SOX2 and Bcl-2 as a Novel Prognostic Value in Hepatocellular Carcinoma Progression

Round 1

Reviewer 1 Report

Overall, the manuscript was well written with great details.

I have following minor comments.

 

1. Sorafenib is one of tyrosine-kinase inhibitor, different from conventional chemotherapy such as doxorubisine or cisplatin. Thus this reviewer suggest to change "chemotherapy" into "sorafenib" throughout the text.

2.   In FIgure 4a, the authors mentioned that cut-off value for low and high SOX2 was mean expression SOX2 level. So, what is mean value? 

 

3. In figure 4a, what is the median overall survival of the two groups (low and high Sox-2)?

Author Response

Response to Reviewer-1 Comments

Dear Editor of Journal of Current Oncology,

 Thank you very much for the review of our manuscript entitled: SOX2 and Bcl-2 as a novel prognostic value in hepatocellular carcinoma progression.

We thank the reviewers for their valuable comments which helped to considerably improve the quality of the manuscript. We addressed all the comments of reviewer-1. Also, the whole manuscript was checked and proofed by a native English speaker, familiar with scientific writing. The detailed answers follow.

Comments and Suggestions for Authors

Overall, the manuscript was well written with great details.

I have following minor comments.

1. Sorafenib is one of tyrosine-kinase inhibitor, different from conventional chemotherapy such as doxorubisine or cisplatin. Thus this reviewer suggest to change "chemotherapy" into "sorafenib" throughout the text.

Response 1: We appreciate the reviewer for pointing this out. According to the reviewer's comment, all the "chemotherapy" in the manuscript which referred to sorafenib, was changed to "sorafenib", (Lines 99, 104, 106, 251, 256) and highlighted using the "Track Changes".

 

2. In FIgure 4a, the authors mentioned that cut-off value for low and high SOX2 was mean expression SOX2 level. So, what is mean value?

Response 2: We would like to thank the reviewer for his/her careful review of our work.

To perform survival analysis, first, the mean expression of SOX2 was calculated in 32 patients (sorafenib-received), as a cut-off point (2.431). Then, patients were divided into two groups: high expression and low expression of SOX2.  Patients with SOX2 expression below this cut-off point were classified as Low SOX2 and those with SOX2 expression above this point were classified as High SOX2 (CYP19A1 gene expression in the peripheral blood of Brazilian women with breast cancer relapse,https://doi.org/10.1186/s12885-020-06978-z).

 

We edited this part to become more fluent (line 252-253) and highlighted using the "Track Changes" as: " The mean expression level of SOX2 in 32 patients (sorafenib-received) was considered as a cut-off point (2.431) ".

 

3. In figure 4a, what is the median overall survival of the two groups (low and high Sox-2)?

Response 3: The median overall survival for low SOX2 was 10.5 months and for high SOX2 was 6.5 months and added to figure-4 legend in the manuscript (line 270) using the "Track Changes".

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

In this manuscript, authors have meticulously designed the study and performed the required experiments. Overall, it is a good effort but needs some improvements.

In the title, authors claim SOX2 and Bcl-2 as novel prognostic markers for hepatocellular carcinoma. It seems to have two problems.

First, in the abstract they have reported a significant correlation between patients with SOX2 over expression and survival rate (but not  for Bcl-2). They have summarized that SOX2 overexpression along with Bcl-2 ‘may' be a potential marker to evaluate the prognosis and response to HCC treatment. Thus, it further diminishes the role of BCL2 as independent novel prognostic marker.

Second, SOX2 and BCL-2 have been long known as prognostic markers for HCC, this the findings are not novel.

Sun, C., Sun, L., Li, Y. et al. Sox2 expression predicts poor survival of hepatocellular carcinoma patients and it promotes liver cancer cell invasion by activating Slug. Med Oncol 30, 503 (2013).

Garcia EJ, Lawson D, Cotsonis G, Cohen C. Hepatocellular carcinoma and markers of apoptosis (bcl-2, bax, bcl-x): prognostic significance. Appl Immunohistochem Mol Morphol. 2002 Sep;10(3):210-7

Statistically, the samples size seems low for survival analysis. It would be better to check and report the power analysis based on the current samples.

Authors have reported, "Quantitative variables were assessed using independent t-test or one-way analysis of variance (ANOVA) if they had a normal distribution. Mann-Whitney U test and Kruskal-Wallis test were used when quantitative data were not normally distributed”. This sounds a generalized statement.

What are the specific comparison where t-test or MWU or KW test were employed? It would be great if authors could provide corresponding distributions plots as supplementary data.

Author Response

Response to Reviewer-2 Comments

Dear Editor of Journal of Current Oncology,

We sincerely appreciate all valuable comments and suggestions, which helped us to improve the quality of the manuscript. Our responses to the Reviewer-2 comments are described below in a point-to-point manner. Appropriated changes, suggested by the Reviewer, have been introduced to the manuscript (highlighted using the "Track Changes" within the text).

Comments and Suggestions for Authors

In this manuscript, authors have meticulously designed the study and performed the required experiments. Overall, it is a good effort but needs some improvements.

1. In the title, authors claim SOX2 and Bcl-2 as novel prognostic markers for hepatocellular carcinoma. It seems to have two problems.

First, in the abstract they have reported a significant correlation between patients with SOX2 over expression and survival rate (but not for Bcl-2). They have summarized that SOX2 overexpression along with Bcl-2 ‘may' be a potential marker to evaluate the prognosis and response to HCC treatment. Thus, it further diminishes the role of BCL2 as independent novel prognostic marker.

Response 1: We thank the reviewer for pointing out this issue and agree with the comment.  The results showed that Bcl-2 expression was higher in patients than in controls (4.16 ± 3.44 vs. 1.27 ± 0.98, p=0.003(. In line with the increased expression of SOX2, high expression levels of Bcl-2 were also associate with tumor grade (p=0.003) and the disease stage (p=0.04). Furthermore, a significant positive correlation was shown between Bcl-2 expression and both ALT (P=0.02) and ALP (p=0.01). Therefore, Bcl-2 may be a potential marker for HCC prognosis.

We revised the abstract (lines 44-45) as follows and highlighted it in the text using the "Track Changes".

 

"Collectively, overexpression of SOX2 and Bcl-2, alone or combined, may be a potential marker to evaluate the prognosis and response to HCC treatment."

 

Second, SOX2 and BCL-2 have been long known as prognostic markers for HCC, this the findings are not novel.

Sun, C., Sun, L., Li, Y. et al. Sox2 expression predicts poor survival of hepatocellular carcinoma patients and it promotes liver cancer cell invasion by activating Slug. Med Oncol 30, 503 (2013).

Garcia EJ, Lawson D, Cotsonis G, Cohen C. Hepatocellular carcinoma and markers of apoptosis (bcl-2, bax, bcl-x): prognostic significance. Appl Immunohistochem Mol Morphol. 2002 Sep;10 (3):210-7

We appreciate the reviewer for this viewpoint and agree that mentioned studies suggesting SOX2 as prognostic markers for HCC. However, our study differs from these studies, which are mentioned below.

The study "Sox2 expression predicts poor survival of hepatocellular carcinoma patients and it promotes liver cancer cell invasion by activating Slug. Sun C et al, Med Oncol 2013, 30: 503.China", showed a correlation between the high levels of SOX2 expression and metastasis and low survival rate of HCC; however, they measured SOX2 expression in cell lines, and 14 pairs of HCC and adjacent tissues using western blot. Furthermore, relationship between overall survival and SOX2 expression in HCC patients was evaluated with immunohistochemistry. While in our study, the expression of SOX2 and apoptotic genes, including Bax and Bcl-2, was evaluated using qRT-PCR in mRNA level in 53 formalin-fixed, paraffin-embedded tissues (FFPET) of HCC patients and 44 control individuals. Moreover, the possible correlations of mRNA SOX2 expression along with Bax and Bcl2 expression and with clinicopathological features, tumor grade, and disease stage of HCC patients were evaluated for the first time. Finally, the relationship between SOX2 mRNA expression and survival rate was determined by Kaplan-Meier survival curves. Therefore, our study has innovation from these points.

In another study, "Hepatocellular carcinoma and markers of apoptosis (bcl-2, bax, bcl-x): prognostic significance. Garcia EJ et al, Appl Immunohistochem Mol Morphol. 2002 ", aimed to determine the frequency of expression of apoptotic markers in HCC and their relationship with prognosis. They evaluated only apoptotic marker expression using Immunohistochemistry. While the purpose of our study was assessment of SOX2 and apoptotic genes (Bax and Bcl2) mRNA expression alone and together and we found a positive correlation between the expression of SOX2 and Bcl-2 in HCC patients and also, higher expression levels of Bcl-2 were associated with tumor grade and the disease stage in line with changes in SOX2 expression.

2. Statistically, the samples size seems low for survival analysis. It would be better to check and report the power analysis based on the current samples.

We appreciate the reviewer’s comment and we agree that increasing the sample size would improve the statistical power of the study. Whereas the incidence of HCC is not very high in Iran (Incidence of Hepatocellular Carcinoma in Southeast Iran, Sodaif Darvish Moghaddam et al. Hepat Mon. 2010; 10(4): 270-274) and also because of restricted entry and exit criteria in our study which only the HCC patients who had been undergoing sorafenib treatment were enrolled for survival analysis, the size of our sample was limited. However, we calculated the power of analysis for 32 sample sizes using G^*Power software, and as the diagram below shows it is 0.8848983.

We revised the manuscript (lines 264-265) as follows and highlighted it in the text using the "Track Changes".

" We calculated the power of analysis using G^*Power software, and it was 0.8848983".

3. Authors have reported, "Quantitative variables were assessed using independent t-test or one-way analysis of variance (ANOVA) if they had a normal distribution. Mann-Whitney U test and Kruskal-Wallis test were used when quantitative data were not normally distributed”. This sounds a generalized statement. What are the specific comparison where t-test or MWU or KW test were employed? It would be great if authors could provide corresponding distributions plots as supplementary data.

We would like to thank the reviewer for his/her careful review of our work, and we agree that the statement is general. We used this sentence in the method part before analyzing the data. After analyzing, all data had normal distribution and we used parametric tests (t-test and ANOVA). According to the reviewer's suggestions, changes were done (line 156) and highlighted using the "Track Changes" as follows.

" All data had normal distribution. Quantitative variables were assessed using independent t-test or one-way analysis of variance (ANOVA) ".

 

Author Response File: Author Response.docx