Exploring Key Biomarkers and Common Pathogenesis of Seven Digestive System Cancers and Their Correlation with COVID-19
Round 1
Reviewer 1 Report
In this study the authors recognized nine genes (CCNA2, CCNB1, 15 CDKN3, ECT2, KIF14, KIF20A, KIF4A, NEK2, TTK) that are common in gastrointestinal cancer and COVID-19 infection. The authors afterthat evaluated the finding using RT-qPCR.
Major points:
1) Did the authors conclude that these nine genes are invariant gene signatures with GIT cancers and COVID-19?
2) What the rationale of selecting these GEO in table 1? The authors selected one GEO/Cancer. The authors must analyze several GEO from same cancer to be sure that these genes are invariant gene signatures or not.
3) RT-qPCR data is not convincing. WHy the authors selected two genes out of 9 genes? then why the authors selected CRC from all GIT cancers? then the analysis and figure presentation (figure 10) should re rephrased, the expression of normal and CRC from each patient should be represented individually as as a dot.
4) What is the severity of COVID-19 in this study and analyzed samples?
5) Patients' criteria in Figure 10 should be included
Moderate language editing
Author Response
1) Did the authors conclude that these nine genes are invariant gene signatures with GIT cancers and COVID-19?
Thank you very much for your hard work and Suggestions. Based on the analysis of bioinformatics, we screened the gene sequencing results of 7 digestive system cancers and COVID-19, and obtained the potential hub genes with consistent expression trends. We also used the widely used "String" online tool and CytoScape software to analyze the interactions between these genes and the importance of their respective genes in the regulatory network, and obtained these 9 hub genes. Then we conducted validation on multiple datasets and in vitro validation in colorectal cancer tissue, all of which obtained consistent validation results. By combining the above analysis and previous research results, we preliminarily identified these 9 genes as potential variant gene signatures with GIT cancer and COVID-19. Our analysis provides a new direction for future research on the common molecular mechanisms of GIT cancer and COVID-19. Further research is needed to determine the common mechanism of these genes in GIT cancer and COVID-19 in the future.
2) What the rationale of selecting these GEO in table 1? The authors selected one GEO/Cancer. The authors must analyze several GEO from same cancer to be sure that these genes are invariant gene signatures or not.
In order to improve the credibility and reference value of our analysis, we searched the available GIT cancer and COVID-19 gene expression datasets in the GEO database and selected the dataset containing as many normal and disease samples as possible for analysis. Therefore, we chose these GEO in table 1. In addition, for each type of cancer, we also selected multiple datasets from GEO and TCGA for at least two validations to ensure the reliability of our analysis results. Some GEO datasets contain two types of cancers, as there are fewer available datasets that meet the requirements for these cancers. However, these cancers are located at the junction of the digestive system, and selecting these datasets also helps to reflect the overall integrity of the digestive system.
3) RT-qPCR data is not convincing. WHy the authors selected two genes out of 9 genes? then why the authors selected CRC from all GIT cancers? then the analysis and figure presentation (figure 10) should re rephrased, the expression of normal and CRC from each patient should be represented individually as as a dot.
In the CytoHubba plugin for searching for hub genes in Cytoscape, multiple algorithms were included, including MCC、DMNC、MNC、Degree、EPC、BottleNeck、EcCentricity、Closeness、Radiality、Betweenness、Stress, and ClusteringCoefficient. We selected the genes with the highest frequency and score of hub genes in the above algorithms. Then CCNA2 and CCNB1 was choosed for validation.
Our team members are doctors in colorectal and anal surgery department. Considering the availability of samples, only CRC was selected for analysis in this study. In future research, we will make greater efforts to obtain more samples of other gastrointestinal cancers and further validate these genes.
We have made modifications to the annotations in Figure 10(lines 332 and 333). The modified expression is “qPCR expression level of CCNA2, CCNB1, and MYC in 25 pairs of colorectal tissues. Each dot represented the expression of normal or CRC sample”.
4) What is the severity of COVID-19 in this study and analyzed samples?
In our analysis, we selected two COVID-19 datasets, with GSE171110 including the normal control group and the severe COVID-19 group, and GSE152418 including the normal control group and the mild/severe COVID-19 group.
5) Patients' criteria in Figure 10 should be included
Patients' criteria in Figure 10 was included in lines158-162
Inclusion criteria: 1. Age older than 18 years old; 2. Patients diagnosed with colorectal cancer for the first time and undergoing surgical resection; 3. The postoperative pathological specimen was diagnosed as colorectal adenocarcinoma by two pathologists. Exclusion criteria; 1. Patients who refused to participate in this study; 2. Patients with postoperative pathological diagnosis of non adenocarcinoma.
Reviewer 2 Report
Exploring key biomarkers and common pathogenesis of seven digestive system cancers and their correlation with COVID-19, looks at the possible molecular level connection among digestive tract cancers along with COVID-19. There is a fine line between madness and genius, and I am not sure which side of the line this paper represents.
General comments-
My first question is, if you leave Covid-19 out of the analysis, how many of the markers and hubs are altered? What is the actual contribution of Covid-19 to the results?
There are 3 possible ramifications from the study. 1. You have just identified or re-identified some very useful molecular markers for the digestive cancers and implemented a blood test for some using a very round-about methodology. Some of this seems to be documented from other studies. 2. Covid-19, in addition to aggravating digestive tract problems might contribute to cancer risk. Is this a possible ramification? I also wonder whether you thought about tying the Covid-19 DEGs to IBD? If Covid-19 presents any down-stream risk for cancer, this would take years to play out. IBD and other inflammatory problems of the digestive track would show up sooner, even manifesting in the current time. 3. You have identified markers for Covid-19 that might help predict complications for the patients, short term, and long term, including aggravation separate from idiopathic IBD and other inflammatory conditions of the digestive tract and cancer and including IBD and cancer.
Specific comments-
Table 3. How good can the AUC analysis be when the number of samples is so low as in the bile duct set and ESC set?
Some of my questions might be answered if the Introduction was more elaborate. And the conclusion could be expanded a bit to include the whole philosophy behind the study and mor about possible ramifications
English is fine.
Author Response
My first question is, if you leave Covid-19 out of the analysis, how many of the markers and hubs are altered? What is the actual contribution of Covid-19 to the results?
Response: First of all, thank you for your Comments and valuable suggestions. Excluding the sample of COVID-19, there are 39 genes in the intersection of seven types of digestive system cancers, of which 33 are up-regulated and 6 are down regulated, with 16 genes increased compared with the original, including THY1, SULF1, CST1, COL5A2, MAD2L1, SPP1, OLFML2B, SPOCK1, CKS1B, DNMT1, CKS2, ALDH6A1, ADH1B, CITED2, DPT and TMPRSS2. These analysis results demonstrate that there is indeed potential commonality in the molecular mechanism of interaction between Covid-19 and digestive system cancers.
There are 3 possible ramifications from the study. 1. You have just identified or re-identified some very useful molecular markers for the digestive cancers and implemented a blood test for some using a very round-about methodology. Some of this seems to be documented from other studies. 2. Covid-19, in addition to aggravating digestive tract problems might contribute to cancer risk. Is this a possible ramification? I also wonder whether you thought about tying the Covid-19 DEGs to IBD? If Covid-19 presents any down-stream risk for cancer, this would take years to play out. IBD and other inflammatory problems of the digestive track would show up sooner, even manifesting in the current time. 3. You have identified markers for Covid-19 that might help predict complications for the patients, short term, and long term, including aggravation separate from idiopathic IBD and other inflammatory conditions of the digestive tract and cancer and including IBD and cancer.
Response: Our analysis results show that there may indeed be a potential common molecular mechanism between Covid-19 and digestive system cancer. Considering the affinity of Covid-19 to the digestive system, this indicates that the imbalance of gene expression caused by Covid-19 infection may have a certain impact on the incidence rate of digestive system cancer, which needs our further research to determine.
Response: After discovering the effect of Covid-19 on the digestive system, we have also considered the impact of Covid-19 on IBD. Currently, relevant studies have found a close relationship between Covid-19 and IBD. Our research topic is the relationship between gastrointestinal cancer and Covid-19, so we did not include any studies related to IBD in this analysis. In future research, we will attempt to further explore the relationship between Covid-19, IBD, and digestive system cancer.
Specific comments-
Table 3. How good can the AUC analysis be when the number of samples is so low as in the bile duct set and ESC set?
Some of my questions might be answered if the Introduction was more elaborate. And the conclusion could be expanded a bit to include the whole philosophy behind the study and mor about possible ramifications
Response: In some GEO datasets of bile duct and esophageal cancer, there are indeed fewer normal samples. Due to the special reasons of bile duct and esophageal cancer surgery, it is difficult to obtain normal normal. When there are fewer normal samples, it will have a certain impact on the accuracy of AUC. We included TCGA and multiple GEO for analysis, hoping to minimize the impact of errors as much as possible.
Round 2
Reviewer 1 Report
No further comments
Moderate language editing
Author Response
Thank you for your advice and guidance. Your review has greatly helped us improve our manuscript.
Reviewer 2 Report
Include the text you addressed to me-“ Due to the special reasons of bile duct and esophageal cancer surgery, it is difficult to obtain normal. When there are fewer normal samples, it will have a certain impact on the accuracy of AUC. We included TCGA and multiple GEO for analysis, hoping to minimize the impact of errors as much as possible.” In the paper at the appropriate spot.
And in the discussion please add your comment-“Our analysis results show that there may indeed be a potential common molecular mechanism between Covid-19 and digestive system cancer. Considering the affinity of Covid-19 to the digestive system, this indicates that the imbalance of gene expression caused by Covid-19 infection may have a certain impact on the incidence rate of digestive system cancer, which needs our further research to determine”.
OK
Author Response
Thank you for your suggestions. We have added the corresponding content of the discussion section according to the modification suggestions（lines 339-344， 352-355）. These contents have been highlighted.
