Title: Circulating miRNA-202-3p is a potential novel biomarker for diagnosis of type 1 gastric
neuroendocrine neoplasms
Version: 1 Date: 15 Jan 2021
Reviewer's report:
In this article, Dou Dou et al. studied the diagnostic potential of circulating levels of
miRNA-202-3p in patients with gastric neuroendocrine neoplasms (gNEN). They evaluated 27
serum samples of patients with gNEN and compared them to 27 sex- and age-matched healthy
controls. As a result, they found a significant increase in miRNA-202-3p levels.
Unfortunately, the manuscript is not well written and therefore needs significant language
revision. Moreover, doubts remain on the methodology and therefore on the analysis of the
results presented.
1. The authors performed standard curve quantification to measure miRNA expression without
using an exogenous or endogenous control/normalization. Unfortunately, this is technically not
correct and not enough for exact and validated measurements. When using the standard curve
quantification, cycle thresholds are converted to copy numbers by drawing a standard curve
using a synthetic chemical standard. For accurate quantification, copy numbers are normalized
according to recovery rations of e.g. spiked-in-cel-miR-39 or cel-miR-238.
(Kook HY, Jin SH, Park PR, Lee SJ, Shin HJ, Kim TJ. Serum miR-214 as a novel
biomarker for ankylosing spondylitis. Int J Rheum Dis. 2019 Jul;22(7):1196-1201. doi:
10.1111/1756-185X.13475. Epub 2019 Feb 6. PMID: 30729703;
Iguchi, T., Niino, N., Tamai, S., Sakurai, K., Mori, K. Absolute Quantification of
Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse
Transcription PCR. J. Vis. Exp. (132), e56850, doi:10.3791/56850 (2018))
2. The authors should explain why they have preferred to use standard curve quantification
since relative qRT-PCR is a reliable and more common method for measuring expression of
circulating miRNA.
3.Given the low number of patients in this study (n=27), the authors should reduce their
conclusions since the lack of statistical value is limited due to their sample size. The authors
should also point out more clearly that further investigations are necessary to investigate the
diagnostic potential of this biomarker.
4. The clinical relevance of miR-222 should be worked out more clearly, e. g by correlating the
biomarker to clinical characteristics such as grading, number of lesions, gender, age, secondary
illnesses (arterial hypertension or diabetes). Moreover, survival data should be included to
investigate the prognostic potential of miRNA 202-3p (e.g.by using Kaplan Meyer analysis).
5. The first two paragraphs of the discussion section should be shortened and included into the
introduction. Moreover, results in the discussion are not confronted enough to previous study
results of circulating miRNA in neuroendocrine neoplasms (also in respect of sample size or
normalization).
Title: Circulating miRNA-202-3p is a potential novel biomarker for diagnosis of type 1 gastric
neuroendocrine neoplasms
Version: 1 Date: 15 Jan 2021
Reviewer's report:
In this article, Dou Dou et al. studied the diagnostic potential of circulating levels of
miRNA-202-3p in patients with gastric neuroendocrine neoplasms (gNEN). They evaluated 27
serum samples of patients with gNEN and compared them to 27 sex- and age-matched healthy
controls. As a result, they found a significant increase in miRNA-202-3p levels.
Unfortunately, the manuscript is not well written and therefore needs significant language
revision. Moreover, doubts remain on the methodology and therefore on the analysis of the
results presented.
1. The authors performed standard curve quantification to measure miRNA expression without
using an exogenous or endogenous control/normalization. Unfortunately, this is technically not
correct and not enough for exact and validated measurements. When using the standard curve
quantification, cycle thresholds are converted to copy numbers by drawing a standard curve
using a synthetic chemical standard. For accurate quantification, copy numbers are normalized
according to recovery rations of e.g. spiked-in-cel-miR-39 or cel-miR-238.
(Kook HY, Jin SH, Park PR, Lee SJ, Shin HJ, Kim TJ. Serum miR-214 as a novel
biomarker for ankylosing spondylitis. Int J Rheum Dis. 2019 Jul;22(7):1196-1201. doi:
10.1111/1756-185X.13475. Epub 2019 Feb 6. PMID: 30729703;
Iguchi, T., Niino, N., Tamai, S., Sakurai, K., Mori, K. Absolute Quantification of
Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse
Transcription PCR. J. Vis. Exp. (132), e56850, doi:10.3791/56850 (2018))
2. The authors should explain why they have preferred to use standard curve quantification
since relative qRT-PCR is a reliable and more common method for measuring expression of
circulating miRNA.
3.Given the low number of patients in this study (n=27), the authors should reduce their
conclusions since the lack of statistical value is limited due to their sample size. The authors
should also point out more clearly that further investigations are necessary to investigate the
diagnostic potential of this biomarker.
4. The clinical relevance of miR-222 should be worked out more clearly, e. g by correlating the
biomarker to clinical characteristics such as grading, number of lesions, gender, age, secondary
illnesses (arterial hypertension or diabetes). Moreover, survival data should be included to
investigate the prognostic potential of miRNA 202-3p (e.g.by using Kaplan Meyer analysis).
5. The first two paragraphs of the discussion section should be shortened and included into the
introduction. Moreover, results in the discussion are not confronted enough to previous study
results of circulating miRNA in neuroendocrine neoplasms (also in respect of sample size or
normalization).
Please review this paper.
user
Title: Circulating miRNA-202-3p is a potential novel biomarker for diagnosis of type 1 gastric
neuroendocrine neoplasms
Version: 1 Date: 15 Jan 2021
Reviewer's report:
In this article, Dou Dou et al. studied the diagnostic potential of circulating levels of
miRNA-202-3p in patients with gastric neuroendocrine neoplasms (gNEN). They evaluated 27
serum samples of patients with gNEN and compared them to 27 sex- and age-matched healthy
controls. As a result, they found a significant increase in miRNA-202-3p levels.
Unfortunately, the manuscript is not well written and therefore needs significant language
revision. Moreover, doubts remain on the methodology and therefore on the analysis of the
results presented.
1. The authors performed standard curve quantification to measure miRNA expression without
using an exogenous or endogenous control/normalization. Unfortunately, this is technically not
correct and not enough for exact and validated measurements. When using the standard curve
quantification, cycle thresholds are converted to copy numbers by drawing a standard curve
using a synthetic chemical standard. For accurate quantification, copy numbers are normalized
according to recovery rations of e.g. spiked-in-cel-miR-39 or cel-miR-238.
(Kook HY, Jin SH, Park PR, Lee SJ, Shin HJ, Kim TJ. Serum miR-214 as a novel
biomarker for ankylosing spondylitis. Int J Rheum Dis. 2019 Jul;22(7):1196-1201. doi:
10.1111/1756-185X.13475. Epub 2019 Feb 6. PMID: 30729703;
Iguchi, T., Niino, N., Tamai, S., Sakurai, K., Mori, K. Absolute Quantification of
Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse
Transcription PCR. J. Vis. Exp. (132), e56850, doi:10.3791/56850 (2018))
2. The authors should explain why they have preferred to use standard curve quantification
since relative qRT-PCR is a reliable and more common method for measuring expression of
circulating miRNA.
3.Given the low number of patients in this study (n=27), the authors should reduce their
conclusions since the lack of statistical value is limited due to their sample size. The authors
should also point out more clearly that further investigations are necessary to investigate the
diagnostic potential of this biomarker.
4. The clinical relevance of miR-222 should be worked out more clearly, e. g by correlating the
biomarker to clinical characteristics such as grading, number of lesions, gender, age, secondary
illnesses (arterial hypertension or diabetes). Moreover, survival data should be included to
investigate the prognostic potential of miRNA 202-3p (e.g.by using Kaplan Meyer analysis).
5. The first two paragraphs of the discussion section should be shortened and included into the
introduction. Moreover, results in the discussion are not confronted enough to previous study
results of circulating miRNA in neuroendocrine neoplasms (also in respect of sample size or
normalization).
assistant