Genomic and Molecular Associations with Preoperative Immune Checkpoint Inhibition in Patients with Stage III Clear Cell Renal Cell Carcinoma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a study on genomic and molecular markers associated with preoperative immune checkpoint inhibitor (ICI) therapy for stage III clear cell renal cell carcinoma (ccRCC). The research design is rigorous, and the methods are advanced, particularly the application of digital spatial analysis technology, which demonstrates a certain degree of innovation.

Below are my comments and suggestions for the authors:

1. Supplement specific information on preoperative ICI treatment regimens: It is recommended to clarify the specific treatment regimen, treatment duration, and efficacy evaluation results for each patient in the supplementary materials, and to analyze whether different treatment regimens have heterogeneous effects on the tumor microenvironment or recurrence outcomes.
2. Explain the potential mechanisms of "improved microenvironment but no survival benefit": The discussion section should further explore the possible reasons for this core finding, incorporating existing literature analysis to enrich the depth of the discussion.
3. In the study, only a few genes showed strong correlations at the RNA and protein levels, with immune checkpoint proteins even exhibiting negative correlations. Further discussion could be added to explore potential mechanisms for this inconsistency and the methodological implications.
4. External validation cohort matching: The NCT02210117 cohort used for validation consisted of patients with metastatic ccRCC, which differs from the Phase III patients in this study. It is recommended to explicitly state this limitation.

Comments on the Quality of English Language

The term 'pre-operative ICI' is used interchangeably with 'peri-operative' in multiple instances; the conclusion paragraph contains excessive long sentences.
Suggestion: Have native editors standardize terminology and use Grammarly or similar tools to split the conclusion into sentences under 25 words.

Author Response

Thank you for your time in reviewing our manuscript; responses to the comments are below.

 

Supplement specific information on preoperative ICI treatment regimens: It is recommended to clarify the specific treatment regimen, treatment duration, and efficacy evaluation results for each patient in the supplementary materials, and to analyze whether different treatment regimens have heterogeneous effects on the tumor microenvironment or recurrence outcomes.

We have updated the supplementary information (Table 1) detailing the treatment regimen details. Two out of the four patients received nivolumab, whereas the other two received pembrolizumab monotherapy versus axitinib with pembrolizumab. Given that that subgroup only included four patients, we hesitate to make broader conclusions regarding specific ICI agents.

 

Explain the potential mechanisms of "improved microenvironment but no survival benefit": The discussion section should further explore the possible reasons for this core finding, incorporating existing literature analysis to enrich the depth of the discussion.

We posit in the limitations section that despite the higher levels of certain CD8+ subtypes that T-cell exhaustion and presence of other immunosuppressive immune cell activity may have contributed to lack of durable response in the patients who received preoperative ICI. Although outside the scope of this study, single-cell transcriptomics and spatially resolved transcriptomics could better yield answers to this topic (lines 358-369).

 

In the study, only a few genes showed strong correlations at the RNA and protein levels, with immune checkpoint proteins even exhibiting negative correlations. Further discussion could be added to explore potential mechanisms for this inconsistency and the methodological implications.

Thank you for this comment. We have expanded our discussion regarding potential mechanisms for these discrepancies (lines 339-345).

 

External validation cohort matching: The NCT02210117 cohort used for validation consisted of patients with metastatic ccRCC, which differs from the Phase III patients in this study. It is recommended to explicitly state this limitation.

Agreed, we have made sure to explicitly state this limitation in the discussion section (lines 346-351).

 

The term 'pre-operative ICI' is used interchangeably with 'peri-operative' in multiple instances; the conclusion paragraph contains excessive long sentences.

Suggestion: Have native editors standardize terminology and use Grammarly or similar tools to split the conclusion into sentences under 25 words.

- We have specified specifically instances where preoperative versus postoperative ICI was relevant (i.e. preoperative for the ICI patients in our cohort, for the PROSPER trial, as opposed to postoperative in the KEYNOTE-564 trial) to standardize terminology. We have also made sentences in the conclusion section shorter to improve readability. 

Reviewer 2 Report

Comments and Suggestions for Authors

It was interesting to study your manuscript. The methodology implemented could be described as ’we prefer quality in front of quantity’. It’s supported due to complexity and costly gene technological analyses when new field is explored to collect preliminary data. I’m leaving only few questions and comments.

Major comments

1. The title of the manuscript describes the time point of treatment as before nephrectomy; ’… preoperative immune checkpoint inhibition…’ However, in the abstract, short summary and the text body the wording ’perioperative’ is used. If the authors agree that the word ’perioperative’ is defined to deal with ”pre-, intra- and postoperative phase” then there is need to check if ’preoperative’ actually is the right word, since immune checkpoint inhibition (ICI) treatment obviously was given to these 19 patients before the nephrectomy. However, it’s to observe that in some sentences the authors may have wanted to refer to the duration of the effect desired by the preoperative ICI, i.e. covering the intra- and postoperative phases of the peripheral immune cells. In the methodology section, it’s proposed to define the words ’preoperative’ and ’perioperative’ in a way that they are used in various sentences in this manuscript. As an example, please, check (and revise if needed), whether in the PROSPER trial, nivolumab was actually only a preoperative treatment (line 73).

 

2. Since RCC is a malignancy seen as an ’immunological tumour type’, meaning that it’s not responding on chemotherapy but on immunologically functioning treatments, a question raises if the authors want to discuss their findings as compared to those in melanoma treatment, also included the same malignancy group as RCC.

Minor comments

1. To improve the message in the abstract, the authors are proposed to include the information that also protein analyses in addition to gene expression profiling was performed.
2. There is at least one abbreviation not explained when used for the first time: IRB (line 89). Please, add the meaning.

Author Response

Thank you for your time in reviewing our manuscript; responses to the comments are below.

The title of the manuscript describes the time point of treatment as before nephrectomy; ’… preoperative immune checkpoint inhibition…’ However, in the abstract, short summary and the text body the wording ’perioperative’ is used. If the authors agree that the word ’perioperative’ is defined to deal with ”pre-, intra- and postoperative phase” then there is need to check if ’preoperative’ actually is the right word, since immune checkpoint inhibition (ICI) treatment obviously was given to these 19 patients before the nephrectomy. However, it’s to observe that in some sentences the authors may have wanted to refer to the duration of the effect desired by the preoperative ICI, i.e. covering the intra- and postoperative phases of the peripheral immune cells. In the methodology section, it’s proposed to define the words ’preoperative’ and ’perioperative’ in a way that they are used in various sentences in this manuscript. As an example, please, check (and revise if needed), whether in the PROSPER trial, nivolumab was actually only a preoperative treatment (line 73).

Thank you, in this case “preoperative” is indeed the most appropriate word for most references to immune checkpoint inhibition in this manuscript. The patients who underwent ICI in this cohort all did so before surgery. We also clarified line 73 to state that nivolumab was given preoperatively to patients in the PROSPER trial. Finally, the data from the NCT02210117 trial we used for external validation also involved ICI preoperatively. “Perioperative” would only come into play given that ICI was administered after surgery during the KEYNOTE-564 trial.

Since RCC is a malignancy seen as an ’immunological tumour type’, meaning that it’s not responding on chemotherapy but on immunologically functioning treatments, a question raises if the authors want to discuss their findings as compared to those in melanoma treatment, also included the same malignancy group as RCC.

Thank you for this comment; this does raise an interesting point re: lack of effiacy of most chemotherapy agents for these two malignancies. I am not sure how applicable/reated those two diseases processes are to each other. We do note in lines 306-307 that overexpression of GZMK in melanoma patients seems to be associated with better immune checkpoint inhibitor response.

To improve the message in the abstract, the authors are proposed to include the information that also protein analyses in addition to gene expression profiling was performed.

- Clarified that both gene and protein expression performed (line 42)

There is at least one abbreviation not explained when used for the first time: IRB (line 89). Please, add the meaning.

- This has been amended to reflect "institutional review board"