Reviewer ’s report  
 
Title: Patient -individual cancer cell lines and tissue analysis de livers no evidence of sequences 
from DNA viruses in colorectal cancer cells  
 
Version: 0 Date: 11 Nov 2019  
Reviewer:  Jörg Fahrer  
Reviewer's report:  
The work by Gock and colleagues addressed the role of viral infections in colorectal 
carcinogenesis. To this end, they used a set of 49 patient -individual CRC cell lines as well as 
primary material obtained from 11 patients. No virus -specific sequences were found using 
endpoint PCR except for a few false -positives that were followed up by qPCR and Sanger 
sequencing. Furthermore, the authors analyzed the susceptibility of the CRC cell line HROC257 
for polyomavirus infection in vitro, prov iding no evidence for viral infection.  
 
In summary, this is an interesting piece of work relevant to the field of colorectal carcinogenesis. 
Nevertheless, I have some concerns as detailed below:  
 
Page 7: Have the authors checked the integrity of the isolat ed RNA?  
Page 8 &amp;  9: Please provide technical details for the performed endpoint PCR (polymerase, 
buffer, annealing temperature, etc. pp)  
Page 8 &amp;  9: Please also include a list of all primers used in this study and indicate the 
supplier  
Page 8 &am p; 9: please include citations/references for the immortalized LCT7 and HROC212 
cells 
Page 8, line 151/152: how does the positive control loop test work?  
Page 9: please provide a rational for using HROC257 and not any other HROC cell line  
Page 10: the MOI used for the infection experiments sounds rather low. Please comment on that.  
Page 14, Fig. 4: the authors mentioned that 2 HROC cells appear to be positive for JCV/BKV 
(hardly visible on the agarose gel, by the way), which was followed up by qPCR. Please include 
the qPCR results for the two HROC cells lines as well as a positive control.  
Page 15: the authors mentioned that the reamplified amplicons were sequenced by the Sanger 
technique. Please provide details in the M &amp; M section  
Page 16 &amp;  17: how w as the experimental set -up for the infection study? Were these cells 
subcultured? If not, how often was the medium exchanged? Is there a quantitative readout 
available regarding cell growth/proliferation of non -infected vs infected cells?  
 
 
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 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 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 manuscr ipt: 
 
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 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 of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied f or 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 compet ing 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 journ al. 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 m y 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 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: Patient -individual cancer cell lines and tissue analysis de livers no evidence of sequences 
from DNA viruses in colorectal cancer cells  
 
Version: 0 Date: 11 Nov 2019  
Reviewer:  Jörg Fahrer  
Reviewer's report:  
The work by Gock and colleagues addressed the role of viral infections in colorectal 
carcinogenesis. To this end, they used a set of 49 patient -individual CRC cell lines as well as 
primary material obtained from 11 patients. No virus -specific sequences were found using 
endpoint PCR except for a few false -positives that were followed up by qPCR and Sanger 
sequencing. Furthermore, the authors analyzed the susceptibility of the CRC cell line HROC257 
for polyomavirus infection in vitro, prov iding no evidence for viral infection.  
 
In summary, this is an interesting piece of work relevant to the field of colorectal carcinogenesis. 
Nevertheless, I have some concerns as detailed below:  
 
Page 7: Have the authors checked the integrity of the isolat ed RNA?  
Page 8 &amp;  9: Please provide technical details for the performed endpoint PCR (polymerase, 
buffer, annealing temperature, etc. pp)  
Page 8 &amp;  9: Please also include a list of all primers used in this study and indicate the 
supplier  
Page 8 &am p; 9: please include citations/references for the immortalized LCT7 and HROC212 
cells 
Page 8, line 151/152: how does the positive control loop test work?  
Page 9: please provide a rational for using HROC257 and not any other HROC cell line  
Page 10: the MOI used for the infection experiments sounds rather low. Please comment on that.  
Page 14, Fig. 4: the authors mentioned that 2 HROC cells appear to be positive for JCV/BKV 
(hardly visible on the agarose gel, by the way), which was followed up by qPCR. Please include 
the qPCR results for the two HROC cells lines as well as a positive control.  
Page 15: the authors mentioned that the reamplified amplicons were sequenced by the Sanger 
technique. Please provide details in the M &amp; M section  
Page 16 &amp;  17: how w as the experimental set -up for the infection study? Were these cells 
subcultured? If not, how often was the medium exchanged? Is there a quantitative readout 
available regarding cell growth/proliferation of non -infected vs infected cells?  
 
 
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 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 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 manuscr ipt: 
 
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 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 of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied f or 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 compet ing 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 journ al. 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 m y 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 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: Patient -individual cancer cell lines and tissue analysis de livers no evidence of sequences 
from DNA viruses in colorectal cancer cells  
 
Version: 0 Date: 11 Nov 2019  
Reviewer:  Jörg Fahrer  
Reviewer's report:  
The work by Gock and colleagues addressed the role of viral infections in colorectal 
carcinogenesis. To this end, they used a set of 49 patient -individual CRC cell lines as well as 
primary material obtained from 11 patients. No virus -specific sequences were found using 
endpoint PCR except for a few false -positives that were followed up by qPCR and Sanger 
sequencing. Furthermore, the authors analyzed the susceptibility of the CRC cell line HROC257 
for polyomavirus infection in vitro, prov iding no evidence for viral infection.  
 
In summary, this is an interesting piece of work relevant to the field of colorectal carcinogenesis. 
Nevertheless, I have some concerns as detailed below:  
 
Page 7: Have the authors checked the integrity of the isolat ed RNA?  
Page 8 &amp;  9: Please provide technical details for the performed endpoint PCR (polymerase, 
buffer, annealing temperature, etc. pp)  
Page 8 &amp;  9: Please also include a list of all primers used in this study and indicate the 
supplier  
Page 8 &am p; 9: please include citations/references for the immortalized LCT7 and HROC212 
cells 
Page 8, line 151/152: how does the positive control loop test work?  
Page 9: please provide a rational for using HROC257 and not any other HROC cell line  
Page 10: the MOI used for the infection experiments sounds rather low. Please comment on that.  
Page 14, Fig. 4: the authors mentioned that 2 HROC cells appear to be positive for JCV/BKV 
(hardly visible on the agarose gel, by the way), which was followed up by qPCR. Please include 
the qPCR results for the two HROC cells lines as well as a positive control.  
Page 15: the authors mentioned that the reamplified amplicons were sequenced by the Sanger 
technique. Please provide details in the M &amp; M section  
Page 16 &amp;  17: how w as the experimental set -up for the infection study? Were these cells 
subcultured? If not, how often was the medium exchanged? Is there a quantitative readout 
available regarding cell growth/proliferation of non -infected vs infected cells?  
 
 
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 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 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 manuscr ipt: 
 
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 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 of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied f or 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 compet ing 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 journ al. 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 m y 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 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