Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a very interesting Phase 1/2 study investigating the addition of SLM to axitinib and pembrolizumab for renal cell carcinoma. Thank you for the opportunity to review this manuscript. However, I have several concerns and would like to offer the following comments.

As this is a Phase 1/2 study, and the dose of SLM used in this study is pharmacologically high, more detailed safety information should be provided. In particular, the authors should present the onset, duration, severity, and selenosis-specific toxicities observed during the initial 14-day period of SLM monotherapy.

There is an inconsistency in the terminology used for the TGF-β1 assessment. The Methods section refers to “tumor TGF-β1,” whereas the Results section refers to “circulating TGF-β1.” It is therefore unclear which samples were actually used to evaluate TGF-β1. If these descriptions refer to the same assessment, the terminology should be clarified and standardized throughout the Methods, Results, and Discussion sections.

Regarding the time-dependent inhibition of TGF-β1, the interpretation requires further clarification. The Day 14 assessment appears to reflect the effect of SLM monotherapy, whereas the later assessment at approximately 3 months was performed after axitinib and pembrolizumab had been added. Therefore, the greater TGF-β1 inhibition observed at the later time point cannot necessarily be attributed to prolonged SLM exposure alone. The potential effects of all three agents should be considered, and the corresponding statements in the Results and Discussion should be interpreted more cautiously.

The manuscript emphasizes the observed response rate, particularly the high CR rate, in comparison with historical trials such as KEYNOTE-426. However, the number of response-evaluable patients is very small, and the study population consists predominantly of patients with favorable or intermediate IMDC risk. Given the inability to adequately adjust for differences in patient characteristics or perform meaningful subgroup analyses in this small cohort, comparisons with historical controls should be interpreted more cautiously.

Author Response

Comment 1. As this is a Phase 1/2 study, and the dose of SLM used is pharmacologically high, more detailed safety information should be provided. In particular, the authors should present the onset, duration, severity, and selenosis-specific toxicities observed during the initial 14-day period of SLM monotherapy.

Response: We have added a dedicated description of adverse events during the 14-day SLM-monotherapy run-in (Results, Section 3.4, “Safety during the SLM run-in”). During this period, adverse events were infrequent. Six of twelve patients had no AE or predominantly grade 1 (dyspnea on exertion, fatigue, cough, dizziness, proteinuria, and hematuria); one patient had a grade 2 infusion-related reaction, and a single grade 3 event (venous thromboembolism, day 14) was attributed to the underlying malignancy rather than to SLM. Importantly, no selenosis-specific toxicities (e.g., garlic or metallic breath, nail or hair changes, selenium dermatitis, or peripheral neuropathy) were observed during SLM monotherapy, and whole-blood selenium reached the pre-specified target range (35–45 µM) by day 14 without selenium-attributable grade ≥3 toxicity. Adverse events increased in frequency only after the addition of axitinib and pembrolizumab on day 15. These data indicate that pharmacologic-dose SLM was well tolerated as monotherapy.

Comment 2. There is an inconsistency in the terminology used for the TGF-β1 assessment. The Methods section refers to “tumor TGF-β1,” whereas the Results section refers to “circulating TGF-β1.” It is unclear which samples were used. The terminology should be clarified and standardized.

Response: We apologize for the inconsistent terminology and have corrected it throughout. Patient TGF-β1 was assessed in plasma (circulating) samples; tumor-tissue TGF-β1 by western blot refers exclusively to the preclinical 786-O ccRCC xenograft model (Figure 1A). We have (i) renamed Section 3.2 to “Plasma TGF-β1 Quantitation” and rewritten it to describe plasma collection at baseline, day 14, and ~3 months, (ii) standardized “circulating (plasma) TGF-β1” in the Abstract, Methods endpoints, and Results, and (iii) corrected the Figure 1 legend to distinguish panel A (xenograft tumor tissue, western blot) from panel B (patient plasma). We have also clarified that paired tumor biopsies were not obtained from patients, which removes the previous inconsistency with the limitations statement.

Comment 3. Regarding the time-dependent inhibition of TGF-β1: the Day 14 assessment reflects SLM monotherapy, whereas the ~3-month assessment was performed after axitinib and pembrolizumab had been added. The greater inhibition at the later time point cannot necessarily be attributed to prolonged SLM exposure alone; the effects of all three agents should be considered, and the statements should be interpreted more cautiously.

Response: We fully agree. Because axitinib and pembrolizumab were initiated on day 15, the greater TGF-β1 suppression observed at ~3 months reflects the combined regimen rather than prolonged SLM exposure alone, and the relative contribution of each agent cannot be isolated in this single-arm study. We have revised the Results (Section 3.1) and the Discussion (including the Future Perspectives) to attribute the later, deeper suppression to continued treatment with SLM in combination with axitinib and pembrolizumab, and we have removed language implying an SLM-specific time effect in patients.

Comment 4. The manuscript emphasizes the high CR rate versus historical trials such as KEYNOTE-426. However, the number of response-evaluable patients is very small, and the population is predominantly favorable/intermediate IMDC risk. Given the inability to adjust for differences or perform meaningful subgroup analyses, comparisons with historical controls should be interpreted more cautiously.

Response: We agree and have tempered all cross-study comparisons in the Results (Section 3.5) and Discussion. We now state explicitly that, with only 10 response-evaluable patients, a population composed predominantly of favorable- and intermediate-risk IMDC disease, and no ability to adjust for baseline differences or perform subgroup analyses, the comparison with KEYNOTE-426 and other first-line regimens is descriptive and hypothesis-generating only. This caution is reinforced in the new “Limitations of the Study” section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Even if the current treatment of clear cell renal cell carcinoma has been considerably improved, in most patients, the clinical response does not correlate with a favourable outcome and, therefore, further new therapy strategies are required. The present research, even if it included only a small group of patients and, at this point, the results cannot be statistically validated, showed a new perspective in limiting the progression of this disease. The methodology was fully explained, and the results were carefully assessed. The conclusions were in accordance with these findings, highlighting the fact that, at this point, this study cannot establish definitive associations between biomarker modulation and clinical outcomes. One minor recommendations, the paragraph describing the limitations of this research should be included as a distinct section entitled Limitations of the study for a better clarity and transparency of this work.

Author Response

Response to Reviewers

Manuscript: “TGF-β1 inhibition, cytokine modulation, and clinical response to Seleno-L-Methionine plus axitinib and pembrolizumab in Treatment-Naïve Clear Cell Renal Cell Carcinoma”

We thank the editor and both reviewers for their careful and constructive evaluation of our manuscript. We have revised the manuscript to address each point, and all changes are highlighted in yellow in the revised file. Our point-by-point responses are provided below; reviewer comments are shown in italics. We have also corrected a misprint of the numbers of patients in the 5000 ug and 6000 ug group (6 and 6 per group, not 3 and 5 in the original). We have also reformatted the paper to editorial standards. 2 papers cited were flagged to review. 10.1158/2767-9764.CRC-25-0447 [25], and 10.1016/S1470-2045(13)70465-0 [31]. To the best of our knowledge, these papers are not retracted and have been cited properly.

Reviewer 2

Comment 1. One minor recommendation: the paragraph describing the limitations should be included as a distinct section entitled “Limitations of the study” for better clarity and transparency.

Response: We thank the reviewer for this helpful suggestion. The limitations are now presented under a dedicated heading, “Limitations of the Study,” in the Discussion.

We believe these revisions have strengthened the manuscript and hope it is now suitable for publication. We thank the reviewers again for their valuable input.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed most of my comments, and I consider the revisions satisfactory.