PONE-D-20-36704

Improving the diagnostic recognition of thoracic endometriosis: Spotlight on a new histo-morphological indicator

PLOS ONE

Dear Dr. Okafor,

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.

This study addressing novel histological features in thoracic endometriosis has been revised by three reviewers. Although they have recognized the interest of the paper, they have raised some concerns that need to be addressed. The Authors should consider all the issues raised, particularly those from Reviewer #3. The paper has too many figures. The Authors should follow suggestions from Reviewer #3 in this regard and consider to list figures as Fig 1, A,B,C ect. More importantly, the statistical analysis has to be better described in Materials and Methods. The Authors have to explain how they mentioned specificity and sensitivity of the various patterns without having calculated them based on ROC curves and AUC analysis. Words related to the statistical analysis need to be more precise.

Please submit your revised manuscript by Mar 04 2021 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Paola Viganò

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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: I Don't Know

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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: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I congratulate the Authors for this interesting paper. Histological diagnosis of thoracic endometriosis in not as common as the clinical diagnosis. I would suggest to the Authors to report in the Discussion section a brief view of the literature about the discrepancy between the clinical and histological diagnosis reported by different centers.

Reviewer #2: The authors reported an interesting study regarding the pathological features of thoracic endometriosis. The paper il well written and interesting. I have some minor points that should be reviewed:

- Introduction, line 46, 47: please, cite some possible theory regarding etiology of TE

- Introduction should be shorten

- Material and methods, from line 106 to 114: this part should be moved in the results part

- Discussion: I would suggest discussing also when TE is impossible to be proved by histology (e.g.: diaphragmatic hernias). For instance, recently it has been reported (Viti A, et al. Endometriosis Involving the Diaphragm: A Patient-Tailored Minimally Invasive Surgical Treatment. World J Surg. 2020 Apr;44(4):1099-1104.) that up to 45% of patient with diaphragmatic endometriosis had no histological diagnosis. Please discuss on this.

Reviewer #3: The article needs some work. And I would like to give you some suggestions.

First of all in endometriosis there are endometrioid glands and endometrioid stroma (not endometrial).

1) periods lines 63-68 are confusing; be concise.

2) lines 68-70

To support this, one study [8] found that less than 50% of laparoscopic biopsies suspicious for endometriosis were really endometriosis.

3) delete lines 76-83

Insert the following:

Histopathologic features of endometriosis include the presence of endometrioid glands, stroma and stroma; hemosiderin-laden macrophages are also commonly present (Flieder 14). However, in contrast to the more common pelvic endometriosis, in thoracic endometriosis the endometrial glands are not always present in biopsy material. In problematic cases immunohistochemical analysis using ER, PR, and CD10 may confirm the endometrioid glands and stroma.

4) Matherial and methods

insert from results:

The interval between the onset of symptoms 239 and definitive histopathological

diagnosis was 49 weeks (range = 1 week to 300 weeks).

5) Figures

There are too many figures.Delete figures 2, 4, and 11. Change the order of the rest:

Fig 1: Typicaltoracic endometriosis simulating eutopic proliferative endometrium.H&E staining, original magnification X300.

Fig 2 (ex Fig 10): Dilated endometrioidgland with intraluminal pseudoxanthoma cells; the surroundinginconspicousendometrioid stroma merges imperceptively into non specificedematous stroma. H&E staining, original magnification X150.

Fig 3 (ex 5): Lymphocytic aggregate and dilated and congested blood vessels in edematous non specificstroma; a distorted endometrioidgland is also present. H&E staining,original magnification X100.

Fig 4 (ex 3): Edematous hyalinized stroma with congested capillaries and

siderophages with golden-brown pigments. H&E staining, original magnification

X150 & X300.

Fig 5 (ex 6): Macrophages with brownishfinely granular cytoplasm (pseudoxantoma cells). H&E staining, original magnification X300.

Fig 6 (ex 7): Foamyhistiocytes in the stroma. H&E staining,original magnification X150.

Fig 7 (ex 8): Crystals andmultinucleated foreign body-type giant cells. H&E staining, original magnification X300.

Fig 8 (ex 9): Bundles of smooth muscle cells.H&E staining, original magnification X150.

Fig 9 (ex12): Pleural fibrosis. H&E staining, originalmagnification X150.

6) Results

Change the order:

Nine notable histological features were identified in varying proportions in the test

(TE) population, namely endometrial glandsand or stroma, lymphoid clusters, ceroid macrophages,siderophages, cholesterol crystals, smooth muscle bundles, capillary congestion,multinucleated giant cells and fibrosis (Figs 1 - 12).

Twenty-four cases (92%) showed endometrial glands but the finding was sometimes

focal and required the examination of several sections to be identified. The glands were either inactive or slightly proliferative; no secretory gland was seen. Occasionally, they appeared atrophic and were lined by a single layer of cuboidal or flat epithelial cells. Hobnail and clear cell metaplasia were the metaplastic changes occasionally seen. Two cases (8%) lacking endometrioid grands were classified as stromal endometriosis. with siderophages, ceroid macrophages, foreign body-type multinucleated macrophages, cholesterol crystals, capillary congestion and fibrosis. In addition these 2 patients complained of catamenial chesty symptoms suggestive of thoracic endometriosis. Bundles of smooth muscle, distinc from the wall of blood vessels, were seen i 4 cases (15%).

Three histological features, namely lymphoid clusters, fibrosis and capillary

congestion, were present in all the 26 test cases (100%) (Fig 13). Overall, there are

6 prevalent features, adding siderophages, endometrioidglands and or stroma and ceroid

macrophages to the earlier 3, which, individually are present in the majority of the

cases (13 or more cases or 50%); all 6 together are present in 16 cases (62%); and

in different permutations are present in all 26 cases (100%). These 6 features, being

prevalent, constitute the backbone for diagnosis of TE and will be referred to as

Group A.

The rest may be ok.

7) Discussion

The histologic diagnosis ofendometriosis is based onthe typical presence of both endometriotic glands and stroma. The glands almost always show typical endometrioid features, usually of proliferative or inactive type; the stroma may be typical with the spiral arteries, which may be engorged with erythrocytes, and provide a helpful initial clue to the identification of the endometriotic nature of the lesion. However, in many cases the stroma may be very subtle, confined to a thin or poorly defined often discontinuous periglandular zone; it can undergo smooth muscle metaplasia or replaced by histiocytes of “foamy type”. Small congested arterioles and capillaries (referred to as capillary congestion) may draw attention to the lesion at scanning magnification. In some cases stroma only is found in biopsies and the preferred terminology is stromal endometriosis (17, 18-20).

Similarly with abdominal or pelvic endometriosis, glandular and stroma breakdown may occur favoringe haemorrhage. Clusters of pigmented histiocytes become common, including ceroid-laden macrophages and haemosiderin-laden macrophages or siderophages. In older lesions fibrosis, lymphoplasmacellular infiltrate, with follicle formation, and foreign body reaction with cholesterol crystals are common (18-20).

A high index of suspicion by the clinician as well as the experience of the pathologist are necessary for a correct diagnosis to avoid [21] erroneous classification as nonspecific pleuritis, fibrinous, pleuritis and similar. In our series, three cases had to be re-examined by the pathologist at the request of the surgeons after the initial diagnosi of nonspecific pleuritis

Summarizing the results of this study in a Venn plot (Fig 16) we can see the overlap

of Groups A, B and C. It is notable that the innermost overlapping segment in the 3

circles contains all the features of Group C. Furthermore, at least 2 Group C features

are present in all TE cases. Given the high sensitivity and specificity to the diagnosis of TE, we recommend that Group C be used as criteria for diagnosis of thoracic endometriosis, as shown in the first column of Table 3, of which any 2 features are diagnostic of the disease. In addition, the identification of any of these 4 Group C features in any thoracic biopsy in a feature should serve as an alarm bell for considering the possibility of TE.

8) Finally I hope that another reviewer will check on statistical analisis.

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Reviewer #1: Yes: Paola Ciriaco

Reviewer #2: Yes: Pietro Bertoglio

Reviewer #3: No

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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-20-36704R1

Improving the diagnostic recognition of thoracic endometriosis: Spotlight on a new histo-morphological indicator

PLOS ONE

Dear Dr. Okafor,

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.

==============================

ACADEMIC EDITOR:

-The Materials and Methods section is still insufficent. The lab protocol needs to be better detailed. How were the various histopathological features identified?

- For the statistical analysis, it is not necessary to explain the tests. The authors have to explain HOW they performed the tests. Results from the paper should be confirmed by other scientists. If others are not able to confirm the results, the study has no value.

- A R script cannot be presented as supplementary table. Again, data need to be understood by everyone. Please transform the R script in something that is understandable. Moreover, correct S1 R code all over the text. - It is unclear how the cut offs for sensitivity and specificity were established. These explanations should be in the statistical analysis section.

- A great limit of the paper is that the reliability of the new diagnostic test should be evaluated in a different cohort. Although collecting a new cohort might be difficult, the Authors should declare this limit of the study in the Discussion section.

This novel approach with new histopathologic findings is interesting but the paper still needs substantial ameliorations.

Please submit your revised manuscript by Apr 30 2021 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,

Paola Viganò

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The materials and methods section is still insufficent. The lab protocol needs to be better detailed. How were the various histopathological features identified? For the statistical analysis, it is not necessary to explain the tests. The authors have to explain how they performed the tests. Results from the paper should be confirmed by other scientists. If others are not able to confirm the results, the study has no value.

A R script cannot be presented as supplementary table. Again, data need to be understood by everyone. Please transform the R script in something that is understandable. Moreover, correct S1 R code all over the text.

It is unclear how the cut offs for sensitivity and specificity were established. These explanations should be in the statistical analysis section.

A great limit of the paper is that the reliability of the new diagnostic test should be evaluated in a different cohort. Although collecting a new cohort might be difficult, the Authors should declare this limit of the study in the Discussion section

This novel aaproach with new histopathologic findings is interesting but the paper still needs substantial ameliorations

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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 #2: 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 #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: Yes

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: (No Response)

Reviewer #2: The authors significantly improved their paper after revisions and they should be commended.

I appreciate their reply to my last comment and I do agree that their paper is about histological features of thoracic endometriosis, but I think it may be worth to briefly discuss that histological proof of endometriosis is not always possible; as a matter of fact, in some cases diagnosis is made on macroscopic details (e.g. diagphragmatic hernias) and patients’ medical history. As suggested by dr. Triponez and colleagues (Triponez F, Alifano M, Bobbio A et al (2010) Endometriosis related spontaneous diaphragmatic rupture. Interact Cardiovasc Thorac Surg 11(4):485–487) diaphragmatic rupture might be the result of the involution of endometriosis tissue. What should the authors suggest to biopsy in these cases of no evidence of macroscopic endometriosis tissue? How did the authors manage similar cases? Maybe authors should also briefly describe in the results the macroscopical features of endometriosic lesions they resected.

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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: Yes: Paola Ciriaco

Reviewer #2: Yes: Pietro Bertoglio

[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.

Improving the diagnostic recognition of thoracic endometriosis: Spotlight on a new histo-morphological indicator

PONE-D-20-36704R2

Dear Dr. Okafor,

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.

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Kind regards,

Paola Viganò

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The Authors have addressed the issues raised

Reviewers' comments: