Reviewer’ s report
Title: Patient-individual cancer cell lines and tissue analysis delivers no evidence of sequences
from DNA viruses in colorectal cancer cells
Version: 0Date: 05 Oct 2019
Reviewer: Robin Mjelle
Reviewer's report:
1)The authors do not list the primer sequences they use which makes it impossible to assess the
specificity of the PCR experiments. The authos says "Primers used for EBV bind to specific EBVnucleotide sequence", however there is no way to know if they indeed analyze specific EBVsequences. Primers must be provided and an in silico analysis showing that the primers do notamplify other species and that they do indeed amplify the desired species. I think that using onlyone primer pair for each species is too little to conclude that virus DNA is not present.2)What Agarose gel electrophoresis percentage did the authors use and what type of gel?3)"To test the susceptibility of CRC cells to the JC and BK virus, HROC257 cells were infectedwith these viruses" - Where did the authors get these viruses, should be described in methods.4)The failure to reamplify HROC296 could be due to problems with the template. How was theHROC296 band purified from the gel?5)How was DNA isolated from the supernatant?6)"This indicates that no viral particles were present in the supernatant." - Particles could bepresents, however, the primer-pairs used did not pick up these particles.7)"In cultured epithelial-derived cells with no contaminating lymphocytes left over from the initialculturing process, a cross-contamination of the PCR results can safely be excluded." - Why does
the author not detect EBV in the primary tissue if the explanation is that EBV is lost during
transformation to cell lines? Other studies have shown that RNA form EBV is readily detectable incolon cancer tissue (PMID:30786859). Maybe the authors could use primers against the EBV-miRNAs as a positive control to check if RNA is presents?8)"To verify these findings, a highly sensitive qPCR (TaqMan259 ®) from HROC348 Met andHROC212 cell lines was performed…. the initial results could thus not be confirmed." Was thetaqman probes similar to the PCR primers, or within the pcr-region? If not, you cannot concludethis using only taqman assays. One way to check if the band at 180 is indeed virus is to addrestriction sites to the PCR primers and clone the product into a plasmid and perform sangersequencing (or other types of sequencing) to investigate what the band is.9)What were the positive controls in the infection-experiments?10)How were the cells followed up during infections? Was the cells washed frequently with PBSand was medium replaced? Was antibiotics used? In the discussion, the authors should comparetheir results with other co-culture studies on CRC if present.11)In the title you say that the study does not show evidence of a role of DNA virus in CRConcogenesis. This study did not try to investigate the role of viruses, but to detect the presence ofvirus DNA. The viruses could have different roles in vivo not captured by the experiments in thisstudy.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
NoDoes 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.
NoAre 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 furtherassessment in your comments to the editors.
I am able to assess the statisticsQuality 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 thismanuscript, either now or in the future?
2. Do you hold any stocks or shares 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 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 interestsI agree to the open peer 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 reportincluding 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 comments
which I do not wish to be included in my named report can be included as confidential commentsto 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 delivers no evidence of sequences
from DNA viruses in colorectal cancer cells
Version: 0Date: 05 Oct 2019
Reviewer: Robin Mjelle
Reviewer's report:
1)The authors do not list the primer sequences they use which makes it impossible to assess the
specificity of the PCR experiments. The authos says "Primers used for EBV bind to specific EBVnucleotide sequence", however there is no way to know if they indeed analyze specific EBVsequences. Primers must be provided and an in silico analysis showing that the primers do notamplify other species and that they do indeed amplify the desired species. I think that using onlyone primer pair for each species is too little to conclude that virus DNA is not present.2)What Agarose gel electrophoresis percentage did the authors use and what type of gel?3)"To test the susceptibility of CRC cells to the JC and BK virus, HROC257 cells were infectedwith these viruses" - Where did the authors get these viruses, should be described in methods.4)The failure to reamplify HROC296 could be due to problems with the template. How was theHROC296 band purified from the gel?5)How was DNA isolated from the supernatant?6)"This indicates that no viral particles were present in the supernatant." - Particles could bepresents, however, the primer-pairs used did not pick up these particles.7)"In cultured epithelial-derived cells with no contaminating lymphocytes left over from the initialculturing process, a cross-contamination of the PCR results can safely be excluded." - Why does
the author not detect EBV in the primary tissue if the explanation is that EBV is lost during
transformation to cell lines? Other studies have shown that RNA form EBV is readily detectable incolon cancer tissue (PMID:30786859). Maybe the authors could use primers against the EBV-miRNAs as a positive control to check if RNA is presents?8)"To verify these findings, a highly sensitive qPCR (TaqMan259 ®) from HROC348 Met andHROC212 cell lines was performed…. the initial results could thus not be confirmed." Was thetaqman probes similar to the PCR primers, or within the pcr-region? If not, you cannot concludethis using only taqman assays. One way to check if the band at 180 is indeed virus is to addrestriction sites to the PCR primers and clone the product into a plasmid and perform sangersequencing (or other types of sequencing) to investigate what the band is.9)What were the positive controls in the infection-experiments?10)How were the cells followed up during infections? Was the cells washed frequently with PBSand was medium replaced? Was antibiotics used? In the discussion, the authors should comparetheir results with other co-culture studies on CRC if present.11)In the title you say that the study does not show evidence of a role of DNA virus in CRConcogenesis. This study did not try to investigate the role of viruses, but to detect the presence ofvirus DNA. The viruses could have different roles in vivo not captured by the experiments in thisstudy.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
NoDoes 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.
NoAre 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 furtherassessment in your comments to the editors.
I am able to assess the statisticsQuality 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 thismanuscript, either now or in the future?
2. Do you hold any stocks or shares 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 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 interestsI agree to the open peer 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 reportincluding 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 comments
which I do not wish to be included in my named report can be included as confidential commentsto 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 delivers no evidence of sequences
from DNA viruses in colorectal cancer cells
Version: 0Date: 05 Oct 2019
Reviewer: Robin Mjelle
Reviewer's report:
1)The authors do not list the primer sequences they use which makes it impossible to assess the
specificity of the PCR experiments. The authos says "Primers used for EBV bind to specific EBVnucleotide sequence", however there is no way to know if they indeed analyze specific EBVsequences. Primers must be provided and an in silico analysis showing that the primers do notamplify other species and that they do indeed amplify the desired species. I think that using onlyone primer pair for each species is too little to conclude that virus DNA is not present.2)What Agarose gel electrophoresis percentage did the authors use and what type of gel?3)"To test the susceptibility of CRC cells to the JC and BK virus, HROC257 cells were infectedwith these viruses" - Where did the authors get these viruses, should be described in methods.4)The failure to reamplify HROC296 could be due to problems with the template. How was theHROC296 band purified from the gel?5)How was DNA isolated from the supernatant?6)"This indicates that no viral particles were present in the supernatant." - Particles could bepresents, however, the primer-pairs used did not pick up these particles.7)"In cultured epithelial-derived cells with no contaminating lymphocytes left over from the initialculturing process, a cross-contamination of the PCR results can safely be excluded." - Why does
the author not detect EBV in the primary tissue if the explanation is that EBV is lost during
transformation to cell lines? Other studies have shown that RNA form EBV is readily detectable incolon cancer tissue (PMID:30786859). Maybe the authors could use primers against the EBV-miRNAs as a positive control to check if RNA is presents?8)"To verify these findings, a highly sensitive qPCR (TaqMan259 ®) from HROC348 Met andHROC212 cell lines was performed…. the initial results could thus not be confirmed." Was thetaqman probes similar to the PCR primers, or within the pcr-region? If not, you cannot concludethis using only taqman assays. One way to check if the band at 180 is indeed virus is to addrestriction sites to the PCR primers and clone the product into a plasmid and perform sangersequencing (or other types of sequencing) to investigate what the band is.9)What were the positive controls in the infection-experiments?10)How were the cells followed up during infections? Was the cells washed frequently with PBSand was medium replaced? Was antibiotics used? In the discussion, the authors should comparetheir results with other co-culture studies on CRC if present.11)In the title you say that the study does not show evidence of a role of DNA virus in CRConcogenesis. This study did not try to investigate the role of viruses, but to detect the presence ofvirus DNA. The viruses could have different roles in vivo not captured by the experiments in thisstudy.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
NoDoes 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.
NoAre 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 furtherassessment in your comments to the editors.
I am able to assess the statisticsQuality 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 thismanuscript, either now or in the future?
2. Do you hold any stocks or shares 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 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 interestsI agree to the open peer 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 reportincluding 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 comments
which I do not wish to be included in my named report can be included as confidential commentsto the editors, which will not be published.
I agree to the open peer review policy of the journal
assistant