Reviewer ’s report  
 
Title: Hepatic Sonic Hedgehog Protein Expressio n Measured by Computer Assisted 
Morphometry  Significantly Correlates with Features of Non -Alcoholic Steatohepatitis  
 
Version: 0 Date: 20 Nov 2018  
 
Reviewer: Naoki Tanaka  
 
Reviewer's report:  
 
Hedgehog system is activated in ballooned hepatocytes, reflecting severity of NAFLD/NASH. 
Estep et al. concluded that quantitation of SHH expression by CAM can provide a tool for 
quantifying changes in hepatocyte injury and assist in una mbiguous staging/grading of NASH. 
Ballooned hepatocytes are important hallmarks of active/severe NASH, but its detection is 
sometimes difficult due to inter -observer's variations and sampling errors. Adding hepatic SHH 
staining might solve this problem.  
 
1. Please add subtitles to the Methods and Results for clearer presentation.  
 
2. It is better to give more information about patients, such as age, gender and race.  
 
3. Circulating SHH is not correlated with hepatic SHH. Are there any methods to measure 
circulating SHH properly? If hepatic SHH can be estimated by circulating indicators, it 
may be useful to assess ballooning severity.  
 
4. The authors should explain figure 2 ore precisely and in more details and show typical 
case quantifying SHH -positive area .  
 
5. Score of fibrosis is basically classified into F0 to F4. The authors should check the 
correlation between fibrosis score and SHH -positive area.  
 
6. It is insufficient to show correlation efficients only. The authors should plot hepatic SHH 
score, M30 , and M65 according to the histological scores (e.g., ballooning 0, 1, and 2,  
steatosis 0, 1, 2, 3, 4, ---). 
 
7. Which is better, hepatic SHH IHC or M65/M30 for detecting ballooning?  
 
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.  
 
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  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 year s 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 that may in a ny 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 u nderstand 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 repor t 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 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: Hepatic Sonic Hedgehog Protein Expressio n Measured by Computer Assisted 
Morphometry  Significantly Correlates with Features of Non -Alcoholic Steatohepatitis  
 
Version: 0 Date: 20 Nov 2018  
 
Reviewer: Naoki Tanaka  
 
Reviewer's report:  
 
Hedgehog system is activated in ballooned hepatocytes, reflecting severity of NAFLD/NASH. 
Estep et al. concluded that quantitation of SHH expression by CAM can provide a tool for 
quantifying changes in hepatocyte injury and assist in una mbiguous staging/grading of NASH. 
Ballooned hepatocytes are important hallmarks of active/severe NASH, but its detection is 
sometimes difficult due to inter -observer's variations and sampling errors. Adding hepatic SHH 
staining might solve this problem.  
 
1. Please add subtitles to the Methods and Results for clearer presentation.  
 
2. It is better to give more information about patients, such as age, gender and race.  
 
3. Circulating SHH is not correlated with hepatic SHH. Are there any methods to measure 
circulating SHH properly? If hepatic SHH can be estimated by circulating indicators, it 
may be useful to assess ballooning severity.  
 
4. The authors should explain figure 2 ore precisely and in more details and show typical 
case quantifying SHH -positive area .  
 
5. Score of fibrosis is basically classified into F0 to F4. The authors should check the 
correlation between fibrosis score and SHH -positive area.  
 
6. It is insufficient to show correlation efficients only. The authors should plot hepatic SHH 
score, M30 , and M65 according to the histological scores (e.g., ballooning 0, 1, and 2,  
steatosis 0, 1, 2, 3, 4, ---). 
 
7. Which is better, hepatic SHH IHC or M65/M30 for detecting ballooning?  
 
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.  
 
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  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 year s 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 that may in a ny 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 u nderstand 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 repor t 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 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: Hepatic Sonic Hedgehog Protein Expressio n Measured by Computer Assisted 
Morphometry  Significantly Correlates with Features of Non -Alcoholic Steatohepatitis  
 
Version: 0 Date: 20 Nov 2018  
 
Reviewer: Naoki Tanaka  
 
Reviewer's report:  
 
Hedgehog system is activated in ballooned hepatocytes, reflecting severity of NAFLD/NASH. 
Estep et al. concluded that quantitation of SHH expression by CAM can provide a tool for 
quantifying changes in hepatocyte injury and assist in una mbiguous staging/grading of NASH. 
Ballooned hepatocytes are important hallmarks of active/severe NASH, but its detection is 
sometimes difficult due to inter -observer's variations and sampling errors. Adding hepatic SHH 
staining might solve this problem.  
 
1. Please add subtitles to the Methods and Results for clearer presentation.  
 
2. It is better to give more information about patients, such as age, gender and race.  
 
3. Circulating SHH is not correlated with hepatic SHH. Are there any methods to measure 
circulating SHH properly? If hepatic SHH can be estimated by circulating indicators, it 
may be useful to assess ballooning severity.  
 
4. The authors should explain figure 2 ore precisely and in more details and show typical 
case quantifying SHH -positive area .  
 
5. Score of fibrosis is basically classified into F0 to F4. The authors should check the 
correlation between fibrosis score and SHH -positive area.  
 
6. It is insufficient to show correlation efficients only. The authors should plot hepatic SHH 
score, M30 , and M65 according to the histological scores (e.g., ballooning 0, 1, and 2,  
steatosis 0, 1, 2, 3, 4, ---). 
 
7. Which is better, hepatic SHH IHC or M65/M30 for detecting ballooning?  
 
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.  
 
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  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 year s 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 that may in a ny 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 u nderstand 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 repor t 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 included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant