Reviewer ’s report  
 
Title: The NETest Liquid Biopsy is Diagnostic for Gastric Neur oendocrine Tumors: 
Observations on the Blood -Based Identification of Microscopic and Macroscopic Residual 
Disease  
 
Version: 0 Date: 12 Jun 2019  
 
Reviewer's report:  
 
This is an original papper entitled: "The NETest Liquid  Biopsy is Diagnostic for Gastric 
Neuroendocrine Tumors: Observations on the Blood -Based Identification of Microscopic and 
Macroscopic Residual Disease" by A. Malczewska et al.  
 
This is the first publication describing the NETest Liquid Biopsy in patients with GNETs, thus 
the results are novel and interesting but some concerns are generated.  
 
Minor Suggestions  
 
I suggest that CAG should be changed to CAG -A (type A).  
NECs should not be considered as Type 3 NETs which are well differentiated (WHO 
classificatio n of tumors of the digestive system, Lyon, 2010, pages 64 -68, Solcia E. et al. 
reference should be included). Table 1 should also be changed accordingly, because NECs are 
included in the same column with GNETs type 3.  
 
Introduction, page 5, lines 11 -13. I s uggest that the meaning should be altered. The follow up is 
neither performed for the possible metastatic potential of small well differentiated GNETs nor 
the risk of bleeding.  The possibility for the development for adeno Ca can remain.  
Patient cohorts, page 7, lines 24 -25. What was the size of the 4 GNETs G1 that underwent partial 
gastrectomy, and how a total gastrectomy is excused in all G2 GNETs (with Ki67 ≤10%), wedge 
shaped resection could not be performed?  
 
The aforementioned WHO ref erence should be included at 2.4, Histological diagnosis.  
 
3.1 Disease extent Lines 9 -13. Can the authors describe more thorough "the abnormality in the 
gastric mucosa that was reported" and the respective findings at gastroscopy? Furthermore, can 
the autho rs describe the result of Ga -PET? Was the uptake focal or diffuse? Strong or weak?  
3.1 Image -negative and histology -positive, page 11, lines 2 -3. What was the size of the GNETs 
described at the pathology report?  
 
Total gastrectomy, page 12. Why FDG PET was not performed in these NEC cases, while FDG 
PET was performed at a well differentiated GNET (page 11, line 2). These cases were performed 
at the authors' center and data should be available to clarify the above.  
Discussion, page 13, lines 5 -6. The way the phrase is written may be misleading for the reader. 
Minute lesions are rarely metastatic when it comes to GNETs type 1, G1.  
How the authors explain that only 5 patients with type 1 GNETs had intestinal metaplasia? This 
number is low compared to literature.  
 
According to the authors the levels of NETest did not differ statistically between the cases that 
were positive despite difference in the size. How do the authors explain the much higher levels 
of NETest at the previously published GNET type 3 case?  
Discu ssion, page 17, lines 11 -18. I have concerns with the postulation of the authors that 
antrectomy is not efficient. There are several publications proving its effectiveness. The risk of 
ECLomas to become autonomous is rather low and only case reports have b een reported. I 
suggest that the authors will rephrase.  
 
Discussion, page 19 -21. The authors present opinions that are not proved by the literature. This 
meaning should be rephrased or omitted.  
 
 
Major issues  
 
Introduction, page 6, lines 4 -5. The patients su ggest that the NETest raises the possibility of 
reconsideration of the "endoscopy for life". The malignant potential of type 1 GNETs is very low 
and the endoscopic follow up is also performed for the risk of adenoCa in the gastric mucosa. 
Thus, the endosco pical follow up is mandatory and is not replaced by NETest. I suggest that the 
authors should rephrase.  
 
I suggest that the authors will describe the protocol followed about biopsies obtained during 
follow up. Was a standardized protocol followed or was it individual for every endoscopist that 
participated? This could cause variations at the interpretation of positive and negative disease as 
at paragraph 3.1 there are described Image -negative and histology -positive cases.  
A cornerstone for the evaluation of GNETs is EUS. Surprisingly the authors present no EUS -
data. At EUS more and deeper located lesions can be identified that are not visible with 
gastroscopy. How the authors address this question?  
 
3.1 Disease extent. Page 10 lines 20 -26 and page 11 lines 1 -3. The authors describe that it can be 
lesions that are image and histology negative or image negative and histology positive. The 
lesions that are described as image and histology negative can possibly be histology positive that 
the authors did not identif y because the tumor was not visible and was not biopsied. EUS could 
be helpful in this situation. How do the authors address this important issue?  
 
Discussion page 19, lines 20 -21. According to the authors the NETest can be used for identifying 
lesions of 2 mm "identifies when dysplasia is transformed to neoplasia". Such small lesions do 
not require resection but follow up, but larger lesions may need treatment. Similarly, at 3.2 
Diagnosis page 11 lines 11 -14: Levels in macroscopic disease were not different to microscopic 
disease. How can the authors distinguish which patients will need endoscopy and which not as 
the test can not identify the ones that need follow up from the ones that need treatment. Thus, the 
economical advantage of NETest in being used ins tead of endoscopy may not be excused.  
 
 
 
 
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 nece ssary 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.  
 
No 
 
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 statis tics 
 
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, 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 s tocks 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 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 compe ting 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' below. If your reply is yes to  any, please give details 
below.  
 
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 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 c omments 
which I do not wish to be included in 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: The NETest Liquid Biopsy is Diagnostic for Gastric Neur oendocrine Tumors: 
Observations on the Blood -Based Identification of Microscopic and Macroscopic Residual 
Disease  
 
Version: 0 Date: 12 Jun 2019  
 
Reviewer's report:  
 
This is an original papper entitled: "The NETest Liquid  Biopsy is Diagnostic for Gastric 
Neuroendocrine Tumors: Observations on the Blood -Based Identification of Microscopic and 
Macroscopic Residual Disease" by A. Malczewska et al.  
 
This is the first publication describing the NETest Liquid Biopsy in patients with GNETs, thus 
the results are novel and interesting but some concerns are generated.  
 
Minor Suggestions  
 
I suggest that CAG should be changed to CAG -A (type A).  
NECs should not be considered as Type 3 NETs which are well differentiated (WHO 
classificatio n of tumors of the digestive system, Lyon, 2010, pages 64 -68, Solcia E. et al. 
reference should be included). Table 1 should also be changed accordingly, because NECs are 
included in the same column with GNETs type 3.  
 
Introduction, page 5, lines 11 -13. I s uggest that the meaning should be altered. The follow up is 
neither performed for the possible metastatic potential of small well differentiated GNETs nor 
the risk of bleeding.  The possibility for the development for adeno Ca can remain.  
Patient cohorts, page 7, lines 24 -25. What was the size of the 4 GNETs G1 that underwent partial 
gastrectomy, and how a total gastrectomy is excused in all G2 GNETs (with Ki67 ≤10%), wedge 
shaped resection could not be performed?  
 
The aforementioned WHO ref erence should be included at 2.4, Histological diagnosis.  
 
3.1 Disease extent Lines 9 -13. Can the authors describe more thorough "the abnormality in the 
gastric mucosa that was reported" and the respective findings at gastroscopy? Furthermore, can 
the autho rs describe the result of Ga -PET? Was the uptake focal or diffuse? Strong or weak?  
3.1 Image -negative and histology -positive, page 11, lines 2 -3. What was the size of the GNETs 
described at the pathology report?  
 
Total gastrectomy, page 12. Why FDG PET was not performed in these NEC cases, while FDG 
PET was performed at a well differentiated GNET (page 11, line 2). These cases were performed 
at the authors' center and data should be available to clarify the above.  
Discussion, page 13, lines 5 -6. The way the phrase is written may be misleading for the reader. 
Minute lesions are rarely metastatic when it comes to GNETs type 1, G1.  
How the authors explain that only 5 patients with type 1 GNETs had intestinal metaplasia? This 
number is low compared to literature.  
 
According to the authors the levels of NETest did not differ statistically between the cases that 
were positive despite difference in the size. How do the authors explain the much higher levels 
of NETest at the previously published GNET type 3 case?  
Discu ssion, page 17, lines 11 -18. I have concerns with the postulation of the authors that 
antrectomy is not efficient. There are several publications proving its effectiveness. The risk of 
ECLomas to become autonomous is rather low and only case reports have b een reported. I 
suggest that the authors will rephrase.  
 
Discussion, page 19 -21. The authors present opinions that are not proved by the literature. This 
meaning should be rephrased or omitted.  
 
 
Major issues  
 
Introduction, page 6, lines 4 -5. The patients su ggest that the NETest raises the possibility of 
reconsideration of the "endoscopy for life". The malignant potential of type 1 GNETs is very low 
and the endoscopic follow up is also performed for the risk of adenoCa in the gastric mucosa. 
Thus, the endosco pical follow up is mandatory and is not replaced by NETest. I suggest that the 
authors should rephrase.  
 
I suggest that the authors will describe the protocol followed about biopsies obtained during 
follow up. Was a standardized protocol followed or was it individual for every endoscopist that 
participated? This could cause variations at the interpretation of positive and negative disease as 
at paragraph 3.1 there are described Image -negative and histology -positive cases.  
A cornerstone for the evaluation of GNETs is EUS. Surprisingly the authors present no EUS -
data. At EUS more and deeper located lesions can be identified that are not visible with 
gastroscopy. How the authors address this question?  
 
3.1 Disease extent. Page 10 lines 20 -26 and page 11 lines 1 -3. The authors describe that it can be 
lesions that are image and histology negative or image negative and histology positive. The 
lesions that are described as image and histology negative can possibly be histology positive that 
the authors did not identif y because the tumor was not visible and was not biopsied. EUS could 
be helpful in this situation. How do the authors address this important issue?  
 
Discussion page 19, lines 20 -21. According to the authors the NETest can be used for identifying 
lesions of 2 mm "identifies when dysplasia is transformed to neoplasia". Such small lesions do 
not require resection but follow up, but larger lesions may need treatment. Similarly, at 3.2 
Diagnosis page 11 lines 11 -14: Levels in macroscopic disease were not different to microscopic 
disease. How can the authors distinguish which patients will need endoscopy and which not as 
the test can not identify the ones that need follow up from the ones that need treatment. Thus, the 
economical advantage of NETest in being used ins tead of endoscopy may not be excused.  
 
 
 
 
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 nece ssary 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.  
 
No 
 
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 statis tics 
 
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, 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 s tocks 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 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 compe ting 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' below. If your reply is yes to  any, please give details 
below.  
 
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 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 c omments 
which I do not wish to be included in 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: The NETest Liquid Biopsy is Diagnostic for Gastric Neur oendocrine Tumors: 
Observations on the Blood -Based Identification of Microscopic and Macroscopic Residual 
Disease  
 
Version: 0 Date: 12 Jun 2019  
 
Reviewer's report:  
 
This is an original papper entitled: "The NETest Liquid  Biopsy is Diagnostic for Gastric 
Neuroendocrine Tumors: Observations on the Blood -Based Identification of Microscopic and 
Macroscopic Residual Disease" by A. Malczewska et al.  
 
This is the first publication describing the NETest Liquid Biopsy in patients with GNETs, thus 
the results are novel and interesting but some concerns are generated.  
 
Minor Suggestions  
 
I suggest that CAG should be changed to CAG -A (type A).  
NECs should not be considered as Type 3 NETs which are well differentiated (WHO 
classificatio n of tumors of the digestive system, Lyon, 2010, pages 64 -68, Solcia E. et al. 
reference should be included). Table 1 should also be changed accordingly, because NECs are 
included in the same column with GNETs type 3.  
 
Introduction, page 5, lines 11 -13. I s uggest that the meaning should be altered. The follow up is 
neither performed for the possible metastatic potential of small well differentiated GNETs nor 
the risk of bleeding.  The possibility for the development for adeno Ca can remain.  
Patient cohorts, page 7, lines 24 -25. What was the size of the 4 GNETs G1 that underwent partial 
gastrectomy, and how a total gastrectomy is excused in all G2 GNETs (with Ki67 ≤10%), wedge 
shaped resection could not be performed?  
 
The aforementioned WHO ref erence should be included at 2.4, Histological diagnosis.  
 
3.1 Disease extent Lines 9 -13. Can the authors describe more thorough "the abnormality in the 
gastric mucosa that was reported" and the respective findings at gastroscopy? Furthermore, can 
the autho rs describe the result of Ga -PET? Was the uptake focal or diffuse? Strong or weak?  
3.1 Image -negative and histology -positive, page 11, lines 2 -3. What was the size of the GNETs 
described at the pathology report?  
 
Total gastrectomy, page 12. Why FDG PET was not performed in these NEC cases, while FDG 
PET was performed at a well differentiated GNET (page 11, line 2). These cases were performed 
at the authors' center and data should be available to clarify the above.  
Discussion, page 13, lines 5 -6. The way the phrase is written may be misleading for the reader. 
Minute lesions are rarely metastatic when it comes to GNETs type 1, G1.  
How the authors explain that only 5 patients with type 1 GNETs had intestinal metaplasia? This 
number is low compared to literature.  
 
According to the authors the levels of NETest did not differ statistically between the cases that 
were positive despite difference in the size. How do the authors explain the much higher levels 
of NETest at the previously published GNET type 3 case?  
Discu ssion, page 17, lines 11 -18. I have concerns with the postulation of the authors that 
antrectomy is not efficient. There are several publications proving its effectiveness. The risk of 
ECLomas to become autonomous is rather low and only case reports have b een reported. I 
suggest that the authors will rephrase.  
 
Discussion, page 19 -21. The authors present opinions that are not proved by the literature. This 
meaning should be rephrased or omitted.  
 
 
Major issues  
 
Introduction, page 6, lines 4 -5. The patients su ggest that the NETest raises the possibility of 
reconsideration of the "endoscopy for life". The malignant potential of type 1 GNETs is very low 
and the endoscopic follow up is also performed for the risk of adenoCa in the gastric mucosa. 
Thus, the endosco pical follow up is mandatory and is not replaced by NETest. I suggest that the 
authors should rephrase.  
 
I suggest that the authors will describe the protocol followed about biopsies obtained during 
follow up. Was a standardized protocol followed or was it individual for every endoscopist that 
participated? This could cause variations at the interpretation of positive and negative disease as 
at paragraph 3.1 there are described Image -negative and histology -positive cases.  
A cornerstone for the evaluation of GNETs is EUS. Surprisingly the authors present no EUS -
data. At EUS more and deeper located lesions can be identified that are not visible with 
gastroscopy. How the authors address this question?  
 
3.1 Disease extent. Page 10 lines 20 -26 and page 11 lines 1 -3. The authors describe that it can be 
lesions that are image and histology negative or image negative and histology positive. The 
lesions that are described as image and histology negative can possibly be histology positive that 
the authors did not identif y because the tumor was not visible and was not biopsied. EUS could 
be helpful in this situation. How do the authors address this important issue?  
 
Discussion page 19, lines 20 -21. According to the authors the NETest can be used for identifying 
lesions of 2 mm "identifies when dysplasia is transformed to neoplasia". Such small lesions do 
not require resection but follow up, but larger lesions may need treatment. Similarly, at 3.2 
Diagnosis page 11 lines 11 -14: Levels in macroscopic disease were not different to microscopic 
disease. How can the authors distinguish which patients will need endoscopy and which not as 
the test can not identify the ones that need follow up from the ones that need treatment. Thus, the 
economical advantage of NETest in being used ins tead of endoscopy may not be excused.  
 
 
 
 
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 nece ssary 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.  
 
No 
 
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 statis tics 
 
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, 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 s tocks 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 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 compe ting 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' below. If your reply is yes to  any, please give details 
below.  
 
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 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 c omments 
which I do not wish to be included in 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