Title: PLAGL1 is associated with prognosis and cell proliferation in pancreatic adenocarcinoma
Version: 0 Date: 16 Sep 2022
Reviewer's report:
The manuscript “Downregulated PLAGL1 is associated with tumor progression and predicts
poor prognosis in pancreatic adenocarcinoma” investigates the expression level of PLAG family
members in tumor tissue of patients with a pancreatic ductal adenocarcinoma. The data were
correlated to clinical parameters and gene association studies were performed. The presentation
of the data in the figures is very well done. PLAGL1 was identified as a new prognostic marker
for PAAD.
There are some point that need to be addressed before publication:
Major points in Material and Methods:
1.) The overall survival time includes also the cases lost to follow-up. Please include the
numbers, how many patients were lost to follow up to estimate the bias on the overall survival
time (see https://doi.org/10.1155/2022/9068214). In the supplementary table 1, change the
follow-up time from years to month and include DFS times.
2.) The description of wet lab techniques is not scientifically accurate. Please describe how you
isolated the RNA out of the Trizol reagent. Change to “cDNA was reverse transcribed based on
1 µg RNA using the PrimerScript RT Reagent Kit”. The SYBRgreen mix was used for qPCR
and not “in the PCR program”. Relative expression levels of target genes are calculated based on
relative expression to the housekeeper gene (#Ct). Whereas normalized gene expression levels
are normalized to e.g. control samples and displayed as fold change compared to controls (##Ct).
Please specify how you analyzed your data.
3.) Were the TMAs not blocked before staining? Please mention the staining reaction that was
used? I am sure, the authors used a microscope with a camera to take pictures, please correct this.
4.) The dataset GSE28735 contained “Microarray gene-expression profiles of 45 matching pairs
of pancreatic tumor and adjacent non-tumor tissues from 45 patients with pancreatic ductal
adenocarcinoma”. Was this cohort used in addition to the TCGA data set? Which data set was
used for the GSEA analysis? Please specify.
Major points in Results and Discussion:
5.) In Figure 1, the mean values of the relative gene expression levels of PLAG1 and PLAGL1
in the GSE28735 cohort are basically the same. Are the authors sure that the data are statistically
significant? Please specify if linear or log2 values were used to plot relative gene expression.
6.) Please mention that you performed the GSEA analysis based on high and low PLAGL1
expression in the TCGA cohort. Please do so also in the discussion.
7.) Please change: “We found that the PLAGL1 level was clearly negatively associated with
the OS of TCGA-PAAD cohort patients” to “We found that a reduced PLAGL1 level was
associated with a worse OS of TCGA-PAAD cohort patients”. In the discussion, please change
also “expression of PLAGL1 were both inversely correlated with the OS of PAAD patients”
accordingly.
8.) Please change the word relationship to association in “while the relationship between
PLAGL1 and disease-free survival (DFS) was not significant (Fig. 4a-4b). The analysis
performed in our PAAD verification cohort 2 and 3 confirmed this relationship as well (Fig.
4c-4f).”
9.) Please restructure the sentence “PLAGL1 can bind imprinted genes and genes regulating
extracellular matrix composition with GC-rich consensus and induce physiological cell cycle
exit with contact inhibition, functioning similar biological role as p53 in different contexts.”;
the context is hard to understand.
10.)  The sentence in the discussion: “In addition, GSEA of cancer gene neighborhoods also
suggested that multiple PLAGL1 nearby genes could regulate cell proliferation” is misleading.
If I remember correctly, the GSEA neighborhood analysis find gene set correlations with a gene
of interest (gene neighbors), it has nothing to do with nearby genes on genome location. Please
correct and specify.
11.) Please specify “Its mortality rate is still approximately 100%, and the five year survival rate
is still less than 10%”; if the survival rate is 10%, the mortality rate cannot be 100%.
Minor points:
12.) What do the authors mean by “anti-oncogene”? A tumor suppressor gene? The term is quite
outdated, please change.
13.) Please change “tumor suppressive gene” into “tumor suppressor gene”.
14.) In Material and Methods, please change “OS” in “overall survival (OS)”, since it is the first
time mentioning the abbreviation.
15.) In the results, please change „paracancerous“ to „tumor-adjacent“.
16.) In the results, please change “ the PLAGL1 expression level was negatively concerned with
T stage ” to “ the PLAGL1 expression level was negatively correlated with T stage”.
17.)  In the results, please change “ in our other two PAAD verification cohorts (Cohort 2
combined 3)” to “ in our other two combined PAAD verification cohorts 2 and 3”. Change it
also in all figures and figure legends.
Title: PLAGL1 is associated with prognosis and cell proliferation in pancreatic adenocarcinoma
Version: 0 Date: 16 Sep 2022
Reviewer's report:
The manuscript “Downregulated PLAGL1 is associated with tumor progression and predicts
poor prognosis in pancreatic adenocarcinoma” investigates the expression level of PLAG family
members in tumor tissue of patients with a pancreatic ductal adenocarcinoma. The data were
correlated to clinical parameters and gene association studies were performed. The presentation
of the data in the figures is very well done. PLAGL1 was identified as a new prognostic marker
for PAAD.
There are some point that need to be addressed before publication:
Major points in Material and Methods:
1.) The overall survival time includes also the cases lost to follow-up. Please include the
numbers, how many patients were lost to follow up to estimate the bias on the overall survival
time (see https://doi.org/10.1155/2022/9068214). In the supplementary table 1, change the
follow-up time from years to month and include DFS times.
2.) The description of wet lab techniques is not scientifically accurate. Please describe how you
isolated the RNA out of the Trizol reagent. Change to “cDNA was reverse transcribed based on
1 µg RNA using the PrimerScript RT Reagent Kit”. The SYBRgreen mix was used for qPCR
and not “in the PCR program”. Relative expression levels of target genes are calculated based on
relative expression to the housekeeper gene (#Ct). Whereas normalized gene expression levels
are normalized to e.g. control samples and displayed as fold change compared to controls (##Ct).
Please specify how you analyzed your data.
3.) Were the TMAs not blocked before staining? Please mention the staining reaction that was
used? I am sure, the authors used a microscope with a camera to take pictures, please correct this.
4.) The dataset GSE28735 contained “Microarray gene-expression profiles of 45 matching pairs
of pancreatic tumor and adjacent non-tumor tissues from 45 patients with pancreatic ductal
adenocarcinoma”. Was this cohort used in addition to the TCGA data set? Which data set was
used for the GSEA analysis? Please specify.
Major points in Results and Discussion:
5.) In Figure 1, the mean values of the relative gene expression levels of PLAG1 and PLAGL1
in the GSE28735 cohort are basically the same. Are the authors sure that the data are statistically
significant? Please specify if linear or log2 values were used to plot relative gene expression.
6.) Please mention that you performed the GSEA analysis based on high and low PLAGL1
expression in the TCGA cohort. Please do so also in the discussion.
7.) Please change: “We found that the PLAGL1 level was clearly negatively associated with
the OS of TCGA-PAAD cohort patients” to “We found that a reduced PLAGL1 level was
associated with a worse OS of TCGA-PAAD cohort patients”. In the discussion, please change
also “expression of PLAGL1 were both inversely correlated with the OS of PAAD patients”
accordingly.
8.) Please change the word relationship to association in “while the relationship between
PLAGL1 and disease-free survival (DFS) was not significant (Fig. 4a-4b). The analysis
performed in our PAAD verification cohort 2 and 3 confirmed this relationship as well (Fig.
4c-4f).”
9.) Please restructure the sentence “PLAGL1 can bind imprinted genes and genes regulating
extracellular matrix composition with GC-rich consensus and induce physiological cell cycle
exit with contact inhibition, functioning similar biological role as p53 in different contexts.”;
the context is hard to understand.
10.)  The sentence in the discussion: “In addition, GSEA of cancer gene neighborhoods also
suggested that multiple PLAGL1 nearby genes could regulate cell proliferation” is misleading.
If I remember correctly, the GSEA neighborhood analysis find gene set correlations with a gene
of interest (gene neighbors), it has nothing to do with nearby genes on genome location. Please
correct and specify.
11.) Please specify “Its mortality rate is still approximately 100%, and the five year survival rate
is still less than 10%”; if the survival rate is 10%, the mortality rate cannot be 100%.
Minor points:
12.) What do the authors mean by “anti-oncogene”? A tumor suppressor gene? The term is quite
outdated, please change.
13.) Please change “tumor suppressive gene” into “tumor suppressor gene”.
14.) In Material and Methods, please change “OS” in “overall survival (OS)”, since it is the first
time mentioning the abbreviation.
15.) In the results, please change „paracancerous“ to „tumor-adjacent“.
16.) In the results, please change “ the PLAGL1 expression level was negatively concerned with
T stage ” to “ the PLAGL1 expression level was negatively correlated with T stage”.
17.)  In the results, please change “ in our other two PAAD verification cohorts (Cohort 2
combined 3)” to “ in our other two combined PAAD verification cohorts 2 and 3”. Change it
also in all figures and figure legends.
Please review this paper.
user
Title: PLAGL1 is associated with prognosis and cell proliferation in pancreatic adenocarcinoma
Version: 0 Date: 16 Sep 2022
Reviewer's report:
The manuscript “Downregulated PLAGL1 is associated with tumor progression and predicts
poor prognosis in pancreatic adenocarcinoma” investigates the expression level of PLAG family
members in tumor tissue of patients with a pancreatic ductal adenocarcinoma. The data were
correlated to clinical parameters and gene association studies were performed. The presentation
of the data in the figures is very well done. PLAGL1 was identified as a new prognostic marker
for PAAD.
There are some point that need to be addressed before publication:
Major points in Material and Methods:
1.) The overall survival time includes also the cases lost to follow-up. Please include the
numbers, how many patients were lost to follow up to estimate the bias on the overall survival
time (see https://doi.org/10.1155/2022/9068214). In the supplementary table 1, change the
follow-up time from years to month and include DFS times.
2.) The description of wet lab techniques is not scientifically accurate. Please describe how you
isolated the RNA out of the Trizol reagent. Change to “cDNA was reverse transcribed based on
1 µg RNA using the PrimerScript RT Reagent Kit”. The SYBRgreen mix was used for qPCR
and not “in the PCR program”. Relative expression levels of target genes are calculated based on
relative expression to the housekeeper gene (#Ct). Whereas normalized gene expression levels
are normalized to e.g. control samples and displayed as fold change compared to controls (##Ct).
Please specify how you analyzed your data.
3.) Were the TMAs not blocked before staining? Please mention the staining reaction that was
used? I am sure, the authors used a microscope with a camera to take pictures, please correct this.
4.) The dataset GSE28735 contained “Microarray gene-expression profiles of 45 matching pairs
of pancreatic tumor and adjacent non-tumor tissues from 45 patients with pancreatic ductal
adenocarcinoma”. Was this cohort used in addition to the TCGA data set? Which data set was
used for the GSEA analysis? Please specify.
Major points in Results and Discussion:
5.) In Figure 1, the mean values of the relative gene expression levels of PLAG1 and PLAGL1
in the GSE28735 cohort are basically the same. Are the authors sure that the data are statistically
significant? Please specify if linear or log2 values were used to plot relative gene expression.
6.) Please mention that you performed the GSEA analysis based on high and low PLAGL1
expression in the TCGA cohort. Please do so also in the discussion.
7.) Please change: “We found that the PLAGL1 level was clearly negatively associated with
the OS of TCGA-PAAD cohort patients” to “We found that a reduced PLAGL1 level was
associated with a worse OS of TCGA-PAAD cohort patients”. In the discussion, please change
also “expression of PLAGL1 were both inversely correlated with the OS of PAAD patients”
accordingly.
8.) Please change the word relationship to association in “while the relationship between
PLAGL1 and disease-free survival (DFS) was not significant (Fig. 4a-4b). The analysis
performed in our PAAD verification cohort 2 and 3 confirmed this relationship as well (Fig.
4c-4f).”
9.) Please restructure the sentence “PLAGL1 can bind imprinted genes and genes regulating
extracellular matrix composition with GC-rich consensus and induce physiological cell cycle
exit with contact inhibition, functioning similar biological role as p53 in different contexts.”;
the context is hard to understand.
10.)  The sentence in the discussion: “In addition, GSEA of cancer gene neighborhoods also
suggested that multiple PLAGL1 nearby genes could regulate cell proliferation” is misleading.
If I remember correctly, the GSEA neighborhood analysis find gene set correlations with a gene
of interest (gene neighbors), it has nothing to do with nearby genes on genome location. Please
correct and specify.
11.) Please specify “Its mortality rate is still approximately 100%, and the five year survival rate
is still less than 10%”; if the survival rate is 10%, the mortality rate cannot be 100%.
Minor points:
12.) What do the authors mean by “anti-oncogene”? A tumor suppressor gene? The term is quite
outdated, please change.
13.) Please change “tumor suppressive gene” into “tumor suppressor gene”.
14.) In Material and Methods, please change “OS” in “overall survival (OS)”, since it is the first
time mentioning the abbreviation.
15.) In the results, please change „paracancerous“ to „tumor-adjacent“.
16.) In the results, please change “ the PLAGL1 expression level was negatively concerned with
T stage ” to “ the PLAGL1 expression level was negatively correlated with T stage”.
17.)  In the results, please change “ in our other two PAAD verification cohorts (Cohort 2
combined 3)” to “ in our other two combined PAAD verification cohorts 2 and 3”. Change it
also in all figures and figure legends.
assistant