quality histology pictures to illustrate the results. Did the authors examine the expression of FMRP in matched primary and metastatic tumors for example lung and brain and in the advancing front of the primary tumors. Are all tumors IDC ,NST or where they other histologic types like invasive lobular carcinoma.

Please add more figures with specific reference to whether whole sections, histologic subtypes and primary versus metastases before consideration.

**********

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

Reviewer #3: 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.

FMRP expression in primary breast tumor cells correlates with recurrence and specific site of metastasis

PONE-D-22-28908R1

Dear Dr. Palombi,

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,

James A.L. Brown, PhD, MA(Ed)

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Congratulations for your work, and for addressing the reviewers comments.

Please take note of the final suggestion of Reviewer 1 to alter the wording in your abstract during the proofing process.

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)

Reviewer #2: All comments have been addressed

**********

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 #2: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: 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: No

Reviewer #2: 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

Reviewer #2: 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 substantially addressed all of the issues that I had raised in the initial review. However, I suggest that the authors make one revision to the abstract that changes "...our study aimed at understanding the correlation..." to ...our study aimed at DESCRIBING the correlation..." since they do not provide any data that represent an attempt to understand the basis of the correlations.

Reviewer #2: The authors have satisfactorily addressed all the previous queries by providing additional data and additional explanation.

**********

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 #2: No

**********