Reviewer ’s report  
 
Title:  High expression of the Claudin -2 in esophageal carcinoma and precancerous lesions is 
significantly associated with the bile salt receptors VDR and TGR5  
 
Version: 0 Date: 04 Oct 2016 
 
Reviewer:  Anna  Casselbrant  
 
Reviewer’s report:  
 
General:  
 
The study by Zhou Z e t al., with the title "High expression of the leaky protein Claudin -2 in 
esophageal carcinoma and precancerous lesions is significantly associated with the bile salt 
receptors VDR and TGR5" is to explore the Claudin -2 expression and its association with VD R 
and TGR5 expression. The paper is well writing and important in this field.  
 
I do however, have the following comments.  
 
The authors have previously showed claudin -2 expression in human colon cancer cells and that 
this claudin -2 expression is a target gene of VDR. They have also showed that VDR and TGR5 
are highly expressed in EAC.  
 
In the present study they just showed the claudin -2 expression. The association or the 
relationship (or correlation?) to the VDR and TGR5 is poorly described. I miss the expression 
analysis of VDR and TGR5 in the results and how the association was made. Moreover, I also 
think the expression of Cl audin -2 is poorly described in the paper. Where did you find claudin -2?  
 
Can you also describe the hypothesis behind the association between claudin -2 and the VDR and 
TGR5 receptors in esophageal carcinoma? Previously paper has described Claudin -2 expressi on 
in basal layer of esophageal squamous epithelium (ref. Bjorkman E, et al, Scand J 
Gastroenterology 2013), whereas the bile acids are coming into esophagus lumen during 
duodenal reflux. Can you describe where the bile acid receptors are and the connectio n to 
Claudin -2 expression?  
 
 
Specific comments:  
 
1. In the abstract: There are abbreviations for VDR and TGR5. Please sign it out!  
 
1. In background: Chronic exposure (lane 25 -26)….induces CDX -2 expression? Can you 
explain and sign it out!  
 
2. In backgrou nd: Bile acids induce inflammatory gene expression (lane 35 -36) .…through 
the bile acid reseptors FXR, RXR and VDR Why not mentioned TGR5 in this sentence?  
3. Material and methods: Why not mentioned the patients for TMA first?  
 
4. Material and methods: Why  describing immunohistochemistry for VDR and TGR5 but 
not showing that in results (see comments above).  
 
5. Results: Describe and show the expression of claudin -2, VDR and TGR5 in the results 
(figure?).  
 
6. Results: There is no figure -reference for figure 2 -4 in the text.  
 
7. Sometime you write "claudin -2", sometime "Claudin2" or sometime "Claudin 2". Use the 
same version.  
 
8. Results: TNM staging! Please sign it out!  
 
9. Discussion: I miss some reference showing claudin expression in GERD (lane 34 -35) 
together  with Mönkemuller et al. and this reference is: Bjorkman E, et al, Scand J 
Gastroenterology 2013 (the same as above).  
 
10. Discussion: Skip the section about the number of sample (lane 4 -21). Just conclude that 
there are differences in number of sample incl uded in these studies.  
 
11. Abbreviations in table 2!  
 
My suggestion is acceptance of the study after revision.  
 
Are the met hods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the aut hors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees,  funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be include d in my named report can be included as confidential comments 
to the editors, which will not be published . 
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title:  High expression of the Claudin -2 in esophageal carcinoma and precancerous lesions is 
significantly associated with the bile salt receptors VDR and TGR5  
 
Version: 0 Date: 04 Oct 2016 
 
Reviewer:  Anna  Casselbrant  
 
Reviewer’s report:  
 
General:  
 
The study by Zhou Z e t al., with the title "High expression of the leaky protein Claudin -2 in 
esophageal carcinoma and precancerous lesions is significantly associated with the bile salt 
receptors VDR and TGR5" is to explore the Claudin -2 expression and its association with VD R 
and TGR5 expression. The paper is well writing and important in this field.  
 
I do however, have the following comments.  
 
The authors have previously showed claudin -2 expression in human colon cancer cells and that 
this claudin -2 expression is a target gene of VDR. They have also showed that VDR and TGR5 
are highly expressed in EAC.  
 
In the present study they just showed the claudin -2 expression. The association or the 
relationship (or correlation?) to the VDR and TGR5 is poorly described. I miss the expression 
analysis of VDR and TGR5 in the results and how the association was made. Moreover, I also 
think the expression of Cl audin -2 is poorly described in the paper. Where did you find claudin -2?  
 
Can you also describe the hypothesis behind the association between claudin -2 and the VDR and 
TGR5 receptors in esophageal carcinoma? Previously paper has described Claudin -2 expressi on 
in basal layer of esophageal squamous epithelium (ref. Bjorkman E, et al, Scand J 
Gastroenterology 2013), whereas the bile acids are coming into esophagus lumen during 
duodenal reflux. Can you describe where the bile acid receptors are and the connectio n to 
Claudin -2 expression?  
 
 
Specific comments:  
 
1. In the abstract: There are abbreviations for VDR and TGR5. Please sign it out!  
 
1. In background: Chronic exposure (lane 25 -26)….induces CDX -2 expression? Can you 
explain and sign it out!  
 
2. In backgrou nd: Bile acids induce inflammatory gene expression (lane 35 -36) .…through 
the bile acid reseptors FXR, RXR and VDR Why not mentioned TGR5 in this sentence?  
3. Material and methods: Why not mentioned the patients for TMA first?  
 
4. Material and methods: Why  describing immunohistochemistry for VDR and TGR5 but 
not showing that in results (see comments above).  
 
5. Results: Describe and show the expression of claudin -2, VDR and TGR5 in the results 
(figure?).  
 
6. Results: There is no figure -reference for figure 2 -4 in the text.  
 
7. Sometime you write "claudin -2", sometime "Claudin2" or sometime "Claudin 2". Use the 
same version.  
 
8. Results: TNM staging! Please sign it out!  
 
9. Discussion: I miss some reference showing claudin expression in GERD (lane 34 -35) 
together  with Mönkemuller et al. and this reference is: Bjorkman E, et al, Scand J 
Gastroenterology 2013 (the same as above).  
 
10. Discussion: Skip the section about the number of sample (lane 4 -21). Just conclude that 
there are differences in number of sample incl uded in these studies.  
 
11. Abbreviations in table 2!  
 
My suggestion is acceptance of the study after revision.  
 
Are the met hods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the aut hors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees,  funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be include d in my named report can be included as confidential comments 
to the editors, which will not be published . 
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title:  High expression of the Claudin -2 in esophageal carcinoma and precancerous lesions is 
significantly associated with the bile salt receptors VDR and TGR5  
 
Version: 0 Date: 04 Oct 2016 
 
Reviewer:  Anna  Casselbrant  
 
Reviewer’s report:  
 
General:  
 
The study by Zhou Z e t al., with the title "High expression of the leaky protein Claudin -2 in 
esophageal carcinoma and precancerous lesions is significantly associated with the bile salt 
receptors VDR and TGR5" is to explore the Claudin -2 expression and its association with VD R 
and TGR5 expression. The paper is well writing and important in this field.  
 
I do however, have the following comments.  
 
The authors have previously showed claudin -2 expression in human colon cancer cells and that 
this claudin -2 expression is a target gene of VDR. They have also showed that VDR and TGR5 
are highly expressed in EAC.  
 
In the present study they just showed the claudin -2 expression. The association or the 
relationship (or correlation?) to the VDR and TGR5 is poorly described. I miss the expression 
analysis of VDR and TGR5 in the results and how the association was made. Moreover, I also 
think the expression of Cl audin -2 is poorly described in the paper. Where did you find claudin -2?  
 
Can you also describe the hypothesis behind the association between claudin -2 and the VDR and 
TGR5 receptors in esophageal carcinoma? Previously paper has described Claudin -2 expressi on 
in basal layer of esophageal squamous epithelium (ref. Bjorkman E, et al, Scand J 
Gastroenterology 2013), whereas the bile acids are coming into esophagus lumen during 
duodenal reflux. Can you describe where the bile acid receptors are and the connectio n to 
Claudin -2 expression?  
 
 
Specific comments:  
 
1. In the abstract: There are abbreviations for VDR and TGR5. Please sign it out!  
 
1. In background: Chronic exposure (lane 25 -26)….induces CDX -2 expression? Can you 
explain and sign it out!  
 
2. In backgrou nd: Bile acids induce inflammatory gene expression (lane 35 -36) .…through 
the bile acid reseptors FXR, RXR and VDR Why not mentioned TGR5 in this sentence?  
3. Material and methods: Why not mentioned the patients for TMA first?  
 
4. Material and methods: Why  describing immunohistochemistry for VDR and TGR5 but 
not showing that in results (see comments above).  
 
5. Results: Describe and show the expression of claudin -2, VDR and TGR5 in the results 
(figure?).  
 
6. Results: There is no figure -reference for figure 2 -4 in the text.  
 
7. Sometime you write "claudin -2", sometime "Claudin2" or sometime "Claudin 2". Use the 
same version.  
 
8. Results: TNM staging! Please sign it out!  
 
9. Discussion: I miss some reference showing claudin expression in GERD (lane 34 -35) 
together  with Mönkemuller et al. and this reference is: Bjorkman E, et al, Scand J 
Gastroenterology 2013 (the same as above).  
 
10. Discussion: Skip the section about the number of sample (lane 4 -21). Just conclude that 
there are differences in number of sample incl uded in these studies.  
 
11. Abbreviations in table 2!  
 
My suggestion is acceptance of the study after revision.  
 
Are the met hods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the aut hors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees,  funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be include d in my named report can be included as confidential comments 
to the editors, which will not be published . 
 
I agree to the open peer review policy of the journal  
assistant