T-Cell Receptor Repertoire as a Predictor of Immune-Related Adverse Events in Renal Cell Carcinoma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this work Kobayashi et al. studied the link between immune–related adverse events (irAEs) and the T cell receptor (TCR) repertoire in patients suffering from renal cell carcinoma (RCC) and treated with dual immune checkpoint inhibition (anti-PD1 plus anti-CTLA-4 mAbs). They found that peripheral blood T lymphocytes from patients with grade 2 irAEs had higher diversity in the TRA and TRB repertoires than T cells from the grade 3 group. Furthermore, patients with grade 3 irAEs showed higher TCRB and TCRA clonality than those from the grade 2 group. The authors suggest the possibility that TCR clonality may be used as a putative biomarker for prognosis of irAF risk in RCC patients receiving immune checkpoint therapy.

Major comments

The work by Kobayashi et al. has been well performed and the results obtained from their studies are interesting and novel. However, data represented on the major Figures 1 and 2 are not very intuitive and difficult to be interpreted as shown, mainly because the huge amount of information presented. Moreover, differences between the expression of V and J genes and their recombination in RCC patients belonging to grade 2 and 3 groups of irAE seem very low and statistically no significant. One of the most important weaknesses of the work is the very small population of RCC patients (n=6, only three of them with grade 2 irAE and three with grade 3 irAF) that has been studied, as acknowledged by the authors. This fact compromises seriously the validity and soundness of results and also the manuscript publication. Further studies with a larger population of patients belonging to grade 2 and grade 3 irAF groups are required to accurately stablish the importance of the relationship between TCR repertoire and irAEs prediction in RCC patients.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. please describe exact numbers regarding treatment not 'half' as the numbers dont make sense. 66% of patients had metastatic disease so how did one of them (to make it 50%) receive adjuvant therapy? Did patient RCC6 have tissue collection etc at the time of localized disease? If so then the designation M1 isnt correct as the readers will assume that the TNM staging is at the time of diagnosis unless stated elsewhere. the patients can't have M1 and receive adjuvant treatment unless the patient had limited M1 and had both nephrectomy plus metastasis directed therapy followed by adjuvant treatment. 

2. Please be exact in the description of adjuvant therapy. Both sunitinib and pembrolizumab are approved as adjuvant therapy. Because of the topic of this manuscript, I am assuming that the patients recevied adjuvant pembro but please be clear on this. 

3. please clarify. Were these blood samples collected before IO treatment or after IO treatment was started? Same question but regarding immune suppressive therapy? Was the blood collected before or afterwards? 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Takuro et al have advocated the use of TCR repertoire features as a predictor of immune related adverse events in renal cell carcinoma. As the authors have indicated, the statistical power of the study is a big shortcoming. There are other major comments that must be addressed before considering the messages from the manuscript.

1.     The manuscript lacks a control group. TCR repertoire features can change over time owing to many different immune related events happening even in a relatively healthy state. These can be affected by for example exposure to environmental pathogens, allergens and even seasonal changes. This strengthens the need for a control group, both healthy and disease controls.

2.     The information on ethnicity of patients is not there in the manuscript. Was the study group homogenous with regard to ethnicity and HLA alleles. HLA strongly drives T-cell responses and variations in HLA can lead to different clones expanding in different patients.

3.     Repertoire diversity must be compared to healthy controls, ideally related family members to see whether the effects observed can actually be attributed to a disease state.

4.     One biggest shortcoming of the study is lack of paired alpha beta TCR repertoire features. It is very difficult specially to infer clonality from alpha and beta chain info where the pairing information is not there.

 

It is very important to control groups in order to make some sense from the results that authors have found here.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In the revised version of this manuscript, the authors have satisfactorily addressed most of my major concerns. Regarding the small sample of RCC patients that has been studied, the authors have convincingly argued about the difficulty of obtaining samples of these specific grades of RCC (which could justify the small sample size) and the importance of the study as preliminary evidence of the link between TCR repertoire and irAEs in RCC patients.

Author Response

Thank you for taking the time to review the revised version of our manuscript. We appreciate your recognition that we have addressed most of your major concerns. Your point on the small sample size of RCC patients was indeed valid. We're glad to know that our explanation regarding the challenges in obtaining these specific grades of RCC samples and the significance of our study as preliminary evidence was convincing. We believe that despite the limitations, our findings can pave the way for future research in this area.

Reviewer 3 Report

Comments and Suggestions for Authors

I appreciate that authors acknowledge the shortcomings, but in my limited understanding, that unfortunatley doesn't help the study. I would strongly recommend inclusion of controls to make some meaning from the study.

Author Response

Thank you for your constructive feedback and the recommendation to include control groups in our study. We understand and agree with the fundamental scientific principle that control groups are essential to enhance the validity of research findings. We acknowledge that the absence of a healthy control group is a limitation of the current study.

However, the focus of this initial study was to specifically identify potential biomarkers and predictors of irAEs severity in a homogeneous cohort of patients with advanced renal cell carcinoma prior to immunotherapy initiation. We selected this study design to focus solely on identifying indicators that might forecast irAEs in patients with advanced renal cell carcinoma. This approach aimed to avoid any potential confusion that could come from comparing these patients with healthy individuals, who do not share the same medical condition.

We believe the findings from this targeted approach still offer valuable preliminary insights that can inform future, more comprehensive studies. In light of your recommendation and in recognition of the study's limitations, we are in the process of designing follow-up research. This subsequent study will include a healthy control group to allow for the comparative analysis that we agree is necessary to fully understand the implications of TCR repertoire differences.

Your critique has provided an important perspective, and we are committed to enhancing the robustness of our research framework. We appreciate the opportunity to clarify our study's objectives and future directions, and we are confident that our follow-up studies will address the gaps identified and contribute meaningful advancements to the field.