Reviewer ’s report  
 
Title: Exosomal miRNA signatures of pancreatic lesions  
 
Version: 2 Date: 20 Feb 2020  
 
Reviewer: Adam Frampton  
 
Reviewer's report:  
 
I have the following comments:  
1. What subtype of IPMN were the 20 cases (as SB and MD a re distinct in prognosis).  
2. Methods: Resuspension in 20ml or 20µl of H2O? The whole paper needs to clarify between 
micro and millilitres  
3. The methods make no sense. You must add chloroform and separate the phases before 
isolating with the Norgen kit. W hy did they choose 1ml of Trizol (which is either too much or 
too little depending on whether it is milli or microlitres).  
4. Were ath -miR-159a, osa -miR-414, and cel -miR-248 synthetic oligos added at the Trizol step 
or later? Which step were these added fo r normalisation and how was normalisation performed. 
Please add.  
5. There's no NTA described in the methods or EM data, so there's no evidence of extracellular 
vesicles at all. Particle count is unknown and the protein amount is incredibly high. The 
Supple mentary figure 2 shows a graph of concentration but this average is not declared in the 
manuscript. The y axis furthermore has the wrong units (it seems to show 1 particle per ml). 
Please correct/explain.  
6. In particular, using 90µg of loading for western blotting seems to indicate a very low 
expression of EV markers. Why was so much used?  
7. Yield of exosomal protein as 9mg per 0.5ml of plasma is very high. See Webber J, Clayton A. 
How pure are your vesic les?. J Extracell Vesicles. 2013;2:10.3402/jev.v2i0.19861. Published 
2013 Jan 10. doi:10.3402/jev.v2i0.19861  
8. Is a linear fold change &gt;1.5 adequate as a threshold for PCR?  
9. Did you use a false discovery rate for Nanostring?  
10. Please be careful with PC or PDAC; PanNET or PET -- --- use the same abbreviations 
please.  
 
 
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.  
 
No 
 
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 shar es in an organisation that 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 rec eived 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 interest s 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.  
 
None  
 
I agree to the open pee r 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 autho rs' 
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 include d 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: Exosomal miRNA signatures of pancreatic lesions  
 
Version: 2 Date: 20 Feb 2020  
 
Reviewer: Adam Frampton  
 
Reviewer's report:  
 
I have the following comments:  
1. What subtype of IPMN were the 20 cases (as SB and MD a re distinct in prognosis).  
2. Methods: Resuspension in 20ml or 20µl of H2O? The whole paper needs to clarify between 
micro and millilitres  
3. The methods make no sense. You must add chloroform and separate the phases before 
isolating with the Norgen kit. W hy did they choose 1ml of Trizol (which is either too much or 
too little depending on whether it is milli or microlitres).  
4. Were ath -miR-159a, osa -miR-414, and cel -miR-248 synthetic oligos added at the Trizol step 
or later? Which step were these added fo r normalisation and how was normalisation performed. 
Please add.  
5. There's no NTA described in the methods or EM data, so there's no evidence of extracellular 
vesicles at all. Particle count is unknown and the protein amount is incredibly high. The 
Supple mentary figure 2 shows a graph of concentration but this average is not declared in the 
manuscript. The y axis furthermore has the wrong units (it seems to show 1 particle per ml). 
Please correct/explain.  
6. In particular, using 90µg of loading for western blotting seems to indicate a very low 
expression of EV markers. Why was so much used?  
7. Yield of exosomal protein as 9mg per 0.5ml of plasma is very high. See Webber J, Clayton A. 
How pure are your vesic les?. J Extracell Vesicles. 2013;2:10.3402/jev.v2i0.19861. Published 
2013 Jan 10. doi:10.3402/jev.v2i0.19861  
8. Is a linear fold change &gt;1.5 adequate as a threshold for PCR?  
9. Did you use a false discovery rate for Nanostring?  
10. Please be careful with PC or PDAC; PanNET or PET -- --- use the same abbreviations 
please.  
 
 
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.  
 
No 
 
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 shar es in an organisation that 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 rec eived 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 interest s 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.  
 
None  
 
I agree to the open pee r 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 autho rs' 
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 include d 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: Exosomal miRNA signatures of pancreatic lesions  
 
Version: 2 Date: 20 Feb 2020  
 
Reviewer: Adam Frampton  
 
Reviewer's report:  
 
I have the following comments:  
1. What subtype of IPMN were the 20 cases (as SB and MD a re distinct in prognosis).  
2. Methods: Resuspension in 20ml or 20µl of H2O? The whole paper needs to clarify between 
micro and millilitres  
3. The methods make no sense. You must add chloroform and separate the phases before 
isolating with the Norgen kit. W hy did they choose 1ml of Trizol (which is either too much or 
too little depending on whether it is milli or microlitres).  
4. Were ath -miR-159a, osa -miR-414, and cel -miR-248 synthetic oligos added at the Trizol step 
or later? Which step were these added fo r normalisation and how was normalisation performed. 
Please add.  
5. There's no NTA described in the methods or EM data, so there's no evidence of extracellular 
vesicles at all. Particle count is unknown and the protein amount is incredibly high. The 
Supple mentary figure 2 shows a graph of concentration but this average is not declared in the 
manuscript. The y axis furthermore has the wrong units (it seems to show 1 particle per ml). 
Please correct/explain.  
6. In particular, using 90µg of loading for western blotting seems to indicate a very low 
expression of EV markers. Why was so much used?  
7. Yield of exosomal protein as 9mg per 0.5ml of plasma is very high. See Webber J, Clayton A. 
How pure are your vesic les?. J Extracell Vesicles. 2013;2:10.3402/jev.v2i0.19861. Published 
2013 Jan 10. doi:10.3402/jev.v2i0.19861  
8. Is a linear fold change &gt;1.5 adequate as a threshold for PCR?  
9. Did you use a false discovery rate for Nanostring?  
10. Please be careful with PC or PDAC; PanNET or PET -- --- use the same abbreviations 
please.  
 
 
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.  
 
No 
 
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 shar es in an organisation that 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 rec eived 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 interest s 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.  
 
None  
 
I agree to the open pee r 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 autho rs' 
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 include d as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant