Combinatorial Analysis of CD4⁺Tregs, CD8⁺Teffs, and Inflammatory Indices Predict Response to ICI in ES-SCLC Patients

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

cancers-4058379: Combinatorial analysis of CD4+Tregs, CD8+Teff and Inflammatory indices predict response to ICI in ES-SCLC patients

In this manuscript, authors investigated the role of Tregs in immunotherapy response prediction for untreated ES-SCLC. Moreover, authors evaluated the potential of inflammatory hematological indices such as neutrophil-to-lymphocyte ratio (NLR) and CRP levels to predict responders. The data of the manuscript indicated a survival benefit in patients harboring low Treg/Teff ratio that was further accompanied with low eosinophil levels and NLR ratio.

Overall, the manuscript suffers from several weaknesses. There is a fictional problem in the planning of the study. Therefore, it is not recommended for publication at its current state. My comments in detail are as following:

• The abstract should be written in an informative style. Please give real values/data, not vague subjective terms and avoid generalizations and nonessential information in the results.
• In the abstract, the aim of the study is written that “We aimed to explore easily obtainable circulating biomarkers, such as CRP, neutrophil-to-lymphocyte ratio (NLR), and the role of regulatory T cells (Tregs), in predicting response to immunotherapy.” However, in the introduction section the aim of the study is written that “we explored the role of Tregs in immunotherapy response prediction for untreated ES-SCLC. Moreover, we evaluated the potential of inflammatory haematological indices such as NLR and CRP levels to predict responders.” The aim of the study is different in different parts of the manuscript. It should be corrected.
• Inclusion and exclusion criteria should be specified for the study population.
• The section titled " 2 Lymphocyte isolation and flow cytometry analysis " mentions a healthy control group. The study population for healthy controls is not presented in the method section (how many people etc.). In addition to this, A reference must be provided for this section.
• Although antibodies against Tregs are presented in section "2.2 Lymphocyte isolation and flow cytometry analysis," the subpopulations shown in presentations such as Fig 1 and Table 2 vary from the method section. This should be presented in a more understandable.
• Figures 4 and 5 should be revised. In particular, it should be specified what is meant by CRP count and eosinophil count (number, %, etc.).
• Authors wrote that "Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and .........and elevated lactate dehydrogenase (LDH) levels." While the manuscript specifically emphasizes the increased LDH levels in SCLC patients, it should be explained why this analysis was not performed.
• The conclusion is very limited and does not reflect the results of the study.
• Cited references in the manuscript are relatively old. It is recommended to change these references by taking into account the current ones.

 

Author Response

Comment 1: The abstract should be written in an informative style. Please give real values/data, not vague subjective terms and avoid generalizations and nonessential information in the results.

Response 1: Thank you for the important comment. In the revised version of this manuscript, the abstract has been written in an informative style and real values/data have been included.

Comment 2: In the abstract, the aim of the study is written that “We aimed to explore easily obtainable circulating biomarkers, such as CRP, neutrophil-to-lymphocyte ratio (NLR), and the role of regulatory T cells (Tregs), in predicting response to immunotherapy.” However, in the introduction section the aim of the study is written that “we explored the role of Tregs in immunotherapy response prediction for untreated ES-SCLC. Moreover, we evaluated the potential of inflammatory haematological indices such as NLR and CRP levels to predict responders.” The aim of the study is different in different parts of the manuscript. It should be corrected.

Response 2: Thank you for this suggestion. The aim of the study has been corrected in the abstract of the revised version of this manuscript so as to be the same with the main part of the text.

Comment 3: Inclusion and exclusion criteria should be specified for the study population.

Response 3: Thank you for this valuable suggestion. Iclusion and exclusion criteria have been specified in the revised form of this manuscript. Please see 2.1. Patients and Blood collection.

Comment 4: The section titled " 2 Lymphocyte isolation and flow cytometry analysis " mentions a healthy control group. The study population for healthy controls is not presented in the method section (how many people etc.). In addition to this, A reference must be provided for this section.

Response 4: Thank you for this helpful suggestion. In the revised version of this manuscript, the number of healthy donors has been included at the section titled “2 Lymphocyte isolation and flow cytometry analysis”. A reference has also been provided.

Comment 5: Although antibodies against Tregs are presented in section "2.2 Lymphocyte isolation and flow cytometry analysis," the subpopulations shown in presentations such as Fig 1 and Table 2 vary from the method section. This should be presented in a more understandable.

Response 5:  Thank you for this suggestion. In the revised version of this manuscript, you can find the subpopulations of Tregs that include the antibodies mentioned in section 2.2. in the figure 1 regent as well as Table 2 legends.

Comment 6: Figures 4 and 5 should be revised. In particular, it should be specified what is meant by CRP count and eosinophil count (number, %, etc.).

Response 6:  Thank you for this important suggestion. Figures 4 and 5 have been revised. CRP and eosinophil count have been now specified.

Comment 7: Authors wrote that "Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and .........and elevated lactate dehydrogenase (LDH) levels." While the manuscript specifically emphasizes the increased LDH levels in SCLC patients, it should be explained why this analysis was not performed.

Response 7: Thank you for the comment. In this manuscript we refer to the most common “easily obtainable” biomarkers for immunotherapy response in SCLC. Although the levels of LDS have been related to response, we were not able to retrieve this information from patients file thereby we were not able to perform this type of analysis.

Comment 8: The conclusion is very limited and does not reflect the results of the study.

Response 8: Thank you for this helpful suggestion.In the revised version of this manuscript the conclusions have been extended.

Comment 9: Cited references in the manuscript are relatively old. It is recommended to change these references by taking into account the current ones.

Response 9: Thank you for this important suggestion. In the revised version of this manuscript relatively new references were included mainly from 2021 to 2025.

Author Response File: Author Response.doc

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates circulating immune cell subsets and inflammatory markers as potential predictors of response to first‑line immunotherapy in extensive‑stage small cell lung cancer (ES‑SCLC). The study is timely and clinically relevant, given the lack of validated biomarkers in this aggressive disease. The authors present interesting observations, particularly the association between a low Treg/Teff ratio and improved survival outcomes. I have following comments for authors -

1 The methodology for defining “high” vs. “low” immune cell populations relies on ROC‑derived cutoffs, but the manuscript does not provide the ROC curves, AUC values, or justification for the chosen thresholds. Including these data—either in the main text or supplementary materials—would strengthen the transparency and reproducibility of the analysis.

2 The study includes 51 ES‑SCLC patients, which is relatively small for multivariable immunological analyses. The authors should discuss the statistical power and potential limitations related to sample size, especially when subdividing patients into groups A/B and C/D. A brief power calculation or acknowledgment of overfitting risk would be appropriate.

3 The manuscript reports a negative correlation between CD8+ Teff and CTLA‑4+ Tregs, but the biological rationale for this relationship is not fully explored. The discussion would benefit from deeper mechanistic interpretation or references to existing literature explaining how these populations interact in SCLC or other high‑inflammation tumor types.

4 The finding that CRP decreases during treatment only in patients with favorable immune profiles is interesting, but the authors should clarify whether CRP changes were adjusted for confounders such as infections, corticosteroid use, or tumor burden. Without this, the interpretation of CRP kinetics as a predictive biomarker remains uncertain.

5 Several figures (e.g., Figures 4 and 5) would benefit from clearer labeling and improved readability. Axis titles, units, and group identifiers should be more explicit. Additionally, the manuscript would be strengthened by including representative flow cytometry gating strategies in the supplementary materials.

Author Response

Comment 1: The methodology for defining “high” vs. “low” immune cell populations relies on ROC derived cutoffs, but the manuscript does not provide the ROC curves, AUC values, or justification for the chosen thresholds. Including these data—either in the main text or supplementary materials—would strengthen the transparency and reproducibility of the analysis.

Response 1: We would like to thank the reviewer for this significant comment. In the revised version of this manuscript ROC curves as well as AUC values for Teff and Tregs have been included in supl. Figure 3.

Comment 2: The study includes 51 ES SCLC patients, which is relatively small for multivariable immunological analyses. The authors should discuss the statistical power and potential limitations related to sample size, especially when subdividing patients into groups A/B and C/D. A brief power calculation or acknowledgment of overfitting risk would be appropriate.

Response 2: We appreciate the comment of the reviewer regarding the number of the patients enrolled in this study. In the revised version of this manuscript we have included a paragraph discussing the limitations of our study mainly related to the small number of the patients.

Comment 3: The manuscript reports a negative correlation between CD8+ Teff and CTLA 4+ Tregs, but the biological rationale for this relationship is not fully explored. The discussion would benefit from deeper mechanistic interpretation or references to existing literature explaining how these populations interact in SCLC or other high inflammation tumor types.

Response 3: Thank you for this valuable suggestion. . In the revised version of this manuscript a discussion regarding the known interactions between Teff and Tregs in cancer has been included.

Comment 4: The finding that CRP decreases during treatment only in patients with favorable immune profiles is interesting, but the authors should clarify whether CRP changes were adjusted for confounders such as infections, corticosteroid use, or tumor burden. Without this, the interpretation of CRP kinetics as a predictive biomarker remains uncertain.

Response 4: Thank you for this important suggestion. We were not able to adjust the reduction of CRP levels with confounders such as infections, corticosteroid use, or tumor burden due to unavailability of patient’s clinical data. In the revised version of this manuscript, we have included a comment on that in the study limitations paragraph at the section “conclusions”.

Comment 5: Several figures (e.g., Figures 4 and 5) would benefit from clearer labeling and improved readability. Axis titles, units, and group identifiers should be more explicit. Additionally, the manuscript would be strengthened by including representative flow cytometry gating strategies in the supplementary materials.

Response 5:  Thank you for this suggestion. Figures 4 and 5 have been revised. CRP and eosinophil count have been now specified. Additionally, representative flow cytometry gating strategy has been included in Sup.Fig.1.

Author Response File: Author Response.doc

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the authors conduct a prospective analysis of peripheral immune cell and inflammation panels as prognostic markers to immune checkpoint therapy in extensive-stage small cell lung cancers (ES-SCLC) patient population, using FACS and routine blood tests. They found that a high ratio of Effector T-cell/Regulator T-cell, along with high baseline lymphocyte/neutrophil ratio are associated better prognostic and overall survival. This study is clinically relevant as it integrates the analysis of immune phenotypes with inflammatory markers specifically in ES-SCLC population   

The following are my comments in this study:

1. This is a relatively small, single-center cohort (n=51), hence it raises concern regarding its statistical power. Ideally, an extra validation cohort should be conducted to cross validate the findings and the proposed prognostic biomarkers. At minimum, the authors should frame the findings as exploratory, single-center analysis, or pilot study.
2. Several typographical and grammatical errors are present throughout the manuscript. E.g
   1. Line 237: Significantelly
   2. Line 280: recentelly

Considering these comments, I recommend a minor revision of this manuscript in this journal.

Author Response

Comment 1: This is a relatively small, single-center cohort (n=51), hence it raises concern regarding its statistical power. Ideally, an extra validation cohort should be conducted to cross validate the findings and the proposed prognostic biomarkers. At minimum, the authors should frame the findings as exploratory, single-center analysis, or pilot study.

Response 1: We appreciate the comment of the reviewer regarding the number of the patients enrolled in this study. In the revised version of this manuscript, we have included a paragraph discussing the limitations of our study mainly related to the small number of the patients. We have additionally mentioned our study as exploratory.

Comment 2: Several typographical and grammatical errors are present throughout the manuscript. E.g

1. Line 237: Significantelly
2. Line 280: recentelly

Response 2: We would like to thank the reviewer for this significant comment. In the revised version of this manuscript typographical and grammatical errors have been corrected.

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is satisfactorily revised.