Title: Feasibility of quantification based on novel evaluation with stool DNA and fecal
immunochemical test for colorectal cancer detection
Version: 4 Date: 24 Dec 2021
Reviewer:  Keita Harada
Reviewer's report:
Recently, as a non-invasive colorectal cancer screening, analyzing fecal DNA has attracted
attention. Since the paper publication by Imperiale et al. (N Engl J Med. 2014 Apr
3;370(14):1287-97), methylation levels of BMP3 and NDRG4 in feces as well as Kras mutation
have been regarded as useful biomarkers for colorectal cancer, and then, test kits named
Cologuard® (Exact Sciences Corporation, WI, USA) have been released.
The novel combination method of stool DNA and fecal immunochemical test was performed
and evaluated in this study. The sensitivity and specificity of the method as CRC screening were
satisfactory. Moreover, it was suggested that it would be useful as monitoring for advanced CRC
treatment. The content of the article was also generally well described. However, there are some
queries for the authors to answer in order to accept the manuscript.
Major points
1. Why the combination of Kras mutation and BMP and NDRG4 methylation was chosen as
sDNA. You have done the PCR yourself instead of using a commercial kit. Did you try several
targets and choose these as the best? Or did you not consider any other options at all?
2. In general, the more factors you combine, the higher the sensitivity and specificity, I think. Is it
possible to get similar results by removing one of Kras mutations, BMP or NDRG4 methylation?
Or did you consider that you could get better results by adding some optional factors?
3. It is difficult to understand how to calculate the combination value. Is it quantitative or
qualitative? If quantitative, I would like to know what percentage of each factor is considered.
Please show a specific formula. I am also not sure how did you determine the threshold of 165
after treatment.
4. The tumors shown in figure 6 were both carcinomas? How did the novel evaluation after
polypectomy? I do not think that it was because of these polyps that the values in two patients
did not decrease after surgery.
Minor points
nothing
Title: Feasibility of quantification based on novel evaluation with stool DNA and fecal
immunochemical test for colorectal cancer detection
Version: 4 Date: 24 Dec 2021
Reviewer:  Keita Harada
Reviewer's report:
Recently, as a non-invasive colorectal cancer screening, analyzing fecal DNA has attracted
attention. Since the paper publication by Imperiale et al. (N Engl J Med. 2014 Apr
3;370(14):1287-97), methylation levels of BMP3 and NDRG4 in feces as well as Kras mutation
have been regarded as useful biomarkers for colorectal cancer, and then, test kits named
Cologuard® (Exact Sciences Corporation, WI, USA) have been released.
The novel combination method of stool DNA and fecal immunochemical test was performed
and evaluated in this study. The sensitivity and specificity of the method as CRC screening were
satisfactory. Moreover, it was suggested that it would be useful as monitoring for advanced CRC
treatment. The content of the article was also generally well described. However, there are some
queries for the authors to answer in order to accept the manuscript.
Major points
1. Why the combination of Kras mutation and BMP and NDRG4 methylation was chosen as
sDNA. You have done the PCR yourself instead of using a commercial kit. Did you try several
targets and choose these as the best? Or did you not consider any other options at all?
2. In general, the more factors you combine, the higher the sensitivity and specificity, I think. Is it
possible to get similar results by removing one of Kras mutations, BMP or NDRG4 methylation?
Or did you consider that you could get better results by adding some optional factors?
3. It is difficult to understand how to calculate the combination value. Is it quantitative or
qualitative? If quantitative, I would like to know what percentage of each factor is considered.
Please show a specific formula. I am also not sure how did you determine the threshold of 165
after treatment.
4. The tumors shown in figure 6 were both carcinomas? How did the novel evaluation after
polypectomy? I do not think that it was because of these polyps that the values in two patients
did not decrease after surgery.
Minor points
nothing
Please review this paper.
user
Title: Feasibility of quantification based on novel evaluation with stool DNA and fecal
immunochemical test for colorectal cancer detection
Version: 4 Date: 24 Dec 2021
Reviewer:  Keita Harada
Reviewer's report:
Recently, as a non-invasive colorectal cancer screening, analyzing fecal DNA has attracted
attention. Since the paper publication by Imperiale et al. (N Engl J Med. 2014 Apr
3;370(14):1287-97), methylation levels of BMP3 and NDRG4 in feces as well as Kras mutation
have been regarded as useful biomarkers for colorectal cancer, and then, test kits named
Cologuard® (Exact Sciences Corporation, WI, USA) have been released.
The novel combination method of stool DNA and fecal immunochemical test was performed
and evaluated in this study. The sensitivity and specificity of the method as CRC screening were
satisfactory. Moreover, it was suggested that it would be useful as monitoring for advanced CRC
treatment. The content of the article was also generally well described. However, there are some
queries for the authors to answer in order to accept the manuscript.
Major points
1. Why the combination of Kras mutation and BMP and NDRG4 methylation was chosen as
sDNA. You have done the PCR yourself instead of using a commercial kit. Did you try several
targets and choose these as the best? Or did you not consider any other options at all?
2. In general, the more factors you combine, the higher the sensitivity and specificity, I think. Is it
possible to get similar results by removing one of Kras mutations, BMP or NDRG4 methylation?
Or did you consider that you could get better results by adding some optional factors?
3. It is difficult to understand how to calculate the combination value. Is it quantitative or
qualitative? If quantitative, I would like to know what percentage of each factor is considered.
Please show a specific formula. I am also not sure how did you determine the threshold of 165
after treatment.
4. The tumors shown in figure 6 were both carcinomas? How did the novel evaluation after
polypectomy? I do not think that it was because of these polyps that the values in two patients
did not decrease after surgery.
Minor points
nothing
assistant