Review for 10.1186/s12876-022-02257-2 (2022)

Title: Identification and validation of EMT-immune-related prognostic biomarkers
CDKN2A, CMTM8 and ILK in colon cancer
Version: 1 Date: 10 Mar 2022
Reviewer's report:
This study aims to explore prognostic biomarkers associated with EMT in correlation with
immune infiltration. This is an in silico analysis of GSE10950 data and TCGA & GTEx
data. Authors identified 3 overlapping EMT-immune mediated genes among the datasets
that were differentially expressed between cancer and normal tissue that affected the disease
prognosis, namely CDKN2A, CMTM8 and ILK. They went on to correlate CDKN2A, CMTM8,
ILK with immune cell infiltration (based on CIBERSORT algorithms) and expression of
immune checkpoint genes. These results, though not definitive, could be considered hypothesis
generating. Addressing the following points could strengthen the manuscript.
- Authors found “the prognostic ROC curves of the three genes were established, the results
showed that the AUC of CDKN2A was 0.68, 0.66, 0.58 for 1, 3, and 5 years respectively, the
AUC of CMTM8 was 0.59, 0.51, 0.57 and the AUC of ILK was 0.71, 0.58, 0.60 (Figure 2C).” In
general, an AUC of 0.5-0.6 suggests a poor discriminative value, meaning these biomarkers had
weak ability to predict survival of CRCs . Usually AUC of >0.7 is more acceptable of introducing
a discriminative biomarker.
-Authors need to discuss the results of their single-cell RNA data, and what those mean. Did
this add anything to their analysis? And if so how?
-ILK is found overexpressed in many cancers including colorectal cancer (CRC) (reviewed
in doi.org/10.3389/fgene.2021.638558) .How do authors explain their findings of ILK “low
expression” in tumors (both gene expression and protein levels) in contrast to the large data
showing otherwise?
- Stain tumors should be characterized and better  explained; were these tumor tissues derived
from cancer patients without prior treatment, vs. metastasized tumors, etc. As the focus of study
was EMT, it would been more informative to stain the same tumor slides with EMT markers
such Vimentin or SNAL etc.
-The correlation results are complex and hard to interpret. For example authors  report that
“Overall, CDKN2A, CMTM8, ILK could regulate the immune infiltration and may result in
immunosuppression to a certain degree.” This is based on their correlation study between
expression of these biomarkers with immune subtypes abundances which actually gave mixed
results. For example, both CDKN2A and ILK  were positively correlated with the infiltration of
macrophages, while they are supposed to have an opposite direction based on their earlier results.
This can be explained if one is associated with M1 and another with M2 subtype. These aren’t
discussed. Similarly correlation of CDKN2A, CMTM8, ILK  with the expression of immune
checkpoints are confusing; again both CDKN2A and ILK  have remarkable overlaps in their
correlation patterns with immune checkpoints. Therefore the conclusion of “We have found
that CDKN2A, CMTM8, ILK may influence the prognosis of COAD patients by leading to
immunosuppression.” is not well supported by their findings.
-It is hard to follow the logics of analyses at times. For example, authors introduced analysis
of competing endogenous RNAs (ceRNAs) networks in the results section. The should have
defined ceRNA, lncRNAs  etc and why they were interested in running this analysis.
Minor:
-Some English editing is needed. Examples”
“To sum up, the expression patterns of the three genes were verified by different omics and they
were conform to the survival results”
“So, we can defined CDKN2A, CMTM8, and ILK as hub genes to conduct the following
analyses”
“Exploration the regulatory mechanism of CDKN2A, CMTM8 and ILK”
Some typos are noted:
“By the resluts above, we know that CDKN2A and CMTM8 were higher expressed in cancer
tissues and the prognosis”
“As a reslut, the expression patterns can be verified by single-cell data.”
“CDKN2A, CMTM8, ILK could affect the immuune microenvironment and EMT progress by
a series of signal pathways.”
“The three hub genes may affect prognosis of COAD patients by the above mechenisms.  “
“  In order to better predicting the porgnosis of COAD patients”
“Genes involved in both biological processes may significant”
“CDKN2A, CMTM8, ILK may affect the prognosis of colon caner patients by regulating the
immune infiltration and EMT process. c should be added to cancer.”
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Housekeeping:
Citations:
“With the same criteria, DEGs between normal (GTEx) and CC (TCGA-COAD) were calculated
by GEPIA 2.0 online tools (http://gepia2.cancer-pku.cn/index.html). Next, we defined immune-
related genes by InnateDB (https://www.innatedb.com/) and Immport (https://immport.org/
shared/home) databases. EMT-related genes were defined by EMTome (www.emtome.org).”
Here, in addition to adding there website, please cite each of them properly. For example, for
import, you should cite as below, for others you can find them in their website:
Bhattacharya S, Dunn P, Thomas CG, Smith B, Schaefer H, Chen J, Hu Z, Zalocusky KA,
Shankar RD, Shen-Orr SS, Thomson E, Wiser J, Butte AJ. ImmPort, toward repurposing
of open access immunological assay data for translational and clinical research. Scientific
data. 2018;5:180015. doi: 10.1038/sdata.2018.15. PubMed PMID: 29485622; PubMed Central
PMCID: PMC5827693.