PONE-D-24-05510High Red Blood Cell Distribution Width Attenuates the Effectiveness of Immune Checkpoint Inhibitor Therapy: An Exploratory Study Using a Clinical Data WarehousePLOS ONE

Dear Dr. Kobayashi,

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Santosh K. Patnaik, MD, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I find the manuscript to be very interesting. The search for prognostic biomarkers related to ICI treatment outcomes is now a highly debated topic, especially with the aim of identifying those patients who may not benefit. In this perspective, the work appears to be well-structured, certainly deserving of publication. I found the statistical analyses performed to be comprehensive. They indicate that RDW could play a prognostic role in predicting shorter ICI therapy duration and PFS.

I would like to make some notes: some are of a purely grammatical nature, and others are more significant regarding the statistical analyses.

- Line 4, Short title: I think it is better to use effectiveness than efficacy.

- Line 31-33, Abstract, Background: It should sound better as "This study utilizes a Clinical Data Warehouse (CDW) to explore the prognostic significance of novel blood-based factors, such as the neutrophil-to-lymphocyte ratio and red cell distribution width (RDW), to enhance the prediction of ICI therapy benefit."

- Line 36, Methods: It misses the extended form of "NSCLC" and "EMR".

- Abstract, Methods: You should mention ROC curve analysis.

- Abstract, Results: You should mention the total number of patients or the number of patients in each cohort.

- Line 51, Abstract, Conclusions: It is better to use "suggests" than "affirms"

- Line 52, Abstract, Conclusions: It is better to use "initiation" than "commencement"

- Line 61, Introduction: Remove "EGFR".

- Line 63, Introduction: ".....prognostically significant." Here, it misses the citation.

- Line 69, Introduction: Remove "rate".

- Line 76, Introduction: You should substitute "predicitve" with "prognostic". These terms have different meanings.

- Line 78, Introduction: You can cite "Maffezzoli M, Santoni M, Mazzaschi G, et al. External validation of a red cell-based blood prognostic score in patients with metastatic renal cell carcinoma treated with first-line immunotherapy combinations. Clin Exp Metastasis. 2024;41(2):117-129. doi:10.1007/s10585-024-10266-6."

- Line 92, Introduction: it is better to use effectiveness than efficacy.

- Line 95, Introduction: You should substitute "predicitve" with "prognostic".

- Line 139, Methods: Introduce "NSCLC" as abbreviation (then use it in the other parts of the manuscript).

- Line 150, Statistical Analysis: Better to use "fewer times" than "less"

- Statistical Analysis: the statistical plan of the study is solid and appropriate. The dual validation approach, logistic regression, ROC curve and correlation analyses, as well as the use of CDW, enhance the robustness and generalizability of the findings. However, there are several areas where the methodology could be strengthened:

1) I think that incorporating time-to-event analysis (Cox proportional hazards models) could provide additional insights about the biomarkers and their association with PFS.

2) I think that exploring additional novel biomarkers or combining multiple markers into a composite score might improve predictive accuracy.

- Table 1: Please, clarify whether the age values are reported as median or mean, and explain ranges in brackets.

- Line 181, Exploration Part: Please, specify that there's no differences in the DISTRIBUTION of age, gender, etc. between the two groups.

- Line 195: blood urea nitrogen (BUN).

- Line 196: Chlorine (Cl)

- Line 203: the reasons for selecting specific variables for the multivariate model should be clearly justified.

- Line 206: The multivariate logistic regression includes multiple predictors, but only RDW remained significant. It might be beneficial to explore interactions between variables and other potential confounders.

Line 208: Please, justify the selection of the RDW threshold (15.5) with more detail (how this cutoff was chosen and its clinical relevance).

- Line 246: You should substitute "predicitve" with "prognostic".

- Validation part 2: It might be beneficial to explore interactions between variables and other potential confounders.

- Line 301: use "NSCLC".

- Line 318: You can cite "Maffezzoli M, Santoni M, Mazzaschi G, et al. External validation of a red cell-based blood prognostic score in patients with metastatic renal cell carcinoma treated with first-line immunotherapy combinations. Clin Exp Metastasis. 2024;41(2):117-129. doi:10.1007/s10585-024-10266-6."

- Discussion: External validation using data from different institutions or a prospective cohort would strengthen the findings' generalizability.

Reviewer #2: The manuscript by Kobayashi et al. is an interesting study that leverages an often-overlooked source of data collection: the Clinical Data Warehouse (CDW). This approach is commendable as it opens new avenues for discovering prognostic biomarkers in cancer treatment, an area where current markers exhibit limited predictive accuracy. The strength of the study lies in the rigorous validation process utilizing two different cohorts to ensure the reliability of the exploratory analysis. By confirming findings in both the CDW and EMR cohorts, the study supports the identification of elevated red cell distribution width (RDW) as a potential for predicting the efficacy of immune checkpoint inhibitor (ICI) therapy and paves the way for future studies on the topic. I recommend this article for publication with a few modifications, as outlined in the following suggestions.

• Abstract

o Line 36: Expand EMR when using for the first time.

o Line 36: Expand NSCLC when using for the first time.

o Clarify the methodology in the abstract: Were there two cohorts? Was the study only done on NSCLC patients? Mention the number of patients in each cohort (n=__). Line 41: "... utilized CDW for discovery and EMR/CDW for validating prognostic biomarkers for ICI treatment using the number of doses of ICI as a proxy." Or restructure the abstract to make it clear that the question is about finding biomarkers to predict ICI response, with an intermediate step being finding a PFS proxy.

• Introduction

o Line 63: Provide a citation for immune-related adverse events. Explain the tail effect and early non-response.

o Line 71: Clarify "various measurement methods"—are they non-standardized? Do they give different results?

• Methodology

o State why the authors selected only lung cancer patients for the validation cohorts.

o Explain how the authors selected the number of doses for early-discontinuation and sustained-treatment.

o Mention the statistical test used for group comparisons in the text as well as in the table captions.

• Results

o Mention the criteria used to select variables for the multivariable regression (Line 203).

o State if RDW was directly compared with PFS, not just the number of pembrolizumab doses, in the first validation cohort. Include additional data if there is an association, as it would strengthen the notion that the number of doses is a good surrogate for PFS.

o Compare patient characteristics of the exploratory cohort and the validation cohorts and identify any possible group differences. Add this in the supplementary materials.

o Table 6: Perform a subgroup comparison for nivolumab, pembrolizumab, and atezolizumab (n%) between the early-discontinuation group and the sustained-treatment group instead of using an overall p-value.

• Discussion

o It is interesting and logical that the number of pembrolizumab doses correlates with progression-free survival. Has any other study reported using the number of doses as a PFS proxy?

o Line 329: Clarify that there is a weak negative correlation between RDW and actual PFS. In the results, it is stated as between RDW and the number of pembrolizumab doses.

• The authors should revise the language to improve readability. Here are a couple of examples:

o Line 37: Use "between" instead of "from."

o Use active voice wherever possible. Split sentences into two instead of using long sentences (Line 60-63).

o Line 300: Remove "the three ICIs."

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Reviewer #1: No

Reviewer #2: No

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High Red Blood Cell Distribution Width Attenuates the Effectiveness of Immune Checkpoint Inhibitor Therapy: An Exploratory Study Using a Clinical Data Warehouse

PONE-D-24-05510R1

Dear Dr. Kobayashi,

We received the revised version of this manuscript. I have examined it and the accompanying response to review letter. The revision appropriately addresses all concerns that were raised by the reviewers.

I am therefore pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Santosh K. Patnaik, MD, PhD

Academic Editor

PLOS ONE