Regulation of Immunity in Clear Cell Renal Carcinoma: Role of PD-1, PD-L1, and PD-L2

Round 1

Reviewer 1 Report

In the manuscript titled Regulation of immunity in clear cell renal carcinoma: role of 4
PD-1, PD-L1, and PD-L2, Spirina et al study the expression pattern of the receptor PD-1 and its ligands PD-L1 and PD-L2 in clear cell RCC patient samples. The aim of the study is to find association between these, and VHL and other growth factors and the extent of metastasis. 

The authors find both PD1 and PD-L2 are reduced during tumor growth. However, during metastasis, PD1 increases while PD-L2 increases. The authors then report an association between PD-1 and many other gene expressions but not VHL. However, VHL total and p-VHL levels change with tumor progression.

In this article, the authors show increase in PD-L2 during metastasis. This, along with changes in VHL total and p-VHL are the novel findings of this paper.

There are several concerns to be addressed before I can recommend the manuscript for publication:

1. The authors have Figure 1 in the article, but I found no mention of it in the text. At the same time, I also strongly suggest improving Figure 1 to make it a model incorporating information that is known and those that are novel to this finding.
2. The increased mRNA expression data needs to be presented better. It was hard to follow the fold changes. The important mRNA expression data needs to be provided in the form of bar graphs.
3. The authors need to provide more detailed legends/explanations for tables 1-3. What are the numbers, and what is inside the parentheses?
4. What is the criterion for dissemination? How do the authors differentiate between disseminated and metastasized?
5. The association between PD-1 and components of the other pathways as shown in table 2 are weak at best (r=0.3). This should be mentioned in the text and discussed.
6. The content of lines 166-174 is essentially repeated in the very next paragraph.
7. In the Western blot showing pVHL, what are lanes 1, 2, 3 and 4? What is quantified? Quantification should be provided as a bar graph.

Author Response

figure 1 was mentioned in the paper. The found correlations show the complex relationships between the molecular markers. They all are novel.

2. The Tables consist of a lot of information. But the formation of bar graphs will repeat the data
3. Legends and explanations to all tables were modified
4. The Criterion of the dissemination – is the formation of distant hematological metastasis. For ccRCC it is the same.
5. The weak associations between the molecular markers were mentioned and discussed in the text.
6. The content of lines 166-174 are repeated in the very next paragraph.
7. The explanation for Western Blot is included in Appendix. We have added the description to the Figures.

Reviewer 2 Report

• The study on the role of PD-1, PD-L1, and PD-L2 in renal cell carcinoma is novel and interesting but needs some revision.

  • Please read the work carefully again and correct some phrases, e.g., on page 5, line 156: "The study revealed associationsbetween the expression of the PD-1 gene was associatedwith the expression of NF-κB..."
  • There is a mess in the Statistical analysis section; it needs to be reworked and rewritten, e.g., Spearman not Spearmen; the mentioned in the chapter non-parametric one-way ANOVA is the Kruskal-Wallis test, which is discussed later in the results, but is not written by name in this chapter; precise the "Mann-Whitney test was used for comparing differences in mean values." It should be noted that not all parameters had normal distribution; hence non-parametric tests were used, U test to compare two variables and Kruskal-Wallis test for more than two variables. Which post-hoc test was used? I recommend the Dunns test.
  • The title suggests that it is more like a review than an original -
  • In Figure 1, it is not clear which of the factors affect which - maybe lines should be replaced by arrows?
  • Inconsistency in naming subsequent subsections, e.g. 

  3.1. PD-1, PD-L1, PD-L2 expression in ccRCC cancers - it is a descriptive title

  3.2. VHL expression and p-VHL content increase in metastatic ccRCC - it is an observational-conclusion title

  • Moreover, the number of respondents is relatively small but acceptable.

   

  The manuscript writing style indicates the little experience of the principal investigator/author in manuscript writing. Still, it does not discriminate between the manuscript for publication after the changes have been made.

Author Response

1. The correction to page 5, line 156 was made.
2. Section “Statistical analysis” was rewritten.
3. The title shows the significance of immunity regulation.
4. Figure 1 shows the correlations. They couldn’t be replaced by arrows.
5. The names of sections were changed
6. We agree the number of respondents are relatively small. The study will be continued.

Round 2

Reviewer 1 Report

Thank you for the responses to the recommendations.