Reviewer ’s report  
 
Title:  HPV positive, wild type TP53, and p16 overexpression correlate with the absence of 
residual tu mors after chemoradiotherapy in anal squamous cell carcinoma.  
 
Version: 0 Date: 17 Nov 2017 
 
Reviewer:  DC Gilbert  
 
Reviewer’s report:  
 
This is an interesting study looking at HPV involvement, p53 mutation and HIV status in Anal 
SCC treated with chemoRT that adds further data to the emerging literature on this topic. This is 
of clinical relevance as at present treatment is determined using tumour stage alone in a one size 
fits all approach - personalisation using biomarkers should improve outcomes.  
 
The study is well presented and well performed - the scientific analyses ar e appropriately carried 
out and described.  
 
My main criticism is that this report only has 6 month data to correlate with the biomarkers 
analysed - 6 month complete response is a meaningful endpoint but is by no means definitive - 3 
year locoregional contr ol rate would be considered the more relevant endpoint with recurrences 
(local and distant) occurring predominately in the first year, but signifcant numbers in year 2 and 
a few in year 3 (ACT 2 data - James et al Lancet Onc 2013).  
 
 
Suggestions:  
 
Title: should make this explicit (and care with 'predict' as this is prognostic not predictive data)    
i.e. something like 'HPV......  and correlations with complete response to chemo -radiotherapy in 
anal squamous cell carcinoma. HIV doesn't correlate so care with this in the title.  
 
Background - para 2, more could be said here (or alternatively in the discussion) with respect to 
the clinical rationale for a better understanding of biomarkers in ASCC (for a review see Jones et 
al BJC Jan 2017). A discussio n around the improved response to HPV+ SCC across tumour sites 
(HNSCC, vulval etc) and the reasons for this would be relevent too - including p53 status in 
HPV+/ - tumours  
 
Methods - FISH for TP53 - why was this done and why only in the samples with mutanti ons 
detected? please explain.  
 
Results para 3 - insert para break after 'and 50% more than control' so 6 month CR is its own 
paragraph. Move the p53 mutation data from this paragraph to the next subheaded paragraph 
('mutational status......).  
 
Discussion - the inverse correlation between p53 mutation and HPV status has also been 
described in HNSCC (Westra Clinical Cancer Research 2008) and Vulval and Penile Cancers 
again supporting a crucial role in HPV - oncogenesis and potentially resistance to treatment  
 
The importance of Immune recognition in ASCC, whether measured systemically or in tumour 
samples could also be included in the discussion (Balermpas et al. Oncoimmunology. 2017 Feb 
6;6(3):e1288331.  Martin D et al. Front Immunol. 2017 Sep 29;8:1225;  Gilbe rt et al Br J Cancer. 
2016 Jan 19;114(2):134 -7) as this links many of the other observations.  
 
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 manuscript or would you recomme nd 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 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 interests  
 
I agree to the open peer review policy of the journ al. 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 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 included 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:  HPV positive, wild type TP53, and p16 overexpression correlate with the absence of 
residual tu mors after chemoradiotherapy in anal squamous cell carcinoma.  
 
Version: 0 Date: 17 Nov 2017 
 
Reviewer:  DC Gilbert  
 
Reviewer’s report:  
 
This is an interesting study looking at HPV involvement, p53 mutation and HIV status in Anal 
SCC treated with chemoRT that adds further data to the emerging literature on this topic. This is 
of clinical relevance as at present treatment is determined using tumour stage alone in a one size 
fits all approach - personalisation using biomarkers should improve outcomes.  
 
The study is well presented and well performed - the scientific analyses ar e appropriately carried 
out and described.  
 
My main criticism is that this report only has 6 month data to correlate with the biomarkers 
analysed - 6 month complete response is a meaningful endpoint but is by no means definitive - 3 
year locoregional contr ol rate would be considered the more relevant endpoint with recurrences 
(local and distant) occurring predominately in the first year, but signifcant numbers in year 2 and 
a few in year 3 (ACT 2 data - James et al Lancet Onc 2013).  
 
 
Suggestions:  
 
Title: should make this explicit (and care with 'predict' as this is prognostic not predictive data)    
i.e. something like 'HPV......  and correlations with complete response to chemo -radiotherapy in 
anal squamous cell carcinoma. HIV doesn't correlate so care with this in the title.  
 
Background - para 2, more could be said here (or alternatively in the discussion) with respect to 
the clinical rationale for a better understanding of biomarkers in ASCC (for a review see Jones et 
al BJC Jan 2017). A discussio n around the improved response to HPV+ SCC across tumour sites 
(HNSCC, vulval etc) and the reasons for this would be relevent too - including p53 status in 
HPV+/ - tumours  
 
Methods - FISH for TP53 - why was this done and why only in the samples with mutanti ons 
detected? please explain.  
 
Results para 3 - insert para break after 'and 50% more than control' so 6 month CR is its own 
paragraph. Move the p53 mutation data from this paragraph to the next subheaded paragraph 
('mutational status......).  
 
Discussion - the inverse correlation between p53 mutation and HPV status has also been 
described in HNSCC (Westra Clinical Cancer Research 2008) and Vulval and Penile Cancers 
again supporting a crucial role in HPV - oncogenesis and potentially resistance to treatment  
 
The importance of Immune recognition in ASCC, whether measured systemically or in tumour 
samples could also be included in the discussion (Balermpas et al. Oncoimmunology. 2017 Feb 
6;6(3):e1288331.  Martin D et al. Front Immunol. 2017 Sep 29;8:1225;  Gilbe rt et al Br J Cancer. 
2016 Jan 19;114(2):134 -7) as this links many of the other observations.  
 
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 manuscript or would you recomme nd 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 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 interests  
 
I agree to the open peer review policy of the journ al. 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 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 included 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:  HPV positive, wild type TP53, and p16 overexpression correlate with the absence of 
residual tu mors after chemoradiotherapy in anal squamous cell carcinoma.  
 
Version: 0 Date: 17 Nov 2017 
 
Reviewer:  DC Gilbert  
 
Reviewer’s report:  
 
This is an interesting study looking at HPV involvement, p53 mutation and HIV status in Anal 
SCC treated with chemoRT that adds further data to the emerging literature on this topic. This is 
of clinical relevance as at present treatment is determined using tumour stage alone in a one size 
fits all approach - personalisation using biomarkers should improve outcomes.  
 
The study is well presented and well performed - the scientific analyses ar e appropriately carried 
out and described.  
 
My main criticism is that this report only has 6 month data to correlate with the biomarkers 
analysed - 6 month complete response is a meaningful endpoint but is by no means definitive - 3 
year locoregional contr ol rate would be considered the more relevant endpoint with recurrences 
(local and distant) occurring predominately in the first year, but signifcant numbers in year 2 and 
a few in year 3 (ACT 2 data - James et al Lancet Onc 2013).  
 
 
Suggestions:  
 
Title: should make this explicit (and care with 'predict' as this is prognostic not predictive data)    
i.e. something like 'HPV......  and correlations with complete response to chemo -radiotherapy in 
anal squamous cell carcinoma. HIV doesn't correlate so care with this in the title.  
 
Background - para 2, more could be said here (or alternatively in the discussion) with respect to 
the clinical rationale for a better understanding of biomarkers in ASCC (for a review see Jones et 
al BJC Jan 2017). A discussio n around the improved response to HPV+ SCC across tumour sites 
(HNSCC, vulval etc) and the reasons for this would be relevent too - including p53 status in 
HPV+/ - tumours  
 
Methods - FISH for TP53 - why was this done and why only in the samples with mutanti ons 
detected? please explain.  
 
Results para 3 - insert para break after 'and 50% more than control' so 6 month CR is its own 
paragraph. Move the p53 mutation data from this paragraph to the next subheaded paragraph 
('mutational status......).  
 
Discussion - the inverse correlation between p53 mutation and HPV status has also been 
described in HNSCC (Westra Clinical Cancer Research 2008) and Vulval and Penile Cancers 
again supporting a crucial role in HPV - oncogenesis and potentially resistance to treatment  
 
The importance of Immune recognition in ASCC, whether measured systemically or in tumour 
samples could also be included in the discussion (Balermpas et al. Oncoimmunology. 2017 Feb 
6;6(3):e1288331.  Martin D et al. Front Immunol. 2017 Sep 29;8:1225;  Gilbe rt et al Br J Cancer. 
2016 Jan 19;114(2):134 -7) as this links many of the other observations.  
 
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 manuscript or would you recomme nd 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 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 interests  
 
I agree to the open peer review policy of the journ al. 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 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 included as confidential comments 
to the editors, which will not be published . 
 
I agree to the open peer review policy of the journal  
assistant