Title: Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal
appearing colonic mucosa from patients with colorectal neoplasia
Version: 1 Date: 29 Oct 2021
Reviewer:  #ukasz Zadka
Reviewer's report:
In this interesting preliminary study, Feddersen et al. showed abnormalities occurring in cancer-
associated normal colonic mucosa for eicosanoid signaling pathway. The molecular disbalances
ongoing in the morphologically unchanged colonic mucosa during carcinogenesis are a logical
destination for many different biomarkers, nevertheless, comparative analyzes in this area are
very rare, therefore the work has additional value in the field of basic research. However, after
carefully reading this manuscript, I have some concerns. Please find below my comments:
Major:
There are missing some important clinicopathological data (section: “Study population”). The
description of patients should be supported (at least) with histopathological diagnosis, G grade
and TNM stage.
The diversity of patients in terms of chronic diseases and medications taken could have a
potential impact on the obtained results.
The gene transcript does not always coincide with the translation mechanism, which means that
the mRNA expression level may differ from the levels of the corresponding proteins. It would be
beneficial for this work to demonstrate the presence of the studied biomarkers in the tissue at the
protein level, not just mRNA. Supplementary to PCR, semi-quantitative protein analysis could
also be an option. I would recommend performing immunoblotting or immunofluorescence
analyses of the examined colonic biopsies for EP1-4 receptors, PGIS/PTGIS and LOXs plus
reference proteins (b-actin or/and GAPDH for WB).
Minor:
1) "CRC carcinogenesis is a multifactorial process, in which an accumulation of mutations leads
to the formation of colorectal neoplasia (CRN), initially as benign adenomas and subsequently
malignant adenocarcinomas (2)."
This is a misleading sentence that generalizes CRC oncogenesis to suggest that all colon cancers
arise from a polyp basis. What about other histological subtypes of cancers (i.e.: Carcinomas
and mesenchymal cancers like colorectal sarcomas) that also meet the CRC definition?
2) "Several theories in form of “field effects” and “mutator pathways” for primary tumor-
induced changes in near and distant gene expression have been forwarded over the last forty
years (18-20)."
This sentence is difficult to build a clear conclusion. Could you please be more precise and
accurately define the meaning of used terms?
3) Authors should provide the exact address details of the Department where the colonoscopy
procedure was performed
4) The numbers of examined cases presented on figures should be better assigned to their charts
(in example: Fig. 3B and 3C, Fig. 4B)
5) Figure 6 caption: please provide the general number of examined cases for both CTRL and
CRN subgroups. Besides of that, it is not clear what the numbers are above the bars in the figure.
If it is the size of the studied groups, it should be presented in a more intuitive way (use n =) or
provide an appropriate explanation in the label.
6) Typos and grammar (selected examples):
a) “Accordingly, we examine eicosanoid-related enzymes and receptors in non-neoplastic
colonic mucosa both from patients with and without CRN.”
b) “Finally, we determine expression levels of 5-, 12-, and 15-LOX enzymes.”
My comment: in these sentences, the past tense should be used
c) “Fig. 6. EP3 and EP4 mRNA levels showed a trend of elevation in CRN patients.”
It would be more accurate to use term “expression” in relation to the mRNA levels
7) Not all abbreviations were explained in the manuscript. For example:
“Following extraction, RNA samples were placed on ice and quantified using a Nanodrop
Spectrophotometer (LabTech International) in accordance with the MIQE guidelines (23).”
<190-192>
Please explain what does the MIQE refer to.
Title: Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal
appearing colonic mucosa from patients with colorectal neoplasia
Version: 1 Date: 29 Oct 2021
Reviewer:  #ukasz Zadka
Reviewer's report:
In this interesting preliminary study, Feddersen et al. showed abnormalities occurring in cancer-
associated normal colonic mucosa for eicosanoid signaling pathway. The molecular disbalances
ongoing in the morphologically unchanged colonic mucosa during carcinogenesis are a logical
destination for many different biomarkers, nevertheless, comparative analyzes in this area are
very rare, therefore the work has additional value in the field of basic research. However, after
carefully reading this manuscript, I have some concerns. Please find below my comments:
Major:
There are missing some important clinicopathological data (section: “Study population”). The
description of patients should be supported (at least) with histopathological diagnosis, G grade
and TNM stage.
The diversity of patients in terms of chronic diseases and medications taken could have a
potential impact on the obtained results.
The gene transcript does not always coincide with the translation mechanism, which means that
the mRNA expression level may differ from the levels of the corresponding proteins. It would be
beneficial for this work to demonstrate the presence of the studied biomarkers in the tissue at the
protein level, not just mRNA. Supplementary to PCR, semi-quantitative protein analysis could
also be an option. I would recommend performing immunoblotting or immunofluorescence
analyses of the examined colonic biopsies for EP1-4 receptors, PGIS/PTGIS and LOXs plus
reference proteins (b-actin or/and GAPDH for WB).
Minor:
1) "CRC carcinogenesis is a multifactorial process, in which an accumulation of mutations leads
to the formation of colorectal neoplasia (CRN), initially as benign adenomas and subsequently
malignant adenocarcinomas (2)."
This is a misleading sentence that generalizes CRC oncogenesis to suggest that all colon cancers
arise from a polyp basis. What about other histological subtypes of cancers (i.e.: Carcinomas
and mesenchymal cancers like colorectal sarcomas) that also meet the CRC definition?
2) "Several theories in form of “field effects” and “mutator pathways” for primary tumor-
induced changes in near and distant gene expression have been forwarded over the last forty
years (18-20)."
This sentence is difficult to build a clear conclusion. Could you please be more precise and
accurately define the meaning of used terms?
3) Authors should provide the exact address details of the Department where the colonoscopy
procedure was performed
4) The numbers of examined cases presented on figures should be better assigned to their charts
(in example: Fig. 3B and 3C, Fig. 4B)
5) Figure 6 caption: please provide the general number of examined cases for both CTRL and
CRN subgroups. Besides of that, it is not clear what the numbers are above the bars in the figure.
If it is the size of the studied groups, it should be presented in a more intuitive way (use n =) or
provide an appropriate explanation in the label.
6) Typos and grammar (selected examples):
a) “Accordingly, we examine eicosanoid-related enzymes and receptors in non-neoplastic
colonic mucosa both from patients with and without CRN.”
b) “Finally, we determine expression levels of 5-, 12-, and 15-LOX enzymes.”
My comment: in these sentences, the past tense should be used
c) “Fig. 6. EP3 and EP4 mRNA levels showed a trend of elevation in CRN patients.”
It would be more accurate to use term “expression” in relation to the mRNA levels
7) Not all abbreviations were explained in the manuscript. For example:
“Following extraction, RNA samples were placed on ice and quantified using a Nanodrop
Spectrophotometer (LabTech International) in accordance with the MIQE guidelines (23).”
<190-192>
Please explain what does the MIQE refer to.
Please review this paper.
user
Title: Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal
appearing colonic mucosa from patients with colorectal neoplasia
Version: 1 Date: 29 Oct 2021
Reviewer:  #ukasz Zadka
Reviewer's report:
In this interesting preliminary study, Feddersen et al. showed abnormalities occurring in cancer-
associated normal colonic mucosa for eicosanoid signaling pathway. The molecular disbalances
ongoing in the morphologically unchanged colonic mucosa during carcinogenesis are a logical
destination for many different biomarkers, nevertheless, comparative analyzes in this area are
very rare, therefore the work has additional value in the field of basic research. However, after
carefully reading this manuscript, I have some concerns. Please find below my comments:
Major:
There are missing some important clinicopathological data (section: “Study population”). The
description of patients should be supported (at least) with histopathological diagnosis, G grade
and TNM stage.
The diversity of patients in terms of chronic diseases and medications taken could have a
potential impact on the obtained results.
The gene transcript does not always coincide with the translation mechanism, which means that
the mRNA expression level may differ from the levels of the corresponding proteins. It would be
beneficial for this work to demonstrate the presence of the studied biomarkers in the tissue at the
protein level, not just mRNA. Supplementary to PCR, semi-quantitative protein analysis could
also be an option. I would recommend performing immunoblotting or immunofluorescence
analyses of the examined colonic biopsies for EP1-4 receptors, PGIS/PTGIS and LOXs plus
reference proteins (b-actin or/and GAPDH for WB).
Minor:
1) "CRC carcinogenesis is a multifactorial process, in which an accumulation of mutations leads
to the formation of colorectal neoplasia (CRN), initially as benign adenomas and subsequently
malignant adenocarcinomas (2)."
This is a misleading sentence that generalizes CRC oncogenesis to suggest that all colon cancers
arise from a polyp basis. What about other histological subtypes of cancers (i.e.: Carcinomas
and mesenchymal cancers like colorectal sarcomas) that also meet the CRC definition?
2) "Several theories in form of “field effects” and “mutator pathways” for primary tumor-
induced changes in near and distant gene expression have been forwarded over the last forty
years (18-20)."
This sentence is difficult to build a clear conclusion. Could you please be more precise and
accurately define the meaning of used terms?
3) Authors should provide the exact address details of the Department where the colonoscopy
procedure was performed
4) The numbers of examined cases presented on figures should be better assigned to their charts
(in example: Fig. 3B and 3C, Fig. 4B)
5) Figure 6 caption: please provide the general number of examined cases for both CTRL and
CRN subgroups. Besides of that, it is not clear what the numbers are above the bars in the figure.
If it is the size of the studied groups, it should be presented in a more intuitive way (use n =) or
provide an appropriate explanation in the label.
6) Typos and grammar (selected examples):
a) “Accordingly, we examine eicosanoid-related enzymes and receptors in non-neoplastic
colonic mucosa both from patients with and without CRN.”
b) “Finally, we determine expression levels of 5-, 12-, and 15-LOX enzymes.”
My comment: in these sentences, the past tense should be used
c) “Fig. 6. EP3 and EP4 mRNA levels showed a trend of elevation in CRN patients.”
It would be more accurate to use term “expression” in relation to the mRNA levels
7) Not all abbreviations were explained in the manuscript. For example:
“Following extraction, RNA samples were placed on ice and quantified using a Nanodrop
Spectrophotometer (LabTech International) in accordance with the MIQE guidelines (23).”
<190-192>
Please explain what does the MIQE refer to.
assistant