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After revisions, all reviewers agreed to publish the manuscript. I also reviewed the manuscript and found no obvious risks to publication. Therefore, I also approved the publication of this manuscript.
The authors have addressed most of my comments.
The authors have addressed most of my comments.
The authors have addressed most of my comments.
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The authors are requested to carefully revise the manuscript and answer the questions raised by the reviewers.
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1. The manuscript uses Cox regression and provides a forest plot with hazard ratios and p-values. While this demonstrates statistical association, it does not evaluate the predictive performance of the markers. To support the claim that the CD8/Foxp3 ratio (or any TIL marker) has potential clinical utility in predicting recurrence, a Receiver Operating Characteristic (ROC) curve analysis should be added. The Area Under the Curve (AUC) will quantify discriminative ability and complement the survival analyses. Ideally, compare AUCs of individual markers (OX40, CD8, Foxp3) and combinations (e.g., CD8/Foxp3, OX40/Foxp3).
2. The authors report using the median value to stratify OX40+, CD8+, and Foxp3+ tumor-infiltrating lymphocytes (TILs) into high and low groups. It is essential to specify whether TIL quantification was based on absolute counts or normalized to the total number of nucleated cells (e.g., OX40+ cells / total cells). If normalization was performed, the definition and method for counting "total cells" should be clearly described, as variability in tissue cellularity can significantly affect the interpretation of immune cell density. Second, the authors should state whether the quantification was conducted manually or through digital pathology, and if manually, how fields were selected and how many were analyzed per sample. Third, the anatomical compartment used for analysis—stromal, intratumoral, or both—should be specified, as immune infiltration patterns can differ across regions. Additionally, summary statistics (e.g., median, interquartile range, or mean ± SD if applicable) for each marker should be provided to give readers a better sense of the data distribution.
3. The authors should clarify the timepoint at which tumor samples were collected for IHC analysis—specifically, whether all specimens were obtained prior to recurrence, or if any were from recurrent tumors. This distinction is critical to ensure the analysis reflects predictive biomarkers rather than immune profiles of already recurrent disease. If samples from both timepoints were included, analyses should be stratified accordingly or at least clearly annotated.
4. Given that validated clinical tools such as the EORTC and CUETO risk scores are widely used for predicting recurrence in NMIBC, it would strengthen the clinical relevance of this work to include a comparison of their proposed immune markers (e.g., CD8/Foxp3 ratio) to these established scoring systems. Even a basic comparison or exploratory integration (e.g., through ROC curves or subgroup stratification) would help position their findings in the context of existing prognostic models and support potential additive value.
5. While the manuscript begins with a focus on OX40, the core finding is that the CD8/Foxp3 ratio is the only independent predictor in multivariate Cox regression. The authors should revise the title, While the manuscript begins with a focus on OX40, the core finding is that the CD8/Foxp3 ratio is the only independent predictor in multivariate Cox regression. The authors should revise the title to reflect this outcome more accurately.
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The authors have utilized tissues from 110 patients (91 males and 19 females) diagnosed with NMIBC to identify the predicting value of OX40+ for NMIBC recurrence. The downstream targets of OX40+(Foxp3 and CD8) were also evaluated. The authors employed histological evaluation and IHC techniques to associate OX40+ CD8+, and Foxp3+ TILs with clinicopathological features.
Overall, this manuscript is fine but needs to clearly how does it differ from other studies in the field. Furthermore, the analyses and method sections need to be more detailed for clarity.
More information might be needed to justify the conclusions.
1. The sex ratio in the sample is not balanced. Did the authors investigate the possible impact on this on the data?
2. During the follow up sessions, were the patients checked for other diseases which could impact the data? What was the rationale behind excluding three patients having connective tissue diseases, acute infection, or another malignancy?
3. More information needs to be provided about why certain statistical tests were performed.
4. At certain places the sentences can be broken down into smaller sentences for better understanding. For example –
• ‘By now, OX40, a dual modulator of effector T cell stimulation and Treg suppression, appears to be an ideal molecule beyond a single or combination of T cell signatures as a promising candidate for predicting survival outcomes’
• ‘The purpose of this retrospective study is to determine whether the distribution of OX40+ TILs can serve as an effective prognostic factor for tumor recurrence in NMIBC, as well as to explore the potential predictive value of CD8+ and Foxp3+ TILs’
5. Is there a possibility of doing some validation studies to support the data?
6. How did the authors account for the limitation of the Foxp3 immunohistochemistry while analyzing the data?
7. The authors mention ‘Five tumor areas with a high density of positive cells (hotspots) (hotspots) for each marker were..’ - How did the authors account for variability and possible bias in the data?
8. How was the data normalized for scoring?
9. How do authors address/improve the limited effectiveness of the risk scores from the European
Organization for Research and Treatment of Cancer (EORTC) and the Spanish Urological Club for Oncological Treatment (CUETO) in their study?
Non-muscle-invasive bladder cancer (NMIBC) frequently recurs and progresses within five years. In this manuscript, the author investigated the potential value of OX40 as a predictor of recurrence risk in patients with NMIBC. The conclusion is that the CD8+ /Foxp3+ tumor-infiltrating lymphocytes ratio can serve as an independent predictor of tumor recurrence in NMIBC. The data analysis and images should be improved.
1. Your introduction needs more detail. I suggest that you improve the description of TILs to provide more justification for your study. Supplement the description of TILs infiltration and the corresponding HE staining images in the results section.
2. The multifocality of non-muscle-invasive bladder cancer (NMIBC) mentioned in the study requires a comprehensive description and quantitative assessment rather than being mere distinguished by yes and no.
3. For Figure 2, areas with comparable TILs infiltration levels should be selected for the evaluation of staining features where feasible.
4. The images need warrant careful examination, particularly since Figure 2C appears to utilize a puncture specimen rather than the surgical specimen mentioned in the article.
5. IHC images should be systematically organized by magnification level, with clear differentiation between distinct magnifications. Superimposing images of varying magnifications is strictly discouraged to prevent visual confusion.
6. There is only one case at Tis stage, potentially compromising statistical power. It is recommended to expand the sample size in this tumor stage to enhance the robustness of the research findings.
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1. Throughout the article, most expressions are marked with '+' superscripts, whereas all figures contain no such notation. I suggest you should maintain this format consistency.
2. Line 175: Associations of OX40+ CD8+, and Foxp3+ TILs, there is a a comma missing after OX40+.
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