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: 1 Date: 28 Dec 2018  
 
Reviewer: Luca Mastracci  
 
Reviewer's report:  
 
I read with interes t the work by Götzel et al and I think it is of great interest the exploration of  
the role played by theWnt/β -catenin pathway in the progression from squamous epithelium to 
adenocarcinoma via Barrett's esophagus and high grade intraepithelial neoplasia.  
 
 
There are some aspect that in my opinion need clarification.  
 
 
In methods section, authors stated (pag 6, lines 10 -12):  Biopsies were placed directly to 
RNAlater® (Sigma -Aldrich, Taufkirchen, Germany) for a maximum of 24h and stored at -80°C 
until further us age.  
 
 
At pag 7 lines 31 -33 authors stated: For isolating RNA from paraffin embedded biopsies, we 
used the AllPrep DNA/RNA FFPE Kit (Qiagen, Hilden, Germany) according to the 
manufacturer's protocol.  
 
 
In this setting I have not understood the following items : 
 
a) human biopsies samples were formalin fixed and paraffin embedded or not?  
 
 
b) If the answer is yes, please specify who performed the diagnosis of Barrett's mucosa, 
high-grade intraepithelial neoplasia, esophageal adenocarcinoma, how this diagnosis was 
confirmed, and if different samples (meaning BE, HGin and EAC) are from the same 
patient or the seven specimens you have collected are from different patients. This is not 
clear also because stated that 'endoscopic biopsies were taken from Barrett's mucosa,  
high-grade intraepithelial neoplasia, esophageal adenocarcinoma, and from normal 
mucosa from each patient with a safety distance of at least 3cm to the Barrett's mucosa', 
so giving the empression that at least normal mucosa and Barrett's metaplasia derive  from 
the same patient. Please also specify which definition was used for Barrett's esophagus 
diagnosis (with intestinal metaplasia?)  
 
 
In surgical specimens from patients with adenocarcinoma developed on a previous Barrett's 
esophagus, is usually not easy to collect clearly separated foci of BE and HGIN which constitute 
the background often completely destroyed by adenocarcinoma. I imagine that this is more 
difficult to perform endoscopically. To understand this part and the result obtained from human 
biops ies I think more details are needed.  
 
 
Moreover, for immunohistochemistry analysis 'Paraffin embedded biopsies from Barrett's 
metaplasia and esophageal adenocarcinoma were cut in 3 μm sections and deparaffinized using a 
series of different concentrated alcoh ols and xylol'. In figure 7 pictures are provided also for 
squamous epithelium, and HGIN. Please  correct or explain what effectively have been done and 
in how many samples. Are these patients and sample the same  cited in the previous section  
'Human biop sy specimens'?  
 
 
Have been positive and negative control for immunohistochemistry used during the antibody test 
and the immunohistochemistry run?  
 
 
While the results obtained on the cell li nes are, in my opinion robust enough to be published, the 
observation on human biopsies are actually poor, starting from the selection of samples, control 
of the diagnosis, number of examined samples for each category  and presence of possible 
confounding factors.  
 
 
Figure 3: the title of the 4th panel is High grade intraepith elial neoplasia, but it probably refers to 
adenocarcinoma.  
 
 
Figure 7: an Ematoxilin eosin staining should be provided, possibly of the same area stained with 
immunohistochemistry, in order to permit the right recognition of different entities. Quality of 
this figure is not high enough and also their orientation (please provide pictures oriented in the 
same way, meaning that the luminal part of epithelium is always on the same side of the figure 
preferably up as for squamous epithelium). It is not clear from these images if the positivity is 
only in the cytoplasm or also in the nucleus of the cells; different localization of the protein in 
other setting also plays a role.  
 
 
 
 
 
 
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Unable to assess  
 
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:  
 
Acceptable  
 
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: 1 Date: 28 Dec 2018  
 
Reviewer: Luca Mastracci  
 
Reviewer's report:  
 
I read with interes t the work by Götzel et al and I think it is of great interest the exploration of  
the role played by theWnt/β -catenin pathway in the progression from squamous epithelium to 
adenocarcinoma via Barrett's esophagus and high grade intraepithelial neoplasia.  
 
 
There are some aspect that in my opinion need clarification.  
 
 
In methods section, authors stated (pag 6, lines 10 -12):  Biopsies were placed directly to 
RNAlater® (Sigma -Aldrich, Taufkirchen, Germany) for a maximum of 24h and stored at -80°C 
until further us age.  
 
 
At pag 7 lines 31 -33 authors stated: For isolating RNA from paraffin embedded biopsies, we 
used the AllPrep DNA/RNA FFPE Kit (Qiagen, Hilden, Germany) according to the 
manufacturer's protocol.  
 
 
In this setting I have not understood the following items : 
 
a) human biopsies samples were formalin fixed and paraffin embedded or not?  
 
 
b) If the answer is yes, please specify who performed the diagnosis of Barrett's mucosa, 
high-grade intraepithelial neoplasia, esophageal adenocarcinoma, how this diagnosis was 
confirmed, and if different samples (meaning BE, HGin and EAC) are from the same 
patient or the seven specimens you have collected are from different patients. This is not 
clear also because stated that 'endoscopic biopsies were taken from Barrett's mucosa,  
high-grade intraepithelial neoplasia, esophageal adenocarcinoma, and from normal 
mucosa from each patient with a safety distance of at least 3cm to the Barrett's mucosa', 
so giving the empression that at least normal mucosa and Barrett's metaplasia derive  from 
the same patient. Please also specify which definition was used for Barrett's esophagus 
diagnosis (with intestinal metaplasia?)  
 
 
In surgical specimens from patients with adenocarcinoma developed on a previous Barrett's 
esophagus, is usually not easy to collect clearly separated foci of BE and HGIN which constitute 
the background often completely destroyed by adenocarcinoma. I imagine that this is more 
difficult to perform endoscopically. To understand this part and the result obtained from human 
biops ies I think more details are needed.  
 
 
Moreover, for immunohistochemistry analysis 'Paraffin embedded biopsies from Barrett's 
metaplasia and esophageal adenocarcinoma were cut in 3 μm sections and deparaffinized using a 
series of different concentrated alcoh ols and xylol'. In figure 7 pictures are provided also for 
squamous epithelium, and HGIN. Please  correct or explain what effectively have been done and 
in how many samples. Are these patients and sample the same  cited in the previous section  
'Human biop sy specimens'?  
 
 
Have been positive and negative control for immunohistochemistry used during the antibody test 
and the immunohistochemistry run?  
 
 
While the results obtained on the cell li nes are, in my opinion robust enough to be published, the 
observation on human biopsies are actually poor, starting from the selection of samples, control 
of the diagnosis, number of examined samples for each category  and presence of possible 
confounding factors.  
 
 
Figure 3: the title of the 4th panel is High grade intraepith elial neoplasia, but it probably refers to 
adenocarcinoma.  
 
 
Figure 7: an Ematoxilin eosin staining should be provided, possibly of the same area stained with 
immunohistochemistry, in order to permit the right recognition of different entities. Quality of 
this figure is not high enough and also their orientation (please provide pictures oriented in the 
same way, meaning that the luminal part of epithelium is always on the same side of the figure 
preferably up as for squamous epithelium). It is not clear from these images if the positivity is 
only in the cytoplasm or also in the nucleus of the cells; different localization of the protein in 
other setting also plays a role.  
 
 
 
 
 
 
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Unable to assess  
 
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:  
 
Acceptable  
 
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: 1 Date: 28 Dec 2018  
 
Reviewer: Luca Mastracci  
 
Reviewer's report:  
 
I read with interes t the work by Götzel et al and I think it is of great interest the exploration of  
the role played by theWnt/β -catenin pathway in the progression from squamous epithelium to 
adenocarcinoma via Barrett's esophagus and high grade intraepithelial neoplasia.  
 
 
There are some aspect that in my opinion need clarification.  
 
 
In methods section, authors stated (pag 6, lines 10 -12):  Biopsies were placed directly to 
RNAlater® (Sigma -Aldrich, Taufkirchen, Germany) for a maximum of 24h and stored at -80°C 
until further us age.  
 
 
At pag 7 lines 31 -33 authors stated: For isolating RNA from paraffin embedded biopsies, we 
used the AllPrep DNA/RNA FFPE Kit (Qiagen, Hilden, Germany) according to the 
manufacturer's protocol.  
 
 
In this setting I have not understood the following items : 
 
a) human biopsies samples were formalin fixed and paraffin embedded or not?  
 
 
b) If the answer is yes, please specify who performed the diagnosis of Barrett's mucosa, 
high-grade intraepithelial neoplasia, esophageal adenocarcinoma, how this diagnosis was 
confirmed, and if different samples (meaning BE, HGin and EAC) are from the same 
patient or the seven specimens you have collected are from different patients. This is not 
clear also because stated that 'endoscopic biopsies were taken from Barrett's mucosa,  
high-grade intraepithelial neoplasia, esophageal adenocarcinoma, and from normal 
mucosa from each patient with a safety distance of at least 3cm to the Barrett's mucosa', 
so giving the empression that at least normal mucosa and Barrett's metaplasia derive  from 
the same patient. Please also specify which definition was used for Barrett's esophagus 
diagnosis (with intestinal metaplasia?)  
 
 
In surgical specimens from patients with adenocarcinoma developed on a previous Barrett's 
esophagus, is usually not easy to collect clearly separated foci of BE and HGIN which constitute 
the background often completely destroyed by adenocarcinoma. I imagine that this is more 
difficult to perform endoscopically. To understand this part and the result obtained from human 
biops ies I think more details are needed.  
 
 
Moreover, for immunohistochemistry analysis 'Paraffin embedded biopsies from Barrett's 
metaplasia and esophageal adenocarcinoma were cut in 3 μm sections and deparaffinized using a 
series of different concentrated alcoh ols and xylol'. In figure 7 pictures are provided also for 
squamous epithelium, and HGIN. Please  correct or explain what effectively have been done and 
in how many samples. Are these patients and sample the same  cited in the previous section  
'Human biop sy specimens'?  
 
 
Have been positive and negative control for immunohistochemistry used during the antibody test 
and the immunohistochemistry run?  
 
 
While the results obtained on the cell li nes are, in my opinion robust enough to be published, the 
observation on human biopsies are actually poor, starting from the selection of samples, control 
of the diagnosis, number of examined samples for each category  and presence of possible 
confounding factors.  
 
 
Figure 3: the title of the 4th panel is High grade intraepith elial neoplasia, but it probably refers to 
adenocarcinoma.  
 
 
Figure 7: an Ematoxilin eosin staining should be provided, possibly of the same area stained with 
immunohistochemistry, in order to permit the right recognition of different entities. Quality of 
this figure is not high enough and also their orientation (please provide pictures oriented in the 
same way, meaning that the luminal part of epithelium is always on the same side of the figure 
preferably up as for squamous epithelium). It is not clear from these images if the positivity is 
only in the cytoplasm or also in the nucleus of the cells; different localization of the protein in 
other setting also plays a role.  
 
 
 
 
 
 
 
 
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Unable to assess  
 
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:  
 
Acceptable  
 
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