PONE-D-20-37191

Adaptor protein XB130 regulates the aggressiveness of cholangiocarcinoma

PLOS ONE

Dear Dr. Ungareevittaya,

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.

At the end of this letter you will find the reports and comments. Please read also carefully the suggested literature.and remind that you are free to choose , which references you will cite or not, if at all.    

Please submit your revised manuscript by Mar 13 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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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.

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We look forward to receiving your revised manuscript.

Kind regards,

Konradin Metze

Academic Editor

PLOS ONE

Additional Editor Comments :

The investigation is original and quite interesting but there are important points to be considerably improved before the paper can be considered for publication:

The authors categorized a lot of variables initially expressed as cardinal numbers (which causes an important loss of information and may induce considerable bias).In particular,  the immunohistochemical quantification should also be expressed in cardinal numbers and not dichotomized. .

We are very aware, that this kind of “data treatment” is still popular among pathologists, but it is a great source of error and publications with low credibility. Therefore, all calculations must be repeated with cardinal numbers. The results might be different.

In a final step, the authors may suggest a score after categorizing of one or another variable, but this must be clearly justified both from a mathematical and a biological point of view and a comparison of the calculi done with and without categorizing must be given. (please see the last paper  for how to do this).

Please consult the following literature:

Dichotomizing continuous predictors in multiple regression: a bad idea. Stat Med 25: 127-141, 2006

Pitfalls in the assessment of prognostic factors. Lancet Oncol. 2011 Nov;12(12):1095-6.

Dichotomizing continuous prognostic factors can cause paradoxical results in survival models. J Am Coll Surg. 2011 Jan;212(1):132-4.

Dichotomization of continuous data--a pitfall in prognostic factor studies. Pathol Res Pract. 2008;204(3):213-4

Breaking up is hard to do: the heartbreak of dichotomizing continuous data. Can J Psychiatry 2002;47:262– 66.

A simple score derived from bone marrow immunophenotyping is important for prognostic evaluation in myelodysplastic syndromes  Sci Rep. 2020 Nov 20;10(1):20281.

Konradin Metze, Academic Editor

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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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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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: 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: The artical is clearly written with minimal spelling and grammatical error.

Very little data regarding mechanism of action is presented. The authors show that a CCA cell line with high XB130 expression has faster proliferation, more motility and more extensive invasiveness than a normal or two CCA cell lines with low XB130 expression using in vitro assays. The authors also show that these properties can be suppressed in the high expresser by siRNA knockdown of XB130 as compared to a scrambled control. What is not presented is why XB130 expression promotes these activities. Several pathways downstream of XB130 are mentioned in the introduction. The authors should inhibit individual downstream pathways (e.g., PI3K/Akt) to test whether such inhibition recapitulates the effect of siRNA knockdown of XB130. The authors should also over-express XB130 in MMNK1 cells and XB130-low CCA cells to test whether this expression is sufficient to confer the proliferative, motility and invasiveness characteristics of XB130-high CCA cells. There is published data indicating that XB130 can promote tumorigenesis via activation of the PI3K/Akt pathway, but it would improve the manuscript to show that in this system. As it is now, too much of the manuscript's mechanistic conclusions rest on a single CCA cell line. More controls and more mechanistic studies are needed for this aspect.

For use as a biomarker, the authors need to report specificity and sensitivity. Discussion should also be provided that compares the sensitivity and specificity of XB130 against existing CCA biomarkers of which there are now a great number.

Table 1 should include definitions of staging features:

N0 (No regional lymph node metastasis)

N1 (Regional lymph node metastasis)

M0 (No distant metastasis)

M1 (Distant metastasis)

T1 (Solitary tumor; no vascular invasion)

T2a (Solitary tumor with vascular invasion)

T2b (Multiple tumors)

T3 (Tumor that has perforated the visceral peritoneum or otherwise demonstrating invasivity)

T4 (Tumor with periductal invasion).

Stage I: T1+N0+M0

Stage II: T2+N0+M0

Stage III: T3+N0+M0

Stage IVA: T4+N0+M0

Stage IVB: T(1-4)+N1 or T(1-4)+M1

Reviewer #2: The authors analyze the expression of the adaptor protein XP130 in 151 cases with gall bladder carcinoma. They note poor survival rates in cases with high XP 130 expression, which they confirm by studies of 4 cell lines. This is a good article and should be published provided that

1. the discrepancies in table 1 (M0, M1<> TNM); (N0, N1<> Lymph node metastases) etc. should be explained 7 corrected

2. What about pTNM?, Tumor size, organ of metastasis (liver, others?)

3. The collection time (2007 - 2017) is quite long; what about changes of the TNM classification during that time?

4. The aspect of tumor cell heterogeity should be briefly adressed (for example see and cite: VULETIĆ, Filip et al. Intratumoral heterogeneity. Diagnostic Pathology, [S.l.], v. 4, n. 1, feb. 2018. ISSN 2364-4893. Available at: <http: 257="" article="" content="" dpath="" index.php="" view="" www.diagnosticpathology.eu="">5. The IHC color classification threshold / method should be explained more in detail (which color space, which color adjustment / image quality analysis has been performed (for details see and cite: KAYSER, Klaus et al. Texture and object related image analysis in microscopic images. Diagnostic Pathology, [S.l.], may 2015. ISSN 2364-4893. Available at: <http: 14="" article="" content="" dpath="" index.php="" view="" www.diagnosticpathology.eu="">.</http:></http:>

Reviewer #3: To the editor/authors:

The study by Poosekeaw et al, deals with the role of the adaptor protein XB130 in cholangiocarcinoma. The authors present data using 5 different cell lines, and one of them expressing XB130. For further experiments, they have used XB130-expressing cell line and via siRNA they knocked down XB130. They could show that XB130 plays a role in proliferation, migration and invasion, at least in this cell line. Additional, they have analysed XB130 expression in 151 CCA tissue samples and could find a correlation of XB130 expression, M classification and LVSI.

Although of interest, following improvements have to be done before it can be considered for publication in Plos One.

Critical points:

1) Figure 1: First, the authors should mention in the fig-legend that tubulin serves as a loading control. Second, please indicate kDa of target protein in brackets. Furthermore, it would also be interesting to know why the other cell do not express XB130 at all. Have the authors checked mRNA expression? Is there a post-transcriptional modification? Are there any mutations or epigenetically silencing in/of XB130 known?

2) Figure 2: General for Fig2A-F. It would be a nice control to see all parameters in untreated KKU-213A (meaning without any transfection). Next point: the authors show a complete down regulation after 48h but, if I understood the data right, the results of proliferation, migration and invasion were analysed after additional 24h? In order to check the efficiency of the XB130 knock down a time course would be helpful – Fig2A: Western Blot after 24, 48 and 72h post-transfection. An additional control experiment would be to use a XB130 non-expressing cell line (e.g. KKU213C).

3) Figure 3: Please give the scale bar! Furthermore, it would be helpful to mark the CCA cells (e.g. with a *), normal bile duct (e.g. with an arrow) and hepatocytes (e.g. with an arrowhead) for non-histologists.

4) Fig 4, legend: please indicate the number of patients (n)

5) Have the authors checked for downstream targets, like PI3K or Akt? These experiments could be easely done by Western Blotting and would underline the specific role of XB130 in tested cell lines and in FFPE material (in this case testing by IHC).

6) A further suggestion/question: are data available for Ki67 level in the FFPE samples?

7) Have the authors checked, both cell lines (WB) and FFPE (IHC), EMT-related targets (N-, E-cadherin, vimentin, or EMT-TFs, e.g. TWIST, SNAI, ZEB)? I think these experiments would improve the paper and underline the conclusions the authors have done!

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-20-37191R1

Adaptor protein XB130 regulates the aggressiveness of cholangiocarcinoma

PLOS ONE

Dear Dr. Ungareevittaya,

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 read carefully my ccomments below !

Please submit your revised manuscript by Jun 03 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. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Konradin Metze

Academic Editor

PLOS ONE

Academic Editor´s comments :

I am not satisfied with the changes. There are still variables, measured originally in cardinal numbera, but tater categorized , such as age. Why  is there a cut point at 50 years, instead of 45, 47 , 55 etc. ?

 This decision is completely arbitraly and therefor unscientific.

Before applying  quartiles, the authors should calculate with the cardinal numbers of raw data  This applies to the correlations, as well as to Cox and logistic regressions !

The  graphic  in the reply to the editor should be i n t e g r a  t e d  into the manuscript.  Regarding this graphhic  please try non-linear curve-fittings , perhaps a logarithmic  or , perhaps still better, a hyperbolic  function  and give the Rsquares as estimates of goodness-of –fit !.  Please discuss this result ! 

In summary : You should calculate all models first with continus cardinal numbers whenever possible.  In a

s e c o n d  step you can establish models  using the quertiles, but always with a l l  other variables measured

in cardinal numbers  n o f categorized.

Please sen a copy m where you highlight your  changes.

Konradin Metze, Academic Editor 

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: (No Response)

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 #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: 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: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

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

Reviewer #1: (No Response)

Reviewer #2: The authors have responded to all recommendations of the reviewers, and the article should be published in its revised form.

Reviewer #3: The authors have answered all my questions and comments. Furthermore, the could nicely show the downstream effects of XB130 in the cell line. To my hand the manuscript can be accepted for publishing in PlosOne.

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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 #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-37191R2

Adaptor protein XB130 regulates the aggressiveness of cholangiocarcinoma

PLOS ONE

Dear Dr. Ungareevittaya,

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 corect according to the suggestions you find below !

Please submit your revised manuscript by Aug 19 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. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Konradin Metze

Academic Editor

PLOS ONE

Additional Editor Comments 

The paper has been improved. Problems are still found in  table 1 . Are these data really all correlations ?Which kind of correlation ? Spearman ? Kendall ? Pearson ? The correlation coefficients are all missing  ! Please add the coefficients together with their confiidence intervals in all cases with p<0.05

Konradin Metze, Academic Editor

[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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

**********

6. Review Comments to the Author

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

Reviewer #1: All of my past concerns were addressed in the prior review.

The current submission addresses questions regarding statistical methodology made by a different reviewer. I am satisfied with the revisions to the statistics provided in the current submission.

All data was and is provided. The new submission additionally has the blots used to generate the figures of the manuscript.

The English is intelligible and written in standard English

Reviewer #2: no comments can be made, a system's error is very likely. Unfortunately, I cannot be of any additional assistance.

Reviewer #3: (No Response)

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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 #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Adaptor protein XB130 regulates the aggressiveness of cholangiocarcinoma

PONE-D-20-37191R3

Dear Dr. Ungareevittaya,

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,

Konradin Metze

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: