Detection of HPV DNA in Saliva of Patients with HPV-Associated Oropharyngeal Cancer Treated with Radiotherapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of Detection of HPV DNA in saliva of patients with HPV-associated oropharyngeal cancer treated with radiotherapy are treating a very important medical condition: OPSCC.

 

Even the authors are mentioning that this is a pilot study, the number of patients followed-up is too small, I think, to assess sensitivity, specificity and disease survival.

Line 32: Should be assessed more precisely, from scientific point of view.

 

Line 71: please mention the inclusion and exclusion criteria of the patients

Line 80: do you have any data of standard curve used for qpCR experiments?

As an overall evaluation: introduction, discussion, reference could be improved.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear Reviewer,

 

Thank you for your comments. I believe your comments helped me improve the significance of the study. Please find author's notes as follows:

 

Even the authors are mentioning that this is a pilot study, the number of patients followed-up is too small, I think, to assess sensitivity, specificity and disease survival.

Thank you for your precise comments. As you pointed out, the limitation of the study is the small number of the patients, therefore it is difficult to make conclusion to assess sensitivity, specificity, and treatment outcome. However, we believe that the technical feasibility has at least been confirmed by this study, we are planning to study more patients in the future.

Line 32: Should be assessed more precisely, from scientific point of view.

Thank you for your comment. We have amended the conclusion to emphasize the technical aspect of the study, as follows:

 This study demonstrates that in addition to detection and quantification of HPV-DNA by RT-PCR, detection by sequencing of HPV DNA using customized cancer panel is technically possible.

 

Line 71: please mention the inclusion and exclusion criteria of the patients

Thank you for your comment.

We added eligibility and exclusion criteria for the study in the methods section.

Inclusion and exclusion criteria

The inclusion criteria were as follows: Patients 1) with histologically confirmed oropharyngeal squamous cell carcinoma, 2) who were scheduled to receive definitive radiotherapy, 3) who were over 20 years old, and 4) who agreed to participate in the study with a written informed consent document.

Patients deemed ineligible by the treating physician to be enrolled was excluded from the study.

 

Line 80: do you have any data of standard curve used for qpCR experiments?

Thank you for the comment.　We added a sentence regarding the construction of the standard curve in the methods section as follows:

Standard curve for RT-PCR was generated using a dilution series constructed from a sample of HPV-DNA which was arbitrarily selected.

As an overall evaluation: introduction, discussion, reference could be improved.

Thank you for the comment. As you and other reviewers pointed out, we had amended the manuscript accordingly.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

I fully accept  the statement present in final part of the article saying that  is “the first technical report of a successful cancer panel incorporating viral DNA sequences”.  However some technical points are to be clarified. Information about tobacco smoking should be included (Table 1 ?). Saliva was collected for 3 times from all (n=9) patients but 24 saliva samples were collected. Discordance. Cancer panel sequencing was performed on 10-saliva samples from 6 patients. Who was eliminated? More, drawing of so many data from saliva is impressive and a good argument for liquid biopsy usage.

Ad meritum:

1.. line 39, standard of care for HPV patients; does it really exist? Compare information  below: lines 40-45,  “efforts are currently on the way…”

2.  The patients are relatively aged that is not common for HPV- associated head and neck cancer Still, thre is nothing to do with it.

3. A small group of patients is highly heterogeneous concerning tumor size, metastasis to adjacent lymph nodes and variable treatment. It makes generalization of results hard to accept. Anyway  decrease of HPV DNA was constantly show  during and after therapy that is promising.

I suggest some revision to make report fully acceptable.

Author Response

Dear Reviewer,

 

Thank you for your comments. I believe your comments helped me improve the significance of the study. Please find author's notes as follows:

 

I fully accept  the statement present in final part of the article saying that  is “the first technical report of a successful cancer panel incorporating viral DNA sequences”.  However some technical points are to be clarified. Information about tobacco smoking should be included (Table 1 ?).

Thank you for the comment. We also believe that information regarding tobacco smoking is very important to predict biological behavior of HPV-OPC. We added a column for Brinkman index in Table 1, and several details regarding smoking history in the results section as follows: Six out of 9 patients had smoking history, with Brinkman index ranging from 0 to 900 (median: 90).

 

Saliva was collected for 3 times from all (n=9) patients but 24 saliva samples were collected. Discordance. Cancer panel sequencing was performed on 10-saliva samples from 6 patients. Who was eliminated? More, drawing of so many data from saliva is impressive and a good argument for liquid biopsy usage.

Thank you for the precise comment. We tried to collect 3 times consecutively from each patient, however, several samples were not collected due to technical problems. We corrected the description in the results section as follows: Because several samples of saliva were not appropriately preserved due to technical problems, 24 saliva samples were collected in total.

Due to our limited cost, we were unable to perform cancer panel sequencing on all saliva samples. We included seven pre-treatment saliva samples because it was anticipated before analysis that the pre-treatment saliva samples would contain the greatest amount of tumor-derived DNA. Three samples obtained after treatment were selected for comparison, however these were selected arbitrarily. We had added the phrase “which were arbitrarily selected.”

 

Ad meritum: 

1.. line 39, standard of care for HPV patients; does it really exist? Compare information  below: lines 40-45,  “efforts are currently on the way…”

Thank you for the comment. Because other treatment options such as induction chemotherapy, concurrent administration cetuximab also constitute standard of care for HPV-OPC, we amended the sentence as follows: Radiotherapy alone or concurrent chemoradiotherapy is currently considered one of the standard of care for patients with HPV-OPC.

We believe that radiotherapy or chemoradiotherapy is one of the standard care because these are listed as standard of care for HPV-OPC in NCCN guidelines.

 

2. The patients are relatively aged that is not common for HPV- associated head and neck cancer Still, thre is nothing to do with it.

Thank you for the comment. This may reflect the demographic background of Japan, which is with relatively low infection rate of HPV in population.

 

3. A small group of patients is highly heterogeneous concerning tumor size, metastasis to adjacent lymph nodes and variable treatment. It makes generalization of results hard to accept. Anyway  decrease of HPV DNA was constantly show  during and after therapy that is promising.

I suggest some revision to make report fully acceptable.

Thank you for the comment. As pointed out also by other reviewers, the limitation of the study is the small number of the patients, therefore it is difficult to make conclusion to assess sensitivity, specificity, and treatment outcome. However, we believe that the technical feasibility has at least been confirmed by this study, we are planning to study more patients in the future.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors used RT-PCR technology and direct sequencing methods to quantitatively explore the feasibility of prognostic analysis using quantitative techniques for HPV DNA in the saliva of patients with human papillomavirus-related or pharyngeal carcinoma (HPV OPC). This research is very valuable. There are a few issues to focus on here.

1. The font size of the table is too small. Also, it's best to use a three-line table.

2. The font size of the figure is too small. In addition, the layout of the figure can be adjusted appropriately. For example, the two-column pattern.

3. Although judging from the author's analysis of the data, there were significant differences between baseline data and assessment levels, but it is advisable for the authors to do medical statistical analysis to better illustrate the significant differences between the baseline and experimental groups with appropriate p-values.

4. Some references lack page numbers or related fields.

Author Response

Dear Reviewer,

 

Thank you for your comments. I believe your comments helped me improve the significance of the study. Please find author's notes as follows:

 

The authors used RT-PCR technology and direct sequencing methods to quantitatively explore the feasibility of prognostic analysis using quantitative techniques for HPV DNA in the saliva of patients with human papillomavirus-related or pharyngeal carcinoma (HPV OPC). This research is very valuable. There are a few issues to focus on here.

1. The font size of the table is too small. Also, it's best to use a three-line table.

Thank you for the comment. We had amended the table according to the instruction from the editorial office.

2. The font size of the figure is too small. In addition, the layout of the figure can be adjusted appropriately. For example, the two-column pattern.

Thank you for the comment. We have enlarged the fonts in figures according to the instruction from the journal.

3. Although judging from the author's analysis of the data, there were significant differences between baseline data and assessment levels, but it is advisable for the authors to do medical statistical analysis to better illustrate the significant differences between the baseline and experimental groups with appropriate p-values.

Thank you for your comment. Although the number of patients enrolled was small, we had compared the correlation between the relative baseline CT values and disease progression. In the results section, we added several sentences as follows: Additionally, the correlation between the baseline relative CT values for HPV DNA and disease progression was examined. The Mann-Whitney U test showed marginal significance (both p=0.070 for HPV E6 and E7) between the baseline relative CT values for HPV DNA and disease progression.

4. Some references lack page numbers or related fields.

 Thank you for the comment. We added page numbers for some of the papers. However, several papers have no volume number because they are independent article published online.