Reviewer ’s report  
 
Title: Use of Immunostaining for the Diagnosis of Lymphovascular Invasion in Superficial  
Barrett’s Esophageal Adenocarcinoma  
 
Version: 0 Date: 20 Feb 2020  
 
Reviewer: B Braden  
 
Reviewer's report:  
 
General  comments  
 
Hosono et al. present a retrospective single center immunohistological re -evaluation of 41 
endoscopic or surgical resections of early Barrett's cancers after D2 -40 and CD31 
immunostaining to improve the detection of lymphovascular involvement. The histological 
specimens were collected over 11 years, most (28) were from oesophagectomies.  
 
Recommended practice is to have two expert pathologist reporting intraepithelial neoplasia or 
difficult cases in Barrett's epithelium. The single pathologist wa s not blinded as the type of 
staining is obvious on the slides. The same pathologist assessed HE and immunostaining and 
therefore was likely biased by knowing the HE findings. The strength of the study would 
increase by evaluation by a second histopatholog ist. Separate assessment of only HE or only 
immunostains by different pathologists would be desirable.  
 
The small study number renders the reliability of prediction of lymph node metastasis and the 
evaluation of survival rates difficult.  
 
The study addresses an interesting topic and the results -if confirmed by larger studies - might 
be relevant for managing patients with submucosal infiltration of endoscopically resected 
early Barrett's cancer. Better prediction of the presence of lymph nod e metastasis will help to 
decide between oesophagectomy and continued surveillance in those patients.  
 
 
Specific comments  
 
Please illustrate the depth of invasion by a figure and also explain SMM superficial and 
DMM deep muscularis mucosae and submucos al infiltration in the text. This terminology 
seems to differ between Eastern and Western world.  
Can you comment on reporting difficulties in presence of duplicated muscularis mucosae?  
 
What kind of cells exactly have been stained? What do the antibodies react with? Please give 
more detail what the two different stains are used for.  
Have both immunostains been used simultaneously? Did both stains perform equally well? 
How long does the immunostaining take? How dependent is the quality of the staining on th e 
preparation method?  
 
Please avoid the abbreviation LSBE and SSBE as it is not frequently used in the manuscript.  
 
 
Page 6 line 38: Please explain the meaning of the sentence in more detail. Lymphovascular 
infiltration was present in 10/41 patients, thus it should be absent in 31/41?  
 
Page 7 line 1: For easier reading, please add: LVI was present in 3 (60%) of patients with 
&lt;500 um submucosal infiltration and in 7 (46.7%) of patients with &gt;500 um 
submucosal infiltration.  
 
Discussion  
Considering the a dditional costs and time for the immunostainig, would it make sense to 
recommend additional immunostaining in patients with submucosal infiltration but not for 
T1a tumours?  
 
According to table 3, only 3 out of 10 patients with lymphovascular infiltration h ad lymph 
node metastasis at surgery and only those with deep submucosal infiltration. Therefore, is 
LVI a reliable predictor or will it increase the number of oesophagectomies with histological 
negative outcome, especially in patients with sm1 who might be  treated conservatively after 
endoscopic resection?  
 
 
Figure 4 Please explain the colour coded graphs in the legend, unfortunately not readable in 
the figure.  
 
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 ar e 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 speci fy 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 t he 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 content o f 
the manuscript?  
4. Have you received reimbursements, fees, funding, or s alary 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 i nterests . 
 
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 for publication, my 
named r eport 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 included i n my named report can be in cluded 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: Use of Immunostaining for the Diagnosis of Lymphovascular Invasion in Superficial  
Barrett’s Esophageal Adenocarcinoma  
 
Version: 0 Date: 20 Feb 2020  
 
Reviewer: B Braden  
 
Reviewer's report:  
 
General  comments  
 
Hosono et al. present a retrospective single center immunohistological re -evaluation of 41 
endoscopic or surgical resections of early Barrett's cancers after D2 -40 and CD31 
immunostaining to improve the detection of lymphovascular involvement. The histological 
specimens were collected over 11 years, most (28) were from oesophagectomies.  
 
Recommended practice is to have two expert pathologist reporting intraepithelial neoplasia or 
difficult cases in Barrett's epithelium. The single pathologist wa s not blinded as the type of 
staining is obvious on the slides. The same pathologist assessed HE and immunostaining and 
therefore was likely biased by knowing the HE findings. The strength of the study would 
increase by evaluation by a second histopatholog ist. Separate assessment of only HE or only 
immunostains by different pathologists would be desirable.  
 
The small study number renders the reliability of prediction of lymph node metastasis and the 
evaluation of survival rates difficult.  
 
The study addresses an interesting topic and the results -if confirmed by larger studies - might 
be relevant for managing patients with submucosal infiltration of endoscopically resected 
early Barrett's cancer. Better prediction of the presence of lymph nod e metastasis will help to 
decide between oesophagectomy and continued surveillance in those patients.  
 
 
Specific comments  
 
Please illustrate the depth of invasion by a figure and also explain SMM superficial and 
DMM deep muscularis mucosae and submucos al infiltration in the text. This terminology 
seems to differ between Eastern and Western world.  
Can you comment on reporting difficulties in presence of duplicated muscularis mucosae?  
 
What kind of cells exactly have been stained? What do the antibodies react with? Please give 
more detail what the two different stains are used for.  
Have both immunostains been used simultaneously? Did both stains perform equally well? 
How long does the immunostaining take? How dependent is the quality of the staining on th e 
preparation method?  
 
Please avoid the abbreviation LSBE and SSBE as it is not frequently used in the manuscript.  
 
 
Page 6 line 38: Please explain the meaning of the sentence in more detail. Lymphovascular 
infiltration was present in 10/41 patients, thus it should be absent in 31/41?  
 
Page 7 line 1: For easier reading, please add: LVI was present in 3 (60%) of patients with 
&lt;500 um submucosal infiltration and in 7 (46.7%) of patients with &gt;500 um 
submucosal infiltration.  
 
Discussion  
Considering the a dditional costs and time for the immunostainig, would it make sense to 
recommend additional immunostaining in patients with submucosal infiltration but not for 
T1a tumours?  
 
According to table 3, only 3 out of 10 patients with lymphovascular infiltration h ad lymph 
node metastasis at surgery and only those with deep submucosal infiltration. Therefore, is 
LVI a reliable predictor or will it increase the number of oesophagectomies with histological 
negative outcome, especially in patients with sm1 who might be  treated conservatively after 
endoscopic resection?  
 
 
Figure 4 Please explain the colour coded graphs in the legend, unfortunately not readable in 
the figure.  
 
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 ar e 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 speci fy 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 t he 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 content o f 
the manuscript?  
4. Have you received reimbursements, fees, funding, or s alary 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 i nterests . 
 
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 for publication, my 
named r eport 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 included i n my named report can be in cluded 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: Use of Immunostaining for the Diagnosis of Lymphovascular Invasion in Superficial  
Barrett’s Esophageal Adenocarcinoma  
 
Version: 0 Date: 20 Feb 2020  
 
Reviewer: B Braden  
 
Reviewer's report:  
 
General  comments  
 
Hosono et al. present a retrospective single center immunohistological re -evaluation of 41 
endoscopic or surgical resections of early Barrett's cancers after D2 -40 and CD31 
immunostaining to improve the detection of lymphovascular involvement. The histological 
specimens were collected over 11 years, most (28) were from oesophagectomies.  
 
Recommended practice is to have two expert pathologist reporting intraepithelial neoplasia or 
difficult cases in Barrett's epithelium. The single pathologist wa s not blinded as the type of 
staining is obvious on the slides. The same pathologist assessed HE and immunostaining and 
therefore was likely biased by knowing the HE findings. The strength of the study would 
increase by evaluation by a second histopatholog ist. Separate assessment of only HE or only 
immunostains by different pathologists would be desirable.  
 
The small study number renders the reliability of prediction of lymph node metastasis and the 
evaluation of survival rates difficult.  
 
The study addresses an interesting topic and the results -if confirmed by larger studies - might 
be relevant for managing patients with submucosal infiltration of endoscopically resected 
early Barrett's cancer. Better prediction of the presence of lymph nod e metastasis will help to 
decide between oesophagectomy and continued surveillance in those patients.  
 
 
Specific comments  
 
Please illustrate the depth of invasion by a figure and also explain SMM superficial and 
DMM deep muscularis mucosae and submucos al infiltration in the text. This terminology 
seems to differ between Eastern and Western world.  
Can you comment on reporting difficulties in presence of duplicated muscularis mucosae?  
 
What kind of cells exactly have been stained? What do the antibodies react with? Please give 
more detail what the two different stains are used for.  
Have both immunostains been used simultaneously? Did both stains perform equally well? 
How long does the immunostaining take? How dependent is the quality of the staining on th e 
preparation method?  
 
Please avoid the abbreviation LSBE and SSBE as it is not frequently used in the manuscript.  
 
 
Page 6 line 38: Please explain the meaning of the sentence in more detail. Lymphovascular 
infiltration was present in 10/41 patients, thus it should be absent in 31/41?  
 
Page 7 line 1: For easier reading, please add: LVI was present in 3 (60%) of patients with 
&lt;500 um submucosal infiltration and in 7 (46.7%) of patients with &gt;500 um 
submucosal infiltration.  
 
Discussion  
Considering the a dditional costs and time for the immunostainig, would it make sense to 
recommend additional immunostaining in patients with submucosal infiltration but not for 
T1a tumours?  
 
According to table 3, only 3 out of 10 patients with lymphovascular infiltration h ad lymph 
node metastasis at surgery and only those with deep submucosal infiltration. Therefore, is 
LVI a reliable predictor or will it increase the number of oesophagectomies with histological 
negative outcome, especially in patients with sm1 who might be  treated conservatively after 
endoscopic resection?  
 
 
Figure 4 Please explain the colour coded graphs in the legend, unfortunately not readable in 
the figure.  
 
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 ar e 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 speci fy 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 t he 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 content o f 
the manuscript?  
4. Have you received reimbursements, fees, funding, or s alary 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 i nterests . 
 
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 for publication, my 
named r eport 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 included i n my named report can be in cluded as 
confidential comments to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant