Reviewer ’s report  
 
Title: In-depth characterization of the Wnt -signaling/ β-catenin pathway in an i n vitro model of 
Barrett's sequence  
 
Version: 0 Date: 07 Aug 2018  
 
Reviewer: Manuele Furnari  
 
Reviewer's report:  
 
I read with great interest the work by Götzel et al which explored the intricate world of cellular 
growth and morphogenesis promoted and regulated by the Wnt pathways. Particularly, they 
investigated the role played by the Wnt/β -catenin pathway in the prog ression from squamous 
epithelium to esophageal metaplasia and eventually adenocarcinoma. In this attempt, they 
performed an elaborate study in in vitro cell culture model of Barrett's esophagus, paraffin 
embedded specimens (metaplasia and ADC) and biopsies  from BE and normal esophagus in 7 
patients. The expression of the FZDs (g -protein receptors), their co -receptors LRP5/6, and 
ligands Wnt3a/5a have been here analysed. Moreover, transcription of several intracellular 
factors have been assessed such as Axin 2 and Cyclin D1.  
 
 
Overall, this is an impressive study of molecular genetics, rigorously conducted, which provides 
new insights into the molecular mechanisms involved in the development of intestinal metaplasia 
and esophageal cancer. However, despite these strong points, the manuscript is difficult to follow 
and the text flow needs to be improved.  
 
 
 
There are some points that deserve major revisions:  
 
- In mammals there are 19 Wnt genes. The authors should briefly explain why Wnt3a and 
Wnt5a have been investig ated.  
 
 
- Expression of Wnt5a suddenly appears in the results section. However, it is mentioned 
only in the abstract before that. It should be mentioned in the materials and methods.  
 
 
- Statistical analysis: data are reported as mean. However, due to the paucity of samples, 
mediam might be considered.  
 
 
- Apparently, paraffin embedded biopsies were collected from metaplasia and 
adenocarcinoma only. Since the purpose of the study is to cla rify Wnt signaling pathway 
in the progression to ADC, why have biopsies from dysplasia been excluded? The 
inclusion of biopsies representing the whole spectrum of transition from normal mucosa 
to metaplasia/dysplasia, and neoplasia might provide further in sight into our 
understanding of the proposed mechanisms.  
 
 
- Regarding the embedded specimens, how many have been analyzed?  
 
 
- The number of patients (7) enrolled in order to obtain biopsies from SC and BE, should 
be easier to catch in the manuscript. Moreove r, a more in -depth characterization of 
patients is lacking. As a fact, different expression of co -receptors between cell -lines and 
specimens could be due to the possible heterogeneity of this poor representative group of 
BE patients, which in turn might be  influenced by several variables (e.g., type and 
severity of GERD, duration of disease, obesity, use of drugs, diet, etc.). Moreover, 
biopsies contain other cell types than Barrett's epithelium. Together with the relatively 
small number of cases (7) this c onstitutes a limit.   
 
 
- Are there different factors that should be addressed as a possible limitation when 
comparing in vivo cells with human specimens  (both frozen and embedded in paraffin).  
 
 
- Limitations of the study have not been highlighted.  
 
 
- Biopsi es have been taken from BE and squamous epithelium from each patient. What are 
the reasons to exclude biopsies from esophageal ADC cases? Despite dysplasia, ADC is 
relatively easier to observe.  
 
 
- WNT3A immunohistochemistry: authors compared staining among SC, BE and ADC…it 
is not clear if BE were obtained from human biopsies (SC and BE) or from embedded 
biopsies (BE and ADC). Moreover, Fig.7 caption needs to be corrected: text is confusing; 
and EC, MC,CC have not been explained before.  
 
 
- Please, check the m anuscript for errors/vagueness.  
 
 
 (i.e. Page 10, line 40 "expressen"  - the type of Wnt3a expression stated at Page 11 line 1? Or 
again, about the expression of LRP5 in specimens at page 10 line 50)  
 
 
 
 
 
Are the methods 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 authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects requ ire 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:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing inter ests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or lose financial ly 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 c ontent 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 understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be po sted 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 included in my named report can be in cluded as confidential comments 
to the editor s, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: In-depth characterization of the Wnt -signaling/ β-catenin pathway in an i n vitro model of 
Barrett's sequence  
 
Version: 0 Date: 07 Aug 2018  
 
Reviewer: Manuele Furnari  
 
Reviewer's report:  
 
I read with great interest the work by Götzel et al which explored the intricate world of cellular 
growth and morphogenesis promoted and regulated by the Wnt pathways. Particularly, they 
investigated the role played by the Wnt/β -catenin pathway in the prog ression from squamous 
epithelium to esophageal metaplasia and eventually adenocarcinoma. In this attempt, they 
performed an elaborate study in in vitro cell culture model of Barrett's esophagus, paraffin 
embedded specimens (metaplasia and ADC) and biopsies  from BE and normal esophagus in 7 
patients. The expression of the FZDs (g -protein receptors), their co -receptors LRP5/6, and 
ligands Wnt3a/5a have been here analysed. Moreover, transcription of several intracellular 
factors have been assessed such as Axin 2 and Cyclin D1.  
 
 
Overall, this is an impressive study of molecular genetics, rigorously conducted, which provides 
new insights into the molecular mechanisms involved in the development of intestinal metaplasia 
and esophageal cancer. However, despite these strong points, the manuscript is difficult to follow 
and the text flow needs to be improved.  
 
 
 
There are some points that deserve major revisions:  
 
- In mammals there are 19 Wnt genes. The authors should briefly explain why Wnt3a and 
Wnt5a have been investig ated.  
 
 
- Expression of Wnt5a suddenly appears in the results section. However, it is mentioned 
only in the abstract before that. It should be mentioned in the materials and methods.  
 
 
- Statistical analysis: data are reported as mean. However, due to the paucity of samples, 
mediam might be considered.  
 
 
- Apparently, paraffin embedded biopsies were collected from metaplasia and 
adenocarcinoma only. Since the purpose of the study is to cla rify Wnt signaling pathway 
in the progression to ADC, why have biopsies from dysplasia been excluded? The 
inclusion of biopsies representing the whole spectrum of transition from normal mucosa 
to metaplasia/dysplasia, and neoplasia might provide further in sight into our 
understanding of the proposed mechanisms.  
 
 
- Regarding the embedded specimens, how many have been analyzed?  
 
 
- The number of patients (7) enrolled in order to obtain biopsies from SC and BE, should 
be easier to catch in the manuscript. Moreove r, a more in -depth characterization of 
patients is lacking. As a fact, different expression of co -receptors between cell -lines and 
specimens could be due to the possible heterogeneity of this poor representative group of 
BE patients, which in turn might be  influenced by several variables (e.g., type and 
severity of GERD, duration of disease, obesity, use of drugs, diet, etc.). Moreover, 
biopsies contain other cell types than Barrett's epithelium. Together with the relatively 
small number of cases (7) this c onstitutes a limit.   
 
 
- Are there different factors that should be addressed as a possible limitation when 
comparing in vivo cells with human specimens  (both frozen and embedded in paraffin).  
 
 
- Limitations of the study have not been highlighted.  
 
 
- Biopsi es have been taken from BE and squamous epithelium from each patient. What are 
the reasons to exclude biopsies from esophageal ADC cases? Despite dysplasia, ADC is 
relatively easier to observe.  
 
 
- WNT3A immunohistochemistry: authors compared staining among SC, BE and ADC…it 
is not clear if BE were obtained from human biopsies (SC and BE) or from embedded 
biopsies (BE and ADC). Moreover, Fig.7 caption needs to be corrected: text is confusing; 
and EC, MC,CC have not been explained before.  
 
 
- Please, check the m anuscript for errors/vagueness.  
 
 
 (i.e. Page 10, line 40 "expressen"  - the type of Wnt3a expression stated at Page 11 line 1? Or 
again, about the expression of LRP5 in specimens at page 10 line 50)  
 
 
 
 
 
Are the methods 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 authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects requ ire 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:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing inter ests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or lose financial ly 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 c ontent 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 understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be po sted 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 included in my named report can be in cluded as confidential comments 
to the editor s, 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: In-depth characterization of the Wnt -signaling/ β-catenin pathway in an i n vitro model of 
Barrett's sequence  
 
Version: 0 Date: 07 Aug 2018  
 
Reviewer: Manuele Furnari  
 
Reviewer's report:  
 
I read with great interest the work by Götzel et al which explored the intricate world of cellular 
growth and morphogenesis promoted and regulated by the Wnt pathways. Particularly, they 
investigated the role played by the Wnt/β -catenin pathway in the prog ression from squamous 
epithelium to esophageal metaplasia and eventually adenocarcinoma. In this attempt, they 
performed an elaborate study in in vitro cell culture model of Barrett's esophagus, paraffin 
embedded specimens (metaplasia and ADC) and biopsies  from BE and normal esophagus in 7 
patients. The expression of the FZDs (g -protein receptors), their co -receptors LRP5/6, and 
ligands Wnt3a/5a have been here analysed. Moreover, transcription of several intracellular 
factors have been assessed such as Axin 2 and Cyclin D1.  
 
 
Overall, this is an impressive study of molecular genetics, rigorously conducted, which provides 
new insights into the molecular mechanisms involved in the development of intestinal metaplasia 
and esophageal cancer. However, despite these strong points, the manuscript is difficult to follow 
and the text flow needs to be improved.  
 
 
 
There are some points that deserve major revisions:  
 
- In mammals there are 19 Wnt genes. The authors should briefly explain why Wnt3a and 
Wnt5a have been investig ated.  
 
 
- Expression of Wnt5a suddenly appears in the results section. However, it is mentioned 
only in the abstract before that. It should be mentioned in the materials and methods.  
 
 
- Statistical analysis: data are reported as mean. However, due to the paucity of samples, 
mediam might be considered.  
 
 
- Apparently, paraffin embedded biopsies were collected from metaplasia and 
adenocarcinoma only. Since the purpose of the study is to cla rify Wnt signaling pathway 
in the progression to ADC, why have biopsies from dysplasia been excluded? The 
inclusion of biopsies representing the whole spectrum of transition from normal mucosa 
to metaplasia/dysplasia, and neoplasia might provide further in sight into our 
understanding of the proposed mechanisms.  
 
 
- Regarding the embedded specimens, how many have been analyzed?  
 
 
- The number of patients (7) enrolled in order to obtain biopsies from SC and BE, should 
be easier to catch in the manuscript. Moreove r, a more in -depth characterization of 
patients is lacking. As a fact, different expression of co -receptors between cell -lines and 
specimens could be due to the possible heterogeneity of this poor representative group of 
BE patients, which in turn might be  influenced by several variables (e.g., type and 
severity of GERD, duration of disease, obesity, use of drugs, diet, etc.). Moreover, 
biopsies contain other cell types than Barrett's epithelium. Together with the relatively 
small number of cases (7) this c onstitutes a limit.   
 
 
- Are there different factors that should be addressed as a possible limitation when 
comparing in vivo cells with human specimens  (both frozen and embedded in paraffin).  
 
 
- Limitations of the study have not been highlighted.  
 
 
- Biopsi es have been taken from BE and squamous epithelium from each patient. What are 
the reasons to exclude biopsies from esophageal ADC cases? Despite dysplasia, ADC is 
relatively easier to observe.  
 
 
- WNT3A immunohistochemistry: authors compared staining among SC, BE and ADC…it 
is not clear if BE were obtained from human biopsies (SC and BE) or from embedded 
biopsies (BE and ADC). Moreover, Fig.7 caption needs to be corrected: text is confusing; 
and EC, MC,CC have not been explained before.  
 
 
- Please, check the m anuscript for errors/vagueness.  
 
 
 (i.e. Page 10, line 40 "expressen"  - the type of Wnt3a expression stated at Page 11 line 1? Or 
again, about the expression of LRP5 in specimens at page 10 line 50)  
 
 
 
 
 
Are the methods 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 authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects requ ire 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:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing inter ests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, f ees, funding, or salary from an 
organisation that may in any way gain or lose financial ly 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 c ontent 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 understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be po sted 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 included in my named report can be in cluded as confidential comments 
to the editor s, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant