PONE-D-24-42448Matching plasma and tissue miRNA expression analysis to detect viable ovarian germ cell tumorsPLOS ONE

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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: Yes

Reviewer #3: Yes

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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: Yes

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: No

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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: Introduction

Please define at least once the acronyms used in the introduction.

Methods

Line 140: Provide the manufacturer and catalog number for the Streck tubes used in the study.

Line 141: Instead of using the term ‘in a standard fashion’, please provide the exact conditions for the centrifugation, e.g., 5,000 rpm for 10 minutes at 4 °C.

Results

Line 204: Please explain the definition of FIGO and include a reference.

Table 1: Please include the definitions of the acronyms at the bottom of the table for better interpretation.

Figure 3: Instead of showing the results in graphs for visual comparison, consider plotting correlation graphs to reach miRNA so that this correlation can be quantifiable.

Figure 4: Have you done statistical analysis comparing the time points assessed? That would be important to show in the graph. Especially the comparison of the first point (Surgical tissue, Pre-OD# 2) to the other time points.

Reviewer #2: Angeles et al present data obtained from 23 patients that have ovarian germ cell tumors which reveals increased expression of miR-371-3p, as well as miR-302/367, within plasma and tumor tissue samples. These miRNAs are known to have higher expression within testicular germ cell tumors. They show increases in the incidence of these miRNAs prior to tumor removal, decreases in their expression after their removal, and return of high expression after a given amount of time after removal. These are all strong evidences that the expression of these miRNAs are, at the very least, correlated with the presence of malignant tumors that originated as OGCT. The manuscript does a good job of transparently presenting their supporting data, however, there are a few points of criticism/potential improvement that need addressing:

1) Lack of data showing relation to mRNA targets of miR-371-3/302/367

a. The evidence of these miRNAs presence and overexpression in OGCT patients, decrease after surgery, and re-increase well-after resection is very well-established in the paper. However, miRNAs do not act alone to cause oncogenicity, and therefore, it would be further solidifying of the authors’ data to show inverse patterns of known targets of these miRNA such as LATS2, TGFBR2, CDKN1A, or PIK3R1 in each of the patients shown in Figures 4 and 5. Although the authors’ intention was not to provide evidence for a putative pathway, it would certainly add to the significance of their findings. To even further concretize the authors' findings, they could create a ceRNA network related to all miRNA in the paper to show possible avenues of action instead of simple correlations.

2) Data-driven comparison of OGCT to TGCT

a. The authors’ rely on the similarity of testicular germ cell tumors within the introduction, and discussion, sections but do not provide any comparative data. Considering that the TGCT are the more well-established tumor to miRNA dysregulation, a comparison to currently available data from theses tumors would further solidify the connection between these primordial sex cells. Additionally, it could provide avenues for further investigation between to the two to find similarities, differences, and correlations.

3) Addressing Tumor Heterogeneity

a. Within the authors’ cohort, there is a wide-range of expression levels of mentioned miRNAs between patients (Figure 3). It would be helpful to delineate in a supplementary and separate figure which miRNAs are most overrepresented in specific tumor types as addressed in the discussion section. It’s one thing to recognize the difference in expression between tumor types, but it’s another, more useful, thing to present those differences quantitatively in graphical form. This could also add to the conclusion that germ cell tumors express specific miRNAs, regardless of sex type.

Overall, this paper contains a good amount of well-analyzed data that could be amplified with minor additions of supporting data as well as the inclusion of new figures that would help clearly show the conclusions of the authors.

Reviewer #3: The manuscript “Matching plasma and tissue miRNA expression analysis to detect viable ovarian germ

cell tumors” by Angeles et. al., investigated role and expression pattern of microRNAs (miRNAs) in ovarian GCTs

(OGCT).

Authors needs to address the following concerns to improve the quality:

1. Authors pointed out abnormal LDH while presenting results. It should be either higher than or lower than normal.

2. Line 212: "AFP at diagnosis was above ULN in 14 of 22 patients." Table 1 says 21 patients, please verify. Authors should consider presenting "Patient Characteristics" section with more clear statements.

3. Authors should use better resolution image for Fig 1 and 2, where labels are almost unreadable.

4. Please point out figure number and sub figure number clearly in the result section for better readability.

5. Conclusion should specifically point out the limitations of this study.

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Reviewer #1: No

Reviewer #2: Yes:  Brian Jorgensen

Reviewer #3: No

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Matching plasma and tissue miRNA expression analysis to detect viable ovarian germ cell tumors

PONE-D-24-42448R1

Dear Dr. Nappi,

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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Kind regards,

Julie Decock, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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 #2: Yes

Reviewer #3: Yes

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

Reviewer #2: 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 #2: 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 #2: Yes

Reviewer #3: Yes

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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 #2: Thank you for your extensive answers. While I understand that you cannot test for mRNA targets due to tissue exhaustion, it is still vital to mention possible avenues of activity for the miRNAs themselves. Yes, there presence is correlated with these tumors but if you do not have a targeted pathway or gene, you have only found a correlation that may not be directly related to the causation of the tumor and could lead to false negatives/positives if used as a diagnostic. This is obviously more for the discussion section, however, future experimentation should require the testing of mRNA levels with miRNA in targeted cell types/in vivo.

Reviewer #3: uthors made significant efforts to address all the concern pointed by the reviewers.

I consider this manuscript is clearly written, well discussed with proper citation. So, I recommend the publication of this manuscript in the esteemed journal PLOS ONE.

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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 #2: Yes:  Brian Glenn Jorgensen

Reviewer #3: No

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