Reviewer ’s report  
 
Title: Exosomal miRNA signatures of pancreatic lesions  
 
Version: 1 Date: 22 Nov 2019  
 
Reviewer: Adam Frampton  
 
Reviewer's report:  
 
Vincentini et al have looked at Exosomal miRNAs in pancreatic  lesions. I have the following 
comments especially regarding the Methods:  
1. In the Intro, what is meant by "digital profiling"? Why not just say NanoString 
profiling?  
2. N=140 is a commendable sample size. However, there weren't any healthy controls? 
Ther e were only 15 CP samples as benign controls. If possible, it would be worth obtaining a 
small cohort of healthy control (HC) plasma exosomes (e.g. 10 to 15 participants) and then 
checking by RT -qPCR the expression of miR -4454, miR -106a -5p and miR -17-5p in  HC vs 
PDAC. As these plasma exosomal miRNAs all seemed down -regulated in PDAC compared to 
other lesions, but the comparison to HC is unknown.  
3. In the Intro, please change to "specific exosomal -miRNA signatures" and "relevance of 
these signatures in PDAC " 
4. "Ampulla of Vater carcinomas (AVC) comprise 30% of pancreaticoduodenectomies 
and 20% of all tumor -related obstructions of the common bile duct" --- I disagree with these 
figures quoted. Please check large pancreaticoduodenectomy series for the percent age of 
Ampullary cancers removed (e.g. Heidelberg, or Johns Hopkins).  
5. Please justify in your Methods using sodium heparin blood tubes, rather than EDTA 
blood tubes, for obtaining plasma. Check and cite studies looking at the best methods for 
measuring p lasma miRNAs / plasma extracellular vesicles and RNA cargo. There must be some 
data on this in Journal of Extracellular Vesicles for example.  
6. Does the ExoQuick system only isolate exosomes or other extracellular vesicles also? 
Again, please look for stu dies to cite to explain this.  
7. As far as I am aware, there is a lack of ribosomal RNA in EVs and the RNA is also 
fragmented and so therefore NanoDrop is unhelpful for assessing RNA purity/quality. Please see 
"Obstacles and opportunities in the functional  analysis of extracellular vesicle RNA an ISEV 
position paper" and comment in your Methods.  
8. For "anti -Alix, anti -TSG101, anti -Calnexin (Sigma), and anti -CD9 (Cell Signaling 
Technology) primary antibodies" please give the concentration of antibodies. Als o, how much 
protein was used for each Western Blot and what was the yield of exosomal protein for 1mL of 
plasma?  
9. Again, for the RT -qPCR Taqman assays. How much starting exosomal -RNA was used 
for each reaction? Please state in Methods. On that point, ho w much exosomal RNA was 
obtained per 1mL of plasma (please give average exosomal RNA yield for each disease group 
and state the volume of elute). Also, if 10mL blood was drawn per patient, how much plasma 
was obtained?  
10. Discussion change to….."In the la st few years, many studies have highlighted…."  
11. In the ISH section, what does it mean "PDAC and PC"?  
 
 
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 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: 1 Date: 22 Nov 2019  
 
Reviewer: Adam Frampton  
 
Reviewer's report:  
 
Vincentini et al have looked at Exosomal miRNAs in pancreatic  lesions. I have the following 
comments especially regarding the Methods:  
1. In the Intro, what is meant by "digital profiling"? Why not just say NanoString 
profiling?  
2. N=140 is a commendable sample size. However, there weren't any healthy controls? 
Ther e were only 15 CP samples as benign controls. If possible, it would be worth obtaining a 
small cohort of healthy control (HC) plasma exosomes (e.g. 10 to 15 participants) and then 
checking by RT -qPCR the expression of miR -4454, miR -106a -5p and miR -17-5p in  HC vs 
PDAC. As these plasma exosomal miRNAs all seemed down -regulated in PDAC compared to 
other lesions, but the comparison to HC is unknown.  
3. In the Intro, please change to "specific exosomal -miRNA signatures" and "relevance of 
these signatures in PDAC " 
4. "Ampulla of Vater carcinomas (AVC) comprise 30% of pancreaticoduodenectomies 
and 20% of all tumor -related obstructions of the common bile duct" --- I disagree with these 
figures quoted. Please check large pancreaticoduodenectomy series for the percent age of 
Ampullary cancers removed (e.g. Heidelberg, or Johns Hopkins).  
5. Please justify in your Methods using sodium heparin blood tubes, rather than EDTA 
blood tubes, for obtaining plasma. Check and cite studies looking at the best methods for 
measuring p lasma miRNAs / plasma extracellular vesicles and RNA cargo. There must be some 
data on this in Journal of Extracellular Vesicles for example.  
6. Does the ExoQuick system only isolate exosomes or other extracellular vesicles also? 
Again, please look for stu dies to cite to explain this.  
7. As far as I am aware, there is a lack of ribosomal RNA in EVs and the RNA is also 
fragmented and so therefore NanoDrop is unhelpful for assessing RNA purity/quality. Please see 
"Obstacles and opportunities in the functional  analysis of extracellular vesicle RNA an ISEV 
position paper" and comment in your Methods.  
8. For "anti -Alix, anti -TSG101, anti -Calnexin (Sigma), and anti -CD9 (Cell Signaling 
Technology) primary antibodies" please give the concentration of antibodies. Als o, how much 
protein was used for each Western Blot and what was the yield of exosomal protein for 1mL of 
plasma?  
9. Again, for the RT -qPCR Taqman assays. How much starting exosomal -RNA was used 
for each reaction? Please state in Methods. On that point, ho w much exosomal RNA was 
obtained per 1mL of plasma (please give average exosomal RNA yield for each disease group 
and state the volume of elute). Also, if 10mL blood was drawn per patient, how much plasma 
was obtained?  
10. Discussion change to….."In the la st few years, many studies have highlighted…."  
11. In the ISH section, what does it mean "PDAC and PC"?  
 
 
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 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: 1 Date: 22 Nov 2019  
 
Reviewer: Adam Frampton  
 
Reviewer's report:  
 
Vincentini et al have looked at Exosomal miRNAs in pancreatic  lesions. I have the following 
comments especially regarding the Methods:  
1. In the Intro, what is meant by "digital profiling"? Why not just say NanoString 
profiling?  
2. N=140 is a commendable sample size. However, there weren't any healthy controls? 
Ther e were only 15 CP samples as benign controls. If possible, it would be worth obtaining a 
small cohort of healthy control (HC) plasma exosomes (e.g. 10 to 15 participants) and then 
checking by RT -qPCR the expression of miR -4454, miR -106a -5p and miR -17-5p in  HC vs 
PDAC. As these plasma exosomal miRNAs all seemed down -regulated in PDAC compared to 
other lesions, but the comparison to HC is unknown.  
3. In the Intro, please change to "specific exosomal -miRNA signatures" and "relevance of 
these signatures in PDAC " 
4. "Ampulla of Vater carcinomas (AVC) comprise 30% of pancreaticoduodenectomies 
and 20% of all tumor -related obstructions of the common bile duct" --- I disagree with these 
figures quoted. Please check large pancreaticoduodenectomy series for the percent age of 
Ampullary cancers removed (e.g. Heidelberg, or Johns Hopkins).  
5. Please justify in your Methods using sodium heparin blood tubes, rather than EDTA 
blood tubes, for obtaining plasma. Check and cite studies looking at the best methods for 
measuring p lasma miRNAs / plasma extracellular vesicles and RNA cargo. There must be some 
data on this in Journal of Extracellular Vesicles for example.  
6. Does the ExoQuick system only isolate exosomes or other extracellular vesicles also? 
Again, please look for stu dies to cite to explain this.  
7. As far as I am aware, there is a lack of ribosomal RNA in EVs and the RNA is also 
fragmented and so therefore NanoDrop is unhelpful for assessing RNA purity/quality. Please see 
"Obstacles and opportunities in the functional  analysis of extracellular vesicle RNA an ISEV 
position paper" and comment in your Methods.  
8. For "anti -Alix, anti -TSG101, anti -Calnexin (Sigma), and anti -CD9 (Cell Signaling 
Technology) primary antibodies" please give the concentration of antibodies. Als o, how much 
protein was used for each Western Blot and what was the yield of exosomal protein for 1mL of 
plasma?  
9. Again, for the RT -qPCR Taqman assays. How much starting exosomal -RNA was used 
for each reaction? Please state in Methods. On that point, ho w much exosomal RNA was 
obtained per 1mL of plasma (please give average exosomal RNA yield for each disease group 
and state the volume of elute). Also, if 10mL blood was drawn per patient, how much plasma 
was obtained?  
10. Discussion change to….."In the la st few years, many studies have highlighted…."  
11. In the ISH section, what does it mean "PDAC and PC"?  
 
 
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 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