PONE-D-19-33270

HIGH EXPRESSION OF TIE-2 PREDICTS POOR PROGNOSIS IN PRIMARY HIGH GRADE SEROUS OVARIAN CANCER

PLOS ONE

Dear Dr. Sopo,

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.

Specifically, the authors should include controls, improve quality of figures and consistency of the presentation of IHC data, details of IHC and editorial corrections including sentences.  

We would appreciate receiving your revised manuscript by Feb 20 2020 11:59PM. When you are 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.

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Surinder K. Batra

Academic Editor

PLOS ONE

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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: No

Reviewer #2: No

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

Reviewer #1: Yes

Reviewer #2: No

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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

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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: 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: In this manuscript, the authors investigate the expression of Ang-2, Tie-1 and Tie 2 in ovarian cancer and their relationship with patient survival, chemo resistance and other clinical parameters. The main conclusion of the study is that low expression of Tie-2 is associated with increased survival of ovarian cancer patients. High expression of Tie-2 was also detected in metastasized ovarian tumors. Surprisingly, Ang-2 expression was low in ovarian tumors and predicted poor overall survival. There is limited data in the literature on the expression of these angiogenic markers in ovarian cancer. The study therefore adds to our understanding of ovarian cancer biology and may also support the targeting of Tie-2 and Ang-1. There are several shortcomings of this manuscript that reduce enthusiasm.

1. Immunohistology is the primary method used to monitor the expression of the angiogenesis markers in ovarian tissues. Therefore, it is important that the IHC data be of high quality. The expression of Tie-2 is diffuse and in some of the images provided by the authors, it is not clear if the expression is sufficiently specific. Authors are requested to demonstrate antibody specificity. At the same time, this study will also benefit from the inclusion of positive controls of human tissues that are known to be positive for Tie-2, Ang-2 and the other markers used in this study.

2. Authors have used the immunoreactivity score (IRS) which takes into account the percent of positive cells and eth intensity of the staining. It is therefore confusing as to why the authors selectively show IRS data in some figures and positive percentage (PP) and specific intensity (SI) for others. It will be best if authors use IRS data for the semi-quantitative analysis. Alternatively, authors may also conduct the quantitation using the accepted H-scoring system.

3. There is no correlation between IHC expression and mRNA expression. Authors point out that this is likely because the RNA samples are derived from the entire tissue and not from cancer, endothelial or stromal cells. This logic makes sense. But it also implies that there is no necessity to include the qPCR results as they only confuse the results.

4. Authors should specify if the samples used for IHC were obtained from treatment naïve patients.

5. Overall, the results are presented in a confusing manner. Often times, in the results section, the number of the figure or the table is not explicitly mentioned. Therefore, it is hard for the reader to determine which data the authors are referring to in the text.

Reviewer #2: This manuscript is focused on analyzing the impact of TIE-2 on the prognosis of serous ovarian cancer. To prove the concept, the authors used 86 primary epithelial ovarian cancer samples and 16 omental metastasis. They observed high epithelial expression of Tie-2 in primary high-grade serous ovarian cancer, which is correlating with survival. Angiopoietin-2 showed low expression in serous ovarian tumors and associated with shorter survival. Additionally, the expression of angiopoietin-2 and Tie-2 showed increased expression in distal omental metastasis compared to primary tumors.

This manuscript needs a major revision for publication.

• Several sentences were incomplete in the manuscript, so extensive language correction is required.

• There is no clear result given the angiopoietin-2 and Tie-2 in other subtypes shown in sample numbers.

• The expression of angiopoietin-2 and Tie-2 in distal omental metastasis is showing an interesting result; however, the discussion is missing.

• Since it is correlated with the omental metastasis, what is the status in other metastasis?

• There is no statistical calculation shown in qRT-PCR of all the molecules. How many mRNA samples were used for this analysis, and how many times, experiments were repeated to calculate significance? Because this is the base result for the manuscript.

• There is no H-score calculated for the intensity of Tie-1, two, and Ang 2 staining in serous ovarian cancer samples. What is the subcellular localization of these molecules?

• Immunohistochemistry images are showing poor quality of staining.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-33270R1

HIGH EXPRESSION OF TIE-2 PREDICTS POOR PROGNOSIS IN PRIMARY HIGH GRADE SEROUS OVARIAN CANCER

PLOS ONE

Dear Dr Sopo,

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.

As only one of the original reviewers was available, two additional reviewers have also read the manuscript. Please address the comments of all reviewers. We would appreciate receiving your revised manuscript by Jun 01 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Elizabeth Christie

Academic Editor

PLOS ONE

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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)

Reviewer #4: (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: Partly

Reviewer #4: No

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

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: No

Reviewer #4: No

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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

Reviewer #4: No

**********

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: Authors have addressed previous critique.

The figures are of good quality and the manuscript has been suitably modified to highlight relevance accurately.

Reviewer #3: 1. The manuscript thematically purports to investigate biomarkers of tumour angiogenesis in ovarian cancer. Ang-2 and Tie-2 protein levels assessed by immunohistochemistry are shown to relate to overall survival; however the predominant expression of both of these proteins is in epithelial tumour cells. Although the predictive value of these markers appears to be strong, it is unclear how this relates to tumour angiogenesis - microvessel density is not assessed in this study. Furthermore, while the Discussion maintains the focus on Ang2/Tie2 in tumour angiogenesis and metastasis, it appears to ignore studies in other tumour types showing that Ang2 signaling through integrins on tumour cells can cell-intrinsically promote invasion and metastasis by inducing epithelial to mesenchymal transition (EMT; e.g. Imanishi et al. 2007 Cancer Research 67(9):4254–63; Dong et al. 2018 Oncotarget 9:12705-12717, and others). This alone could explain the observation of higher Ang2 (and possibly Tie2) expression in metastases than in primary tumours. The authors should acknowledge promotion of EMT by Ang2 as a possible mechanism, and acknowledge the limitations of their data in making conclusions regarding tumour angiogenesis and systemic metastasis.

2. In Figure 1 it is not clear what values are displayed – are these median values of IRS? Epithelial, stromal or overall? Presuming that an IRS value was calculated for each tumour specimen, and the data are non-normally distributed, the data could be better presented as a box-and-whisker plot (or similar) with individual data points. Currently there is no indication of the spread of the data.

3. Please provide images of the positive and negative tissue controls for Ang-2, Tie-2 and Tie-1 as Supporting Information, along with references indicating expected patterns of staining in positive control tissues for each protein. It is not sufficient merely to state that the negative controls were negative. Were antibody isotype controls included? If Western Blots have been performed to validate the specificity of the antibodies used for IHC, please present this data as well.

4. Some of the data concerning Ang-2 expression are counterintuitive. Low Ang-2 expression is associated with poor overall survival in spite of higher Ang-2 immunoreactivity in metastases, and in contrast with studies in other tumour types showing that Ang-2 promotes tumour invasiveness (ref. 22). The negative correlation between Ang-2 immunoreactivity and mRNA levels is also perplexing. Although there are potential explanations for these discrepancies, it does raise suspicion about the specificity of the Ang-2 antibody used, and underscores the importance of independent validation of antibody specificity by investigators. Furthermore, the authors state in the Abstract that relative expression of Ang-2 determined by qRTPCR correlated significantly with IHC protein levels, which is misleading – the correlation is statistically significant, but negative.

5. Line 235: the authors state that “the correlation [between Ang-2 and Tie-1] was significant among other histological types”. If so, please present r and p values in the text or a table. Please clarify in the Methods section which statistical test was used to measure this correlation.

6. In Tables 2 and 3, the authors seem to only present data for analyses that yielded statistically significant results. Did all analyses performed yield statistically significant results? Please provide the results of all analyses for completeness. Were all parameters analysed for their relationship to overall survival also assessed regarding PFS? If so, please present these data as well.

7. Similarly, if correlation analyses between IRS and qRTPCR results were performed for Tie1 and Tie2, please present these as well. Given that the authors have removed most of the qRTPCR data from the previous version of their manuscript, they should consider toning down claims of their study’s importance/uniqueness related to this.

8. The authors state (line 97) that “there are no studies investigating the tissue expressions of Ang-2 and its receptors in ovarian cancer”, referencing Table S1, yet this Table contains several examples where such investigations appear to have been published, e.g. ref. 19. Please revise this statement.

9. Table S1: several references within this table do not appear within the main References list. Please add a list of full Supplementary references to the Supporting information file to cover these.

10. Statistical analysis: I am not familiar with the use of a Mann-Whitney U test as a post-hoc test for pairwise comparisons subsequent to the overarching Kruskal-Wallis test. In my understanding Dunn’s post-hoc test is more appropriate. Please provide a reference validating the appropriateness of this approach, or seek expert statistical advice.

11. Do the omental metastases examined reflect systemic or local/in transit metastasis?

12. S1 Table 1st row data, 3rd column: “Post-oper” – word incomplete

13. Line 326: “angiogenetic” should be “angiogenic”

Reviewer #4: 1) Figure 1 – Figure 1 and the Figure 1 legend both lack sufficient detail to interpret the immunohistochemistry data. Readers should be able to determine the distribution of staining scores across the cohort, which is not possible with data presented as a histogram. Please change histograms to violin plots, which will allow you to show the median score in each group, the individual values per patient, their distribution and the density. The figure or legend must also state the number of patients included in each group. A statistical test should also be used to compare expression values for each marker between primary tumors and metastases, and P values should be reported for each comparison. The y-axis also should be labelled. Please also state in the legend whether the IRS was calculated from the tumor epithelium or stroma.

2) Results – Throughout the text the authors refer to staining patterns in primary high grade serous and endometrioid cancer. This data is not shown. For example, lines 205-206 – The authors report that “the stromal staining of Ang-2 was mostly weak in 74% of cases in primary high grade serous and endometrioid tumors”. No Figure or Table is referred to. When referring to staining intensity, perhaps the authors mean to refer to Table S2? However, Table S2 contains staining intensity for all histotypes combined. Where is the data presented for selected primary high grade serous and endometrioid tumors?

3) Results, lines 224-225 – “stromal staining was strong in 64% of cases, emphasizing stromal fibroblasts and vascular endothelium (Figs 1 and 2c-d)”. Figures 1 and 2 do not show that 64% of cases have strong stromal staining. Authors need to carefully check that each results statement is correctly linked to the appropriate Figure and/or Table, and that the Figures and/or Tables referred to actually contain that data.

4) Table S2 – Given the amount of key data presented in Table S2, this should be changed to a main Table within the manuscript.

5) Table S2 – It is not clear whether the P values are calculated for just the paired primary and metastatic samples from the same patients, or unpaired groups of all primary and all metastases. Consider doing both. The range of IRS values are missing from the table.

6) Results, lines 234-237 – “The level of Tie-1 expression was moderately, but significantly, correlated with its ligand Ang-2 (r = 0.5, p < 0.001) in primary serous ovarian tumors, and the correlation was also significant among other histological types. The expression level of Tie-2 showed a weak, but significant correlation with Ang-2 (r = 0.3, p = 0.012) and moderate correlation with Tie-1 (r = 0.5, p < 0.001) in primary cancer.” Which values were used to test these correlations and in which cellular compartment?

7) Results, lines 246-247 – “The level of Tie-1 expression did not differ between primary and metastatic lesions (Fig 1, 2 and S2 Table)”. However table S2 shows that the stromal expression of Tie-2 does differ between primary and metastatic lesions (p-value 0.046. Please clarify.

8) Table S3 – Please indicate whether the values were calculated from epithelial or stromal cells.

9) Results, lines 251-270 – Throughout this section the authors are investigating the association between biomarker expression and various clinical parameters. However, we are not shown the expression data. For example, “The low level epithelial expression of Ang-2 in primary tumors associated significantly with the ovarian cancer recurrence and with the greater residual tumor size (� 1cm) after primary surgery (p = 0.018, p = 0.012)”. For this comparison, the authors should show the median IRS for tumours with residual tumor > 1cm, compared to IRS of tumours with residual tumor < 1cm and no residual tumour. Therefore, in addition to the P values in Table S3, please include the median IRS and PP values for each group comparison.

10) Results, lines 254-256 – “In high grade serous tumors, high levels of Ang-2 staining were related to the resistance to platinum chemotherapy (p = 0.017) (S3 Table).” Platinum resistance and high-grade serous tumors are both not shown in Table S3.

11) Results, line 284-286 – The authors need to explain how high Tie-2 is associated with shorter overall survival, and low Ang-2 is associated with poor survival, and yet Tie-2 and Ang-2 expression are positively correlated (as stated in Results lines 236-237). Presumably these are different populations of patients? Please explore and explain this result.

12) Figure 3 – In the figure, please indicate the number of individuals at risk at each major time interval under each Kaplan-Meier plot.

13) Figure 3 and Table 3 – How was high and low assigned for each biomarker, and what was the cut-off used for high and low?

14) qRT-PCR is mentioned in the Abstract, the Methods and Discussion, however not mentioned in the Results. Please clarify.

15) Table S4 – Please include a table description indicating that Ang-2 IRS and PP is being correlated with qRT-PCR, and how the P values were calculated, and the numbers of samples tested. Also, please include the data for Tie-1 and Tie-2.

16) Grammar needs to be corrected throughout the manuscript.

17) Table 3 – Table 3 shows 48 patients with low Ang-2 IRS and 82 patients with high Ang-2, which added together equals 130 patients. However, there are only 86 women described in the Materials and Methods. Please clarify. Also, Tie-1 is missing from this Table.

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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

Reviewer #4: 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.

PONE-D-19-33270R2

HIGH EXPRESSION OF TIE-2 PREDICTS POOR PROGNOSIS IN PRIMARY HIGH GRADE SEROUS OVARIAN CANCER

PLOS ONE

Dear Dr. Sopo,

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 all of the points raised during the review process by both reviewers.

Please submit your revised manuscript by Aug 08 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Elizabeth Christie

Academic Editor

PLOS ONE

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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 #3: (No Response)

Reviewer #4: (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 #3: Yes

Reviewer #4: No

**********

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

Reviewer #3: Yes

Reviewer #4: No

**********

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 #3: No

Reviewer #4: No

**********

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 #3: Yes

Reviewer #4: No

**********

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 #3: The manuscript is overall improved, but there are some important points remaining to be addressed. Please see below, with numbers corresponding to the original questions raised. All points not specifically mentioned have been satisfactorily addressed.

1. The authors have acknowledged the potential role of Ang2 in EMT and metastasis, but have done so very superficially – in fact the statement I made regarding this in my initial review has been copied almost word for word into the manuscript, which is not acceptable. Please add your own insight. In asking for the authors to consider the limitations of the conclusions that can be drawn from their data re angiogenesis, I intended a more balanced assessment of the different possible mechanisms at play, rather than the blunt statement that has been inserted. Although the Discussion is overall appropriate, it remains unclear to me whether the observed relationship between tumour cell-expressed Tie2/Ang2 and survival/metastasis has anything to do with angiogenesis, and if so, how. A correlation analysis between tumour cell-expressed Tie2/Ang2 and blood vessel density would have been informative in this regard, but perhaps this must wait for a future study. See also point 11.

3. Thankyou for adding the images in S1 Figure. Please add detail in the caption to describe what tissues are stained in each image, and what the negative control represents – negative control tissue, or negative control antibody?

4. These results are still somewhat perplexing, but they appear to be experimentally sound and sufficiently explained.

6. Tables 3 and 4 still have data missing. Please present all multivariate hazard ratios, confidence intervals and p values for all variables assessed in Table 3, and all HRs, CIs and p values in Table 4. There should be no blank spaces in the tables – all data should be presented regardless of significance so readers can transparently assess the results. Statistically significant results can be highlighted with asterisks or bolding of numbers if desired – if so please make sure this is done consistently across all tables.

7. Thankyou for presenting complete data in Table S3. However there still appears to be data missing from Table S2. Please fill in the blanks as in point 6. Also, in table 2, some entries contain two numbers separated by a comma. What is meant by this? Should the comma be a decimal point?

8. Please cite the four studies in the text on p4.

9. Please ensure all references in the Supplementary materials and the main manuscript are formatted the same way, consistent with PLoS One’s standards. Some references have authors’ first names while most have only initials.

11. The Discussion regarding omental metastases (e.g. p21 lines 389-406) could benefit from more detail as to how omental metastases occur, if this is known. Do these secondary tumours form as in-transit metastases along blood vessels or lymphatics? Or are they simply the result of local invasion through the parenchyme or along the surface of the omentum, only engaging blood vessels when the deposit becomes large enough to be hypoxic? This also relates back to point 1.

Reviewer #4: 1) Figure 1 – The median is difficult to see when represented as a larger dot of the same colour as all the other data points. Please use a horizontal line of a different colour to indicate the median IRS. Also, please use grey transparent dots for each data point, to allow readers to distinguish data points that are close together.

2) Throughout the manuscript, the authors refer interchangeably to “high-grade serous ovarian tumors”, and “high grade serous and endometrioid tumors”, and “primary high grade cancer”, and “primary ovarian tumors”. This makes the text difficult to follow. According to Table 1, high-grade serous tumors make up 45 of the 86 ovarian carcinomas tested in this study. If the authors would like to draw specific conclusions about high-grade serous (e.g. lines 201-202), please refer to figures or tables that display results for those specific 45 high-grade serous tumors. Or equally, if the authors would like to make conclusions about high-grade serous and high-grade endometrioid combined (e.g. lines 205-207), please refer to figures or tables in which those 45 high-grade serous have been combined with the 13 high-grade endometrioid tumors, and provide a rationale for doing so. Or if the authors would like to present results pertaining to all ovarian carcinomas combined (e.g. Figure 1 and Table 2), please clearly state this in the text.

3) Related to the above issue, throughout the text, many statements in the Results section are still not linked to a Figure or Table. There are too many to list.

4) According to the Methods, ten randomly selected microscopic fields were examined per tumor section. Please include in the Methods a statistical measure on how consistent the scores were between microscopic fields, and state whether the final scores for each tumor were an average of all 10 fields.

5) Table 2 – How the p values relate to the data presented in the table is still unclear. If the paired Wilcoxon test was used to compare expression of the paired primary and metastatic high-grade serous tumours (as stated by the authors response to reviewer), the table should show the IRS, PP and SI results for the 16 primary high-grade serous tumours alongside their matched 16 high-grade serous metastases, as that is the data that the Wilcoxon test is being applied to. In addition, all the other unpaired primary tumour IRS, PP and SI results should also be presented in a separate column. Also, Wilcoxon seems appropriate to compare paired IRS and PP scores, but how would that be applied to SI? The SI results are presented in the table as a proportion of tumours that are weak, moderate or strong, so wouldn’t a chi-square test be more appropriate?

6) Table S2 – This table is hard to follow as currently presented. Why is SI reported under Residual tumor, and not the other clinical characteristics, and how is SI reported here as values between 62 and 88, when in the methods it was scored as “0, negative; 1, weak; 2, moderate; 3, strong”? In addition, for some clinical characteristics there appear to be too many p values reported, whilst under Histology the PP Tie-1 p value is missing. Please edit this table to be easier to follow.

7) Results, lines 266-268, the authors state that “The low epithelial expression of Ang-2 in primary tumors associated significantly with the ovarian cancer recurrence and with the greater residual tumor size (� 1cm) after primary surgery (p = 0.018, p = 0.012).” However, in Table S2 the median IRS of Ang-2 is 6 both in patients with recurrence and without recurrence, and the median IRS of Ang-2 is also 6 regardless of residual tumor status, which seems to indicate there is no significant difference between any of these groups. This calls into question the conclusions drawn from this part of the analysis.

8) Figure 3 – Kaplan Meier curves are shown for HGSC patients for Tie-2 (n=45), but not for all ovarian (n=86). Whereas K-M curves are shown for all patients for Ang-2 (n=86), but not HGSC (n=45). Please show the omitted Kaplan-Meier curves.

9) If the authors have noted in their correspondence to reviewers that Tie-2 and Ang-2 are correlated in non-HGSC tumours, and do not correlate in HGSC, please state this explicitly in the text of the manuscript for readers, and use the Discussion to explore what this might mean.

10) Please state in the Methods how the high and low cut-offs were assigned for each biomarker.

11) Table S3 - The qRT-PCR data needs to be mentioned in the Results, even if the Table was relocated to the Supporting Information. This data does call into question the specificity of the antibodies, or the specificity of the PCR, considering the Ang-2 correlation was significantly negatively correlated, and the Tie-1 and Tie-2 correlations were considerably weak.

**********

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 #3: No

Reviewer #4: 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.

PONE-D-19-33270R3

HIGH EXPRESSION OF TIE-2 PREDICTS POOR PROGNOSIS IN PRIMARY HIGH GRADE SEROUS OVARIAN CANCER

PLOS ONE

Dear Dr. Sopo,

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 address the comments made by the reviewers. Additionally, please note that Reviewer #4 has said that not all data underlying the findings in the manuscript are fully available (see point 4 below) - please ensure this is addressed in your response.

Additionally, Reviewer #3 has noted a number of typographical and grammatical errors. PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous, therefore we suggest you thoroughly copyedit your manuscript for language usage, spelling and grammar. If you do not know anyone who can help you with this, you may wish to consider employing a professional scientific editing service. 

Please submit your revised manuscript by Oct 18 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Elizabeth Christie

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 #3: (No Response)

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: No

**********

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 #3: Yes

Reviewer #4: 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 #3: 1. This section of the Discussion is much improved. However, in acknowledgement that this study does not provide any direct conclusions regarding the relationship between Ang/Tie expression and angiogenesis in ovarian cancer, please delete the word “angiogenic” from the conclusion in line 452, and insert the word “potentially” before “reflecting the more active angiogenesis” on line 449. Possible effects of tumour-expressed Tie2 and Ang2 on pro-angiogenic signalling through endothelial-expressed receptors can be inferred and discussed but they have not been shown directly in this study.

2. Original Q3: Thankyou for the additional detail in the caption for Figure S1, but please be more precise with the descriptions. In the Methods the authors have defined the negative controls as tissues stained with no primary antibody, whereas in the caption the negative controls are described as “negative control tissue”. “Negative control tissue” would imply an image derived by staining a tissue that does not endogenously express a protein with a specific antibody for that protein. Panels b), d) and f) are probably better described as “ovarian carcinoma negative control staining (primary antibody omitted)”. A more stringent standard for IHC controls would be to perform specific antibody staining and negative control staining on serial sections of the same (positively staining) tissue; thus it is not ideal that the Tie1 positive control is tonsil and the negative control is ovarian carcinoma.

3. Original Q4: The fact that IHC and qRT-PCR results for Ang2 correlate negatively in one tissue and positively in another is confusing. The authors may therefore wish to consider removing this information from the abstract.

4. Original Q6: It will be important for the authors to check the final formatting of Table 3 by the journal to ensure the results are read correctly and the results and parameter headings are in the correct hierarchy. I would suggest adding a subheading “immunohistochemical staining” above the IRS results and e.g. “clinical features” above stage/ascites and then un-bolding the IRS parameters and stage/ascites to bring these into better alignment with the other sections. This will make it clearer which comparisons relate to overall survival and which to progression free survival when both sets of results are presented within the one table.

5. Original Q7: The commas in Tables 2 and S3 appear not to have been changed to decimal points. Please amend. Also the presentation of the data in Table S3 is still confusing. Please clarify in the table caption if the data presented here represent epithelial staining, stromal staining or both. The text (p15 line 285) states that low Tie-1 expression was associated with a greater residual tumour size (>1cm) after primary surgery (p = 0.008), but in Table S3 the p value 0.008 is placed corresponding to zero residual tumour. Similarly I cannot find the stated p = 0.006 corresponding to significantly lower Tie1 expression in high grade tumours, and there are several other examples where the p values cited in the text cannot be found against the corresponding variables in Table S3. Why is this? I assume the remaining blank spaces against a parameter in this table (e.g. for >1 cm residual tumour, Yes recurrence) imply that that parameter was used as the reference for the comparison (with the exception of Histology where there is no obvious reference). Should the 0 residual tumour be the reference condition in Table S3 as it is in Table 3? Please confirm this and ensure that the results are correctly aligned within the table. Also change “0” residual tumour in Table S3 to “None” to be consistent with Table 3. In Table 4 the reference conditions are explicitly labelled but I don’t think this is the norm. Be consistent throughout all tables.

6. Original Q8: OK

7. Original Q9: OK

8. Original Q11: Thankyou, this part of the discussion is much improved.

Other comments and Typographical/grammatical errors:

I have roughly proofread the manuscript for English expression. Note that this journal does not provide copyediting, so the authors must please proofread all text, figures and tables very carefully.

Figures and Tables should be referred to as “Figure X” or “Table X” throughout – this appears to only be the wrong way around for the supplementary figures and tables.

P2 line 37: add comma after “ovarian cancer”

P2 line 39 and line 41: change “expressions” to “expression”, and delete “the” before “angiopoietin-2” on line 39.

P3 line 64: add comma after “angiogenesis”

P3 line 70: There are many endothelial growth factor systems. Sentence should read “Other endothelial growth factor systems, such as the angiopoietin-Tie complex, have not been as extensively studied.”

P3 line 81: replace “speeded up” with “accelerated”; remove “the” before “targeted”

P4 lines 89-90: amend to read “has prolonged median progression free survival (PFS)”

P4 line 100: delete “the” before “dissemination”

P4 lines 103, 104: amend “expressions” to “expression

P8 line 143: delete “the” before “written”

p11 line 238: amend to “between expression in”

p14 lines 255-257: section heading could be amended to “Expression of angiogenic factors in primary high grade serous tumours as compared to related metastases”. First sentence could be amended to “Expression of both Ang-2 and Tie-2 …. was”

p14 lines 265-272: the concept of “qRT-PCR levels” doesn’t make sense – qRT-PCR is a technique that measures mRNA levels. I would suggest amending this section to the following:

Correlation of qRT-PCR results with immunohistochemical staining

Ang-2 IRS was significantly negatively correlated to the mRNA levels of Ang-2 measured by qRT-PCR in primary ovarian cancer (r = -0.64, p = 0.002). The correlation was even stronger when only high grade serous tumors were counted (r = -0.868, p < 0.001) (Table S2). In metastatic tumors Ang-2 PP was strongly correlated to the corresponding mRNA levels (r = 0.752, p = 0.005) (Table S2). Tie-1 and Tie-2 qRT-PCR values correlated strongly to each other (r = 0.919, p < 0.001), but did not have statistically significant correlations to the respective immunohistochemical expression. In metastases, mRNA levels of Ang-2 correlated strongly with Tie-2 mRNA (r= 0.61, p= 0.016).

Likewise in the caption:

S2 Table.

Correlation of qRT-PCR results with immunohistochemical staining. Ang-2 IRS and PP were correlated with relative mRNA levels as measured by qRT-PCR. There were 22 primary tumor samples and 15 metastatic samples in each group.

P14 lines 276-277: delete “the” before “ovarian” and “greater”

P15 line 280: delete “the” before “resistance”

P15 line 297: delete “the” before “ovarian”

P16 line 324: delete “the” before “significance”

P19 line 354: amend “expressed widely” to “widely expressed”

P21 line 390: add comma after “in addition”

P21 line 398: amend to “Expression of Ang-2 and Tie-2”

P22 line 414-415: delete “the” before “omental”; amend “exfoliating” to “exfoliation”; add “the” before “omental surface”

P22 lines 425-427: delete “the” before “lower” and “larger residual tumour”. Also every instance of “the” could be deleted in the sentence beginning “The expression of Tie-1”.

P23 line 434: amend to “Ang-2 specific andtibody and VEGF blocker aflibercept more strongly reduced tumour growth”

P23 line 453: delete “the” before “OS”.

Reviewer #4: (No Response)

**********

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 #3: No

Reviewer #4: 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.

PONE-D-19-33270R4

HIGH EXPRESSION OF TIE-2 PREDICTS POOR PROGNOSIS IN PRIMARY HIGH GRADE SEROUS OVARIAN CANCER

PLOS ONE

Dear Dr. Sopo,

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 Nov 07 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Elizabeth Christie

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 #3: (No Response)

Reviewer #4: (No Response)

**********

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 #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: No

**********

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 #3: Yes

Reviewer #4: 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 #3: All of my previous comments have been satisfactorily addressed. However there are still some problems with Table S3.

Original Q7: a) some of the data in Table S3 still seems to be misaligned. Should all of the p values cited for the Stage analyses be aligned with the III-IV rows rather than the I-II rows? Similarly, should the p values related to Recurrence all be aligned with the Yes rows?

b) Thankyou for your explanation of the reporting of p values for serous vs total tumours in Table. S3. Please explain this more clearly in the legend, e.g. “* p value reported for serous tumours when only comparison for this subgroup was statistically significant”. However, if this is the case, why are the p values 0.938 and 0.605 corresponding to Tie1 staining with residual tumour <1cm also marked with *? Do these p values represent the whole study population, or serous tumours only? To avoid confusion, please present the all p values (significant or not) for the whole study population in Table S3 to ensure consistency throughout the table. For the instances where the p value was only statistically significant for serous tumours, include this value with * and in brackets after the value for the whole study population; e.g. 0.###(0.008*).

Reviewer #4: Please carefully review the PLOS Data Policy https://journals.plos.org/plosone/s/data-availability

The following points extracted from the Policy are particularly relevant:

"PLOS journals require authors to make all data necessary to replicate their study’s findings publicly available without restriction at the time of publication. When specific legal or ethical restrictions prohibit public sharing of a data set, authors must indicate how others may obtain access to the data."

"Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods... For example, authors should submit the following data: The values behind the means, standard deviations and other measures reported;

The values used to build graphs..."

"Stating ‘data available on request from the author’ is not sufficient."

"PLOS recognizes that, in some instances, authors may not be able to make their underlying data set publicly available for legal or ethical reasons. This data policy does not overrule local regulations, legislation or ethical frameworks. Where these frameworks prevent or limit data release, authors must make these limitations clear in the Data Availability Statement at the time of submission... For studies involving human research participant data or other sensitive data, we encourage authors to share de-identified or anonymized data. However, when data cannot be publicly shared, we allow authors to make their data sets available upon request. If there are ethical or legal restrictions on sharing a sensitive data set, authors should provide the following information within their Data Availability Statement upon submission: Explain the restrictions in detail (e.g., data contain potentially identifying or sensitive patient information); Provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent."

The authors have stated in their Data Availability Statement that "All relevant data are within the manuscript and its Supporting Information files." This is not the case, as all data necessary to replicate the various analyses, tables and figures is currently not in the Supporting Information files. Please refer to the PLOS Data policy.

**********

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 #3: No

Reviewer #4: 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.

HIGH EXPRESSION OF TIE-2 PREDICTS POOR PROGNOSIS IN PRIMARY HIGH GRADE SEROUS OVARIAN CANCER

PONE-D-19-33270R5

Dear Dr. Sopo,

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,

Elizabeth Christie

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 #3: All comments have been addressed

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: (No Response)

Reviewer #4: (No Response)

**********

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 #3: No

Reviewer #4: No