Chemoradiotherapy and Lymph Node Metastasis Affect Dendritic Cell Infiltration and Maturation in Regional Lymph Nodes of Laryngeal Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors Introduction - line 34, voice rehabilitation protocol should be discussed to evaluate QoL of the patients outcomes. cite doi:10.1016/j.jvoice.2021.09.040

- Provide more context on the epidemiology and burden of NSCLC globally and locally

- Discuss limitations of current first-line treatments and need for personalized strategies

- Elucidate the hypothesized roles of CTCs in metastasis and treatment response. cite doi:10.1016/j.ejca.2012.05.007.

- Clearly define the knowledge gaps around CTC enumeration/prognostic potential

- Explicitly state the study objectives to evaluate association of CTCs with outcomes.

Methods
- Describe patient cohort characteristics and selection criteria in greater detail  

- Validate choice and protocol registration for CTC measurement technique

- Explicitly state definition of CTC positivity and cut-off values applied

- Fully explain statistical analysis plan for between/within group comparisons

- Acknowledge limitations of retrospective design and potential confounders  

- Clearly define primary and secondary outcomes to address objectives

- State protocols for ensuring accurate, reproducible measurement and analysis

Results

- Present demographic/clinical characteristics of patient subgroups numerically    

- Include figures/tables to report CTC counts and statistical analysis findings

- Consider subgroup analyses by factors like stage, line of therapy, biomarker status

Discussion

- Critically appraise strengths/limitations of study design, patient cohorts, techniques   

- line 216, discuss epidemiological and gender difference for laryngeal cancer. cite doi:10.1007/s00405-022-07466-9
- Interpret findings in context of existing literature on CTCs in NSCLC

- Discuss proposed mechanisms linking CTC levels to outcomes

- Evaluate clinical applicability and need for prospective validation

- Acknowledge lack of follow up data limits interpretation of prognostic relevance

- Identify limitations in ability to determine causality from retrospective analysis

- Propose optimized study designs to address unanswered questions prospectively

- Suggest potential clinical utility pending validation in larger cohorts

Discussion

- line 216, epidemiology of laryngeal cancer should be considered, with different outcomes according to gender. cite doi:10.1007/s00405-022-07466-9

 

Comments on the Quality of English Language

none

Author Response

Responses to reviewers

We would like to thank all the reviewers for their careful reading of our manuscript and for providing such useful feedback, which was a great help in improving the clarity of our manuscript. We have responded to all of the queries and made all the requested changes in the revised manuscript. All changes based on the reviewer’s comments are highlighted. The English usage and grammar of the revised manuscript have been kindly checked by a senior editor at Oxford University who is familiar with its contents.

 

Our point-by-point responses to the reviewer’s comments are given below.

 

Reviewer 1

Query/Comment 1: Introduction- line 34, voice rehabilitation protocol should be discussed to evaluate QoL of the patients’ outcomes. cite doi:10.1016/j.jvoice.2021.09.040

Response: According to the reviewer’s suggestion, we have added the documentation regarding voice rehabilitation and cited the recommended reference (revised manuscript, page 1, line 34-36).

 

Query/Comment 2: Provide more context on the epidemiology and burden of NSCLC globally and locally.

Response: Sorry, we could not understand what is“NSCLC”meaning for (non-small cell lung cancer?). Instead of that, we have added a description regarding epidemiology of laryngeal cancer globally and locally in the revised manuscript, page 1-2, line 37-44.

 

Query/Comment 3: Discuss limitations of current first-line treatments and need for personalized strategies.

Response: According to the reviewer’s comment, documentation regarding first-line chemotherapy and therapeutic strategies for laryngeal cancer has been added to the revised manuscript (page 2, lines 58-64).

 

Query/Comment 4: Elucidate the hypothesized roles of CTCs in metastasis and treatment response. cite doi:10.1016/j.ejca.2012.05.007.

Response: The present study was not focused on CTC (Circulating Tumor Cell).

 

Query/Comment 5: Clearly define the knowledge gaps around CTC enumeration/prognostic potential.

Response: The present study was not focused on CTC.

 

Query/Comment 6: Explicitly state the study objectives to evaluate association of CTCs with outcomes.

Response: The present study was not focused on CTC.

 

Query/Comment 7: Describe patient cohort characteristics and selection criteria in greater detail.

Response: Similar comments has also provided by Reviewer 2. We have now described detailed selection criteria in “4.1. Patients” section. The detailed patient cohort characteristics are listed in Table 1. To help readers better understand the patient selection criteria, we have added Figure 4, which is a flow chart for case selection, to the revised manuscript (see page 11).

 

Query/Comment 8: Validate choice and protocol registration for CTC measurement technique.

Response: The present study was not focused on CTC.

 

Query/Comment 9: Explicitly state definition of CTC positivity and cut-off values applied.

Response: The present study was not focused on CTC.

 

Query/Comment 10: Fully explain statistical analysis plan for between/within group comparisons.

Response: Similar comments were also made by Reviewer 2. We would like to sincerely thank the reviewers for their appropriate comments. We had mistaken the description of the statistical analysis for group comparisons in a previous manuscript. Pearson’s chi-squared test and a linear regression analysis were not used in the present study. Student's t-test (two-tailed) was employed for comparison of patient age and Fisher’s exact test (two-tailed) was used for comparison of other factors. Univariate analyses were performed using the Cox proportional hazard model. These issues have been amended and described in the revised manuscript (page 12, line 347-349).

 

Query/Comment 11: Acknowledge limitations of retrospective design and potential confounders

Response: As the reviewer has pointed out, the retrospective nature of the study is one of its limitations. We have added appropriate text regarding this matter to the revised manuscript (page 10, line 268-269).

 

Query/Comment 12: Clearly define primary and secondary outcomes to address objectives.

Response: The primary and secondary outcomes of this study were disease-specific survival (DSS) and overall survival (OS). The definitions of DSS and OS are clearly described in the “4.4. Statistical analysis” section.

 

Query/Comment 13: State protocols for ensuring accurate, reproducible measurement and analysis.

Response: The detailed protocol of immunohistochemistry (IHC) of CD1a and S-100 is given in the “4.2. Immunohistochemistry” section. The IHC of CD1a and S-100 are usually performed in daily pathological practice worldwide and the values are readily reproducible

 

Query/Comment 14: Present demographic/clinical characteristics of patient subgroups numerically.

Response: The clinicopathological characteristics per subgroup of CD1a-DC and S100-DC infiltration are presented in Table 2 and Table 3.

 

Query/Comment 15: Include figures/tables to report CTC counts and statistical analysis findings.

Response: This study is not focused on CTC.

 

Query/Comment 16: Consider subgroup analyses by factors like stage, line of therapy, biomarker status.

Response: We initially performed the subgroup analyses according to the CRT status. However, we have deleted these data because too many tables complicated the content of the manuscript and obscured the purpose of our study. Because our cohort of patients was relatively small the data were not suitable to conduct subgroup analyses.

 

Query/Comment 17: Critically appraise strengths/limitations of study design, patient cohorts, techniques.

Response: The limitations of the present study are described on page 10, lines 268-270. The strength of the study findings are documented in the conclusion section.

 

Query/Comment 18: Discuss epidemiological and gender difference for laryngeal cancer. cite doi:10.1007/s00405-022-07466-9.

Response: Documentation for epidemiological and gender differences in laryngeal cancer have now been added to the revised manuscript with an appropriate citation (page 1-2, line 39-44).

 

Query/Comment 19: Interpret findings in context of existing literature on CTCs in NSCLC.

Response: The present study did not focus on CTC or NSCLC.

 

Query/Comment 20: Discuss proposed mechanisms linking CTC levels to outcomes.

Response: Our study did not focus on CTC.

 

Query/Comment 21: Evaluate clinical applicability and need for prospective validation.

Response: Clinical applicability (potential of application for immunotherapies) and the need for validation by accumulation of clinicopathological and basic research are now documented in the conclusion section of the revised manuscript.

 

Query/Comment 22: Acknowledge lack of follow up data limits interpretation of prognostic relevance.

Response: The maximum follow-up period in our study was 120 months and the median follow-up time 45.0 months. As the reviewer has pointed out, lack of follow-up data is a limitation of the present study. That issue is addressed in the revised manuscript (page 10, line 269).

 

Query/Comment 23: Identify limitations in ability to determine causality from retrospective analysis.

Response: The limitations of the retrospective nature of the analysis is considered in the revised manuscript (page 10, line 268-269).

 

Query/Comment 24: Propose optimized study designs to address unanswered questions prospectively.

Response: The need for accumulation of further clinicopathological and basic research data are now documented in the revised conclusion section. To elucidate the role and function of DCs in cancer tissue is a big topic and requires research from many angles. Thus, we have refrained from proposing a specific research design in our manuscript.

 

Query/Comment 25: Suggest potential clinical utility pending validation in larger cohorts.

Response: Validation studies of larger cohorts will provide useful knowledge in this field. We have added documentation regarding this issue in the conclusion section of the revised manuscript (page 11, line 308-309).

Once again, we would like to thank all the reviewers for the time and effort they put into greatly improve the clarity of our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The main question addressed by the research is the role of DCs in tumor immunity in laryngeal cancer, focusing on their infiltration and maturation in primary tumors and regional LNs. The study aims to elucidate the impact of chemoradiotherapy (CRT) on DC induction in cancer cells and the surrounding stroma.

- Change abstract form and split it into chategories (Objectives, Material and methods,...

-The structure of the manuscript is chaotic. Please rearrange it according to editors's suggestions

- The authors should provide more details on patient selection criteria and the rationale behind excluding certain cases.

- The cut-off values for high and low DC infiltration could be explained more thoroughly, considering their crucial role in the analysis.

- Statistical methods should be described in greater detail, including the rationale for choosing specific tests.

- The conclusions are generally consistent with the evidence presented. The results suggest that DC maturation in metastatic LNs plays a significant role in tumor immunity, and CRT appears to impact DC infiltration differently in metastatic and non-metastatic LNs. However, further clarification on the statistical significance of some findings would enhance the overall consistency of the conclusions.

Author Response

Responses to reviewers

We would like to thank all the reviewers for their careful reading of our manuscript and for providing such useful feedback, which was a great help in improving the clarity of our manuscript. We have responded to all of the queries and made all the requested changes in the revised manuscript. All changes based on the reviewer’s comments are highlighted. The English usage and grammar of the revised manuscript have been kindly checked by a senior editor at Oxford University who is familiar with its contents.

 

Our point-by-point responses to the reviewer’s comments are given below.

 

Reviewer 2

Query/Comment 1: Change abstract form and split it into categories (Objectives, Material and methods,...

Response: We have prepared the abstract following the Journal’s instructions (https://www.mdpi.com/journal/ijms/instructions#front) that specified a structured abstract without headings.

 

Query/Comment 2: The structure of the manuscript is chaotic. Please rearrange it according to editors’ suggestions.

Response: The structure of the manuscript follows the Journal’s instructions, and the revised version of our manuscript has been rearranged according to the editor’s guidelines.

 

Query/Comment 3: The authors should provide more details on patient selection criteria and the rationale behind excluding certain cases.

Response: Similar comments were made by Reviewer 1. We have described detailed selection criteria in the “4.1. Patients” section and detailed patient cohort characteristics are given in Table 1. To help readers better understand our patient selection criteria, we have added Figure 4, which is flow chart for case selection, to the revised manuscript (page 11).

 

Query/Comment 4: The cut-off values for high and low DC infiltration could be explained more thoroughly, considering their crucial role in the analysis.

Response: The cut-off values for high and low DC infiltration were determined by the median values of DCs infiltrating into the primary tumor. These cut-off values were used for analyses of LNs. To avoid reader misunderstanding, we have added an explanation of the cut-off values to the revised manuscript (page 5, lines 161 and 166, and page 6, lines 179 and 185-186).

 

Query/Comment 5: Statistical methods should be described in greater detail, including the rationale for choosing specific tests.

Response: Similar comments were also made by Reviewer 1. We would like to sincerely thank the reviewers for their perceptive and appropriate comments. We mistook the description of statistical analysis for group comparisons in a previous manuscript. Pearson’s chi-squared test and a linear regression analysis were not used in the present study. Student's t-test (two-tailed) was used for comparisons of age and Fisher’s exact test (two-tailed) was used for comparison of other factors. Univariate analyses were per-formed using a Cox proportional hazard model. These matters have now been addressed and described in the revised manuscript (page 12, line 347-349).

 

Query/Comment 6: The conclusions are generally consistent with the evidence presented. The results suggest that DC maturation in metastatic LNs plays a significant role in tumor immunity, and CRT appears to impact DC infiltration differently in metastatic and non-metastatic LNs. However, further clarification on the statistical significance of some findings would enhance the overall consistency of the conclusions.

Response: We quite agree with the reviewer’s opinion. Further studies using a larger cohort would likely enhance statistical significance and therefore the overall strength of our conclusions. We have added documentation regarding validation studies using larger cohorts to the conclusion section of the revised manuscript (page 11, line 308-309).

 

Once again, we would like to thank all the reviewers for the time and effort they put into greatly improve the clarity of our manuscript.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I recommend it for publication