Multiplex profiling of 16 immune checkpoints identifies novel serum biomarker panels for breast cancer detection and TNBC stratification: A case-control study

Mouna Stayoussef; Azza Habel; Weili Xu; Mariem Bessaad; Hanen Bouaziz; Mouna Ayadi; Wassim Y. Almawi; Anis Larbi; Besma Yacoubi-Loueslati

doi:10.1371/journal.pone.0348953

Peer Review History

Original SubmissionJanuary 3, 2026
16 Mar 2026 Decision Letter - Rishi Jaiswal, Editor -->PONE-D-25-65128-->-->Multiplex Profiling of 16 Immune Checkpoints Identifies Novel Serum Biomarker Panels for Breast Cancer Detection and TNBC Stratification: A Case-Control Study-->-->PLOS One Dear Authors, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ================================== Thank you for submitting your manuscript entitled “Multiplex Profiling of 16 Immune Checkpoints Identifies Novel Serum Biomarker Panels for Breast Cancer Detection and TNBC Stratification: A Case-Control Study” to PLOS ONE. After careful evaluation of the manuscript and the reviewers’ comments, I believe that the study addresses an interesting and potentially important topic. 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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. --> Reviewer #1: Yes Reviewer #2: Yes ******** -->2. Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: Yes Reviewer #2: Yes ****** -->3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.--> Reviewer #1: Yes Reviewer #2: Yes ****** -->4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.--> Reviewer #1: Yes Reviewer #2: Yes ****** -->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: Wassim Y. Almawi and al took advantage of a multiplex imunoassay with 16 checkpoint imuno proteins of the serum to define a BC disgnostic signature as well as a TNBC classification signature. The paper is clear and well presented. The results points seven checkpoint proteins as a signature of BC and 8 checkpoint proteins where capable of distinguishing TNBC from non TNBC. Authors points in the discussion section that although these results are strong and important as biomarkers easy to detect they have not been validated in a larger and heterogeneous cohort. However there is a very important point that should be highlighted. These biomarkers have been identified in patients with a BC diagnostic and the important is to find biomarkers for early diagnosis in the population. Moreover biomarkers for early diagnosis must be easy to measure in routine blood tests and must have a very low coast. I recommend that authors add these comments in their discussion section. Reviewer #2: Summary The authors present a case control study evaluating 16 soluble immune checkpoint proteins in serum samples of 88 treatment naïve breast cancer (BC) patients and 49 healthy controls. Using a 16 plex MILLIPLEX panel, they examine differential expression, develop a diagnostic seven protein panel (AUC=0.89), identify an eight protein TNBC signature, and explore associations with chemotherapy response and short term PFS. The study claims novelty in being the first comprehensive serum based multiplex profiling of 16 checkpoint molecules for BC detection and TNBC stratification. Key scientific and conceptual strengths • Novelty and Scope: First study to evaluate a 16 checkpoint multiplex serum assay, expanding beyond the commonly studied PD 1/PD L1 and CTLA 4. This is indeed an underexplored area. • Minimally Invasive Diagnostic Potential: Serum biomarkers offer clear translational relevance, especially for early detection and TNBC stratification. • Rigorous Assay Validation: The authors provide extensive details on assay precision, linearity, CV thresholds, and quality control — unusually thorough for biomarker studies. • Statistical Breadth: Multiple analyses performed: ROC, logistic regression, PCA, Cox regression, correlation networks — giving a multidimensional view. • Clear identification of clinically relevant signatures • Seven-protein diagnostic panel • Eight-protein TNBC classifier • Associations with response and short term PFS Critical Issues which need to be addressed: Manuscript has a novelty component, however there are some areas where it needs improvement. Study Design Limitations 1. Cross-sectional design: Claims around prediction, treatment monitoring, and clinical utility are overstated given that all measurements are baseline. 2. Single-center, modest sample size: Although adequately powered for discovery, it is insufficient for firm conclusions regarding predictive biomarkers, especially for TNBC (n=23) and progression (14 events). 3. Short PFS follow-up (6 months): BC outcomes require longer follow-up; current survival conclusions are preliminary. Methodological and Analytical Issues 1. Controls are only “healthy”. It would be great to include other controls to justify Real-world diagnostic comparisons: • benign breast lesions • inflammatory conditions • autoimmune diseases These can alter soluble immune checkpoints, risking inflated specificity. 2. Potential pre-analytical variability not fully addressed: Despite QC efforts, the massive difference in LAG 3 concentrations between controls and patients raises questions. 3. Logistic regression and model generalizability • No external validation • No internal cross-validation (e.g., bootstrap, k fold) • Risk of model overfitting, especially with small TNBC numbers 4. ROC analyses lack reporting of confidence intervals for cut-offs and performance metrics. Interpretation Concerns 1. Causal language is used without justification E.g., authors imply clinical utility for monitoring, immune profiling, and treatment decisions. 2. Discussion overinterprets findings Some conclusions exceed evidence (e.g., recommending panels for early detection and TNBC management without validation). 3. Limited mechanistic insight The discussion heavily cites literature but does not integrate the biological meaning of the patterns observed (downregulated vs. upregulated ICs). The study is promising, innovative, and technically rigorous. However, several substantial issues must be addressed before it is suitable for publication. With robust revision, the manuscript could become acceptable for PLOS ONE. Major Comments: 1. Please temper claims about diagnostic/predictive clinical utility and emphasize the need for prospective validation. 2. Provide internal validation for logistic regression models to reduce risk of overfitting. 3. Re-examine LAG 3 and other extreme values to rule out technical artifacts. 4. Add FDR adjustments and report corrected p-values clearly. 5. Improve alignment among Figures/Tables—IDO and CD86 appear inconsistently. 6. Move mechanistic interpretations into Discussion. 7. Expand limitations (single center, modest sample, short follow up). Minor Comments: 1. Several typographical errors and grammar issues need polishing. 2. Simplify overly long sentences for clarity. 3. Add 95% CIs to AUCs, sensitivity, and specificity. 4. Clarify whether chemotherapy regimens were standardized across patients. 5. Consider supplementing with a flowchart showing sample selection and exclusions. ****** -->6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.--> Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0348953.r001
Revision 1
14 Apr 2026 Author Response Reviewer #1: Reviewer: Authors points in the discussion section that although these results are strong and important as biomarkers easy to detect they have not been validated in a larger and heterogeneous cohort. Authors: Although the immune‑checkpoint panels show strong diagnostic potential, their clinical use requires confirmation in larger and more diverse cohorts, as stated by the Reviewer. Changes: We revised the Discussion to emphasize the absence of external validation as a key limitation, to clarify that the current results are exploratory, and to outline next steps for multi‑center and prospective studies. Reviewer: These biomarkers have been identified in patients with a BC diagnostic and the important is to find biomarkers for early diagnosis in the population. Moreover biomarkers for early diagnosis must be easy to measure in routine blood tests and must have a very low coast. I recommend that authors add these comments in their discussion section. Authors: In the revised Discussion, we clarify that our results are based on already diagnosed breast cancer cases, reflecting a diagnostic enrichment setting more than population screening. We outline the requirements for early-detection biomarkers, including feasibility within standard blood tests and cost considerations, and place our multiplex immune‑checkpoint panel within this translational context, noting both its promise and its current limitations. Changes: Appropriate modifications consistent with the reviewer’s comment were made in the Discussion and Conclusion sections. Reviewer #2: Reviewer: 1. Cross-sectional design: Claims around prediction, treatment monitoring, and clinical utility are overstated, given that all measurements are baseline. Authors: Given the cross‑sectional design and reliance on baseline measurements, we agree that the findings should be interpreted as associative rather than predictive. These changes made to the revised version will align the conclusions with the study design and current standards for biomarker research. Changes: We revised the Results, Discussion, and Conclusion sections to remove any causal or prognostic wording. We also clarified that the analyses are exploratory and hypothesis‑generating and expanded the limitations to note that prospective studies are required before any clinical application can be inferred. Reviewer: 2. Single-center, modest sample size: Although adequately powered for discovery, it is insufficient for firm conclusions regarding predictive biomarkers, especially for TNBC (n=23) and progression (14 events). Authors: It is true that the overall sample size is adequate for exploratory analyses, and not sufficient to draw firm conclusions about predictive biomarkers, particularly in smaller subgroups such as TNBC (n=23) and progression events (n=14). Changes: We have “softened” statements implying predictive value, clarified the limitations related to single‑centre recruitment and subgroup size, and emphasized the exploratory nature of the work. We have also added text noting that larger, multi‑centre studies with adequate event numbers are needed to validate these associations. Reviewer: 3. Short PFS follow-up (6 months): BC outcomes require longer follow-up; current survival conclusions are preliminary. Authors: A point well-taken and appreciated. Changes: We revised the manuscript (Results, Discussion, and Conclusion), stating that the short follow‑up is a key limitation that moderates any prognostic interpretations, and clearly framing the survival results as preliminary and hypothesis‑generating. We also emphasized the need for extended prospective follow‑up to substantiate the observed associations with PFS. Reviewer: Methodological and Analytical Issues 1. Controls are only “healthy”. It would be great to include other controls to justify Real-world diagnostic comparisons: • benign breast lesions • inflammatory conditions • autoimmune diseases These can alter soluble immune checkpoints, risking inflated specificity. Authors: We used healthy controls to establish baseline differences and to enable initial biomarker discovery. We acknowledge that this may overestimate specificity, as soluble immune checkpoint levels can be altered in non-malignant inflammatory or immune-mediated conditions. Changes: We revised the Discussion and Conclusion sections, recognizing this limitation and emphasizing the need for future studies that incorporate disease-control groups to better evaluate real-world diagnostic performance. Reviewer: Methodological and Analytical Issues. 2. Potential pre-analytical variability not fully addressed: Despite QC efforts, the massive difference in LAG 3 concentrations between controls and patients raises questions. Authors: We agree with the Reviewer that the marked difference in LAG-3 concentrations warrants careful consideration of potential pre-analytical and analytical sources of variability. Changes: We modified the Methods section, expanding the description of pre‑analytical standardization procedures (sample collection timing, processing, storage, and quality control). We also revised the Results, acknowledging that residual variability or matrix effects may influence absolute concentration differences, and clarified that the LAG‑3 signal is supported by consistent effect sizes, internal assay validation, and sensitivity analyses. In addition, we modified the Discussion emphasizing that the magnitude of the observed differences should be interpreted carefully and independently validated. Reviewer: Methodological and Analytical Issues. 3. Logistic regression and model generalizability • No external validation • No internal cross-validation (e.g., bootstrap, k-fold) • Risk of model overfitting, especially with small TNBC numbers Authors: We acknowledge the absence of external validation and internal resampling approaches, which limit the generalizability of the regression models, thus raising the possibility of overfitting, particularly in subgroup analyses such as TNBC. Changes: Appropriate modifications were introduced into the Methods, Results, Discussion, and Conclusion sections. Reviewer: Methodological and Analytical Issues. 4. ROC analyses lack reporting of confidence intervals for cut-offs and performance metrics. Authors: We acknowledge that the need for reporting confidence intervals (CIs) for ROC-derived performance metrics is essential for the appropriate interpretation of diagnostic accuracy. Changes: We have revised the Methods and Results to report 95% confidence intervals (CIs) for AUC values (already partially included), clarify the statistical method used for AUC estimation (DeLong), and indicate that CIs for sensitivity, specificity, and related diagnostic indices at optimal cut-offs were computed. Reviewer: Interpretation Concerns. 1. Causal language is used without justification. E.g., authors imply clinical utility for monitoring, immune profiling, and treatment decisions. Authors: We acknowledge the concern of the reviewer that causal inferences and statements implying established clinical utility are not justified, given the cross-sectional design and baseline-only measurements. Changes: The manuscript was systematically revised to replace causal and deterministic language with association-based terminology, remove or temper statements suggesting immediate clinical application (including treatment monitoring and decision-making), and explicitly frame the findings as exploratory and hypothesis-generating. We also clarified in the Discussion and Conclusion that clinical implementation requires prospective and longitudinal validation. Reviewer: 2. Discussion overinterprets findings. Some conclusions exceed evidence (e.g., recommending panels for early detection and TNBC management without validation). Authors: In response to the Reviewer’s comment, we revised the manuscript to remove prescriptive or definitive claims regarding early detection, TNBC management, and clinical implementation. We replaced those with cautious, association-based language and framed the results as exploratory and hypothesis-generating. We also strengthened the limitations and added clear statements emphasizing the need for prospective validation, independent replication, and evaluation in pre-diagnostic and clinically diverse populations. Changes: The Discussion and Conclusion were extensively revised, as suggested by the Reviewer. Reviewer: 3. Limited mechanistic insight. The discussion heavily cites literature but does not integrate the biological meaning of the patterns observed (downregulated vs. upregulated ICs). Authors: We revised the Discussion to integrate the biological meaning of downregulated versus upregulated ICs, organizing the interpretation into a dedicated subsection that links the observed patterns (e.g., reduced LAG-3, BTLA, CD80, GITRL vs. increased GITR) to established immunological functions (co-inhibition, co-stimulation, T-cell exhaustion, and regulatory T-cell dynamics). We also clarified that these interpretations are hypothesis-generating and do not imply causality. Changes: A new paragraph outlining the mechanisms underlying the consequences of altered ICs was added to the revised Discussion. Additional modifications were also incorporated. Reviewer: The study is promising, innovative, and technically rigorous. However, several substantial issues must be addressed before it is suitable for publication. With robust revision, the manuscript could become acceptable for PLOS ONE. Authors: We thank the reviewer for the positive assessment of the study’s innovation and technical rigor, as well as for the constructive feedback. Changes: We have thoroughly revised the manuscript to address all methodological, analytical, and interpretative issues raised by both reviewers, and we are confident that the revisions and accompanying responses fully resolve their concerns. Major Comments: Reviewer: 1. Please temper claims about diagnostic/predictive clinical utility and emphasize the need for prospective validation. Authors: Will do. Changes: We have already revised the manuscript to temper any diagnostic or predictive claims and now clearly emphasize the need for prospective validation, as detailed in our earlier response. Reviewer: 2. Provide internal validation for logistic regression models to reduce risk of overfitting. Authors: Formal internal validation procedures, including bootstrap or cross-validation, were not performed, given the exploratory nature of this study. Changes: Appropriate modifications were made in the Methods (Statistical Analysis), Results, Discussion, and Conclusion sections. Reviewer: 3. Re-examine LAG 3 and other extreme values to rule out technical artifacts. Authors: We re-examined the dataset and assay performance, including inspections for outliers, batch effects, and consistency across duplicate measurements. We confirmed that the observed LAG-3 values were not influenced by isolated outliers or batch-specific effects and remained consistent after sensitivity analyses. These included log-transformation and exclusion of extreme percentile values. Changes: The Methods and Results sections were revised to clarify the quality control procedures. A cautionary statement was also added to the Discussion acknowledging that extreme values, while robust in this dataset, require independent validation using alternative platforms. Reviewer: 4. Add FDR adjustments and report corrected p-values clearly. Authors: Will do. Changes: FDR adjustments (Benjamini–Hochberg method) were added to Table 2. Corrected p values were also adjusted. Reviewer: 5. Improve alignment among Figures/Tables—IDO and CD86 appear inconsistently. Authors: We apologize for the poor quality of the figures in the initial submission. Changes: Fig. 1 and Fig. 2 were redrawn for clarity and sharpness, and saved as TIFF images. Reviewer: 6. Move mechanistic interpretations into Discussion. Authors: Will do. Changes: The manuscript was revised by removing mechanistic and biological explanatory statements (Results section) and relocating them to the Discussion. The Discussion was also restructured to include a subsection on mechanistic interpretation that links our findings to known immune checkpoint biology and breast cancer pathophysiology. A new reference was added to the references list. Reviewer: 7. Expand limitations (single center, modest sample, short follow up). Authors: Noted. Changes: The limitations section has been reorganized, and the list of potential shortcomings has been expanded to encompass clinical, methodological, and statistical considerations. Minor Comments: Reviewer: 1. Several typographical errors and grammar issues need polishing. Authors: Thank you for noting the typographical and grammatical issues. Changes: We have carefully reviewed the full manuscript and corrected all identified errors, ensuring consistency, clarity, and adherence to the journal's style throughout. Reviewer: 2. Simplify overly long sentences for clarity. Authors: Will do. Changes: Long sentences were restructured in the revised text. Reviewer: 3. Add 95% CIs to AUCs, sensitivity, and specificity. Authors: Done. Changes: 95% CIs to AUCs, sensitivity, and specificity were added. Reviewer: 4. Clarify whether chemotherapy regimens were standardized across patients. Authors: Standardized chemotherapy regimens for breast cancer in Tunisia generally follow international evidence‑based protocols. We provide details on the types of regimens administered and clarify the timing of blood sampling relative to treatment initiation. Changes: The Methods, Results and Discussion sections were modified as per the Reviewer’s comment. Reviewer: 5. Consider supplementing with a flowchart showing sample selection and exclusions. Authors: While we agree that a flowchart improves transparency of sample selection, detailed counts for each exclusion category were not systematically recorded at the time of data collection. As such, exclusions are presented in aggregate form. Changes: None for now. Attachments Attachment Submitted filename: Mouna 16-plex PLoS One Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0348953.r002
24 Apr 2026 Decision Letter - Rishi Jaiswal, Editor Dear Dr. Almawi, I hope you are doing well. I am pleased to inform you that your manuscript entitled “Multiplex Profiling of 16 Immune Checkpoints Identifies Novel Serum Biomarker Panels for Breast Cancer Detection and TNBC Stratification: A Case-Control Study” (Manuscript Number: PONE-D-25-65128R1) has been accepted for publication in PLOS ONE. Thank you for your submission. The manuscript will now proceed to the production stage, and you will be contacted regarding the next steps. Best regards, Dr. Rishi Kumar Jaiswal Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0348953.r003
Formally Accepted
Acceptance Letter - Rishi Jaiswal, Editor PONE-D-25-65128R1 PLOS One Dear Dr. Almawi, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Rishi Jaiswal Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0348953.r004

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