Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An In Silico Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The research work led by Tesfaye Wolde et al, under the title " Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An in silico Approach" focuses on addressing the clinical challenges associated with endometrial carcinoma of the uterine corpus uteri (UCEC). This type of cancer has a significant incidence and poor prognosis, exacerbated by the lack of effective screening methods.

The study is distinguished by its innovative approach, using a multi-omics analysis that combines RNA sequencing data and clinical information from various databases, including The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA). The central objective is to evaluate the prognostic value of MLH1 gene expression in UCEC.

The results reveal that MLH1 dysregulation is associated with unfavorable prognostic outcomes and inhibition of immune cell infiltration. Through detailed analysis, the involvement of MLH1 in immunological processes is explored, as well as its correlation with tumor mutational burden (TMB) and microsatellite instability (MSI). This comprehensive study contributes to the field by establishing MLH1 as a biomarker with potential for predicting prognosis, response to immunotherapy and drug sensitivity in UCEC. However, I wish to express certain pertinent observations regarding the manuscript, consideration of which could result in optimizing its scientific robustness and content.

1.       In the introduction section the authors should provide more relevant information regarding endometrial uterine corpus carcinoma, such as a description of the disease, epidemiology, clinical significance and relevance, and a summary description of the pathophysiology of the disease. The authors should improve the rationale for why the results of this article focused on the study of MLH1 in UCEC. Since they also report drug-susceptible biomarkers in different selected cancers such as ccRCC, GBM, HNSCC, and LUAD.

2.       The authors refer to the prognostic implications of mRNA expression levels of the 11 DEGs in section 2.5. Prognostic Gene Expression Levels Associated with Gemcitabine Chemoresistance in UCEC referring to Supplementary Figure 4A-H, the authors refer to SUPPLEMENTARY FIGURES 3: KM Curves for Prognostic values and expression levels of the genes related to gemcitabine resistance, what is shown in the supplementary materials? There appear to be errors regarding the number of figures presented in the supplementary materials.

3.       The authors describe in section 2.7. MLH1 Expression Negatively Correlated with the Tumor Immune Microenvironment in UCEC, that results obtained from the CIBERSORT database showed that MLH1 expression had a negative correlation with CD8+ T cell infiltration but showed positive correlations with B cells, CD4+ T cells, macrophages, neutrophils and dendritic cells. However, according to Figure 6A it can be seen that MLH1 expression had a negative correlation with CD4+ T cells, how do these results affect the conclusions obtained by the authors?

4.       The authors report that TISIDB analysis comprehensively revealed the correlation between MLH1 mRNA expression and immunokines, immune receptors and tumor infiltrating lymphocytes. Thus, positive correlations in MLH1 expression were observed with IL10 and IL10RB immunoinhibitors, CD40 and CXCL12 immunostimulators, and HLA-DOA and HLA-DMB MHC genes. TISIDB analysis provides more extensive information on the expression levels of the mRNA of interest and expression of tumor-infiltrating lymphocytes (TILs), immunostimulators, immunoinhibitors, MHC molecules, chemokines, and receptors. Therefore, I suggest that the authors provide information on the correlation between MLH1 mRNA and each of the TILs, immunostimulators, immunoinhibitors, MHC molecules, chemokines, and receptors in UCEC.

5.       I suggest that the authors provide the date of consultation of each of the databases used in this study as well as the version used, because some of these databases are constantly being updated.

Author Response

Dear reviewer,

We appreciate the time and effort you dedicated to reviewing our manuscript titled "Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An in silico Approach." Your insightful observations are invaluable, and we have carefully considered each point raised. Below, we address your concerns and provide clarification:

1. Introduction section:

We acknowledge the importance of providing a more comprehensive background on endometrial uterine corpus carcinoma, including its epidemiology, clinical significance, and pathophysiology. We have enhanced the introduction in lines 32-63 to better justify our focus on MLH1 in UCEC, particularly in comparison to other cancer types, such as ccRCC, GBM, HNSCC, and LUAD.

2. Supplementary Figures:

We apologize for the confusion regarding Supplementary Figure references. We have revised and corrected the numbering discrepancies in the supplementary materials, ensuring accurate alignment with the text.

3. Correlation of MLH1 Expression with Tumor Immune Microenvironment:

Your observation about the discrepancy between the description of CIBERSORT results and Figure 6A is duly noted. We have carefully reevaluated the data and rectify any inaccuracies. The impact of these findings on our conclusions has been thoroughly addressed in the revised lines 346 to 348.

4. TISIDB Analysis:

We appreciate your suggestion to provide more extensive information on the correlation between MLH1 mRNA expression and individual components such as TILs, immunostimulators, immunoinhibitors, MHC molecules, chemokines, and receptors in UCEC. In the process of designing our study, we aimed for a balance between comprehensiveness and relevance. Recognizing that TISIDB encompasses a vast array of immune markers, we carefully screened and included only those markers that were deemed significant or directly pertinent to the subject matter under investigation. We intended to focus on markers with potential implications for MLH1 expression, prognosis, and immunotherapeutic efficacy in Uterine Corpus Endometrial Cancer (UCEC).

We understand the importance of transparency in reporting our methodology, and we will enhance the manuscript to explicitly state the criteria used for marker selection, emphasizing the significance and relevance of the chosen markers. This clarification will better inform readers about our decision-making process and the considerations taken into account.

We appreciate your guidance in ensuring the clarity and accuracy of our work. If you have any further concerns or suggestions, please feel free to communicate, and we will address them diligently in the revised manuscript.

5. Database Information:

Your point about specifying the date of database consultation and versions used is well-taken. In the revised manuscript, we have included this essential information in the methodology section.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor,

I would like to express my deep thanks to you for giving me the opportunity to review this valuable manuscript "Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An in silico Approach".

This thorough research identifies MLH1 as a possible biomarker for accurately predicting prognosis, responsiveness to immunotherapy, and sensitivity to drugs in UCEC. These findings provide valuable insights into the clinical management of patients.

The paper needs the following changes:

1.      The whole manuscript needs careful proofreading for minor spacing, syntax, and language corrections in some places.

2.       The authors need to expand on the pathophysiology of Uterine Corpus Endometrial Cancer in the introduction section

3.       The authors need to include updated statistical data about the occurrence and death rate in Metastatic endometrial cancer patients worldwide in the introduction section

4.       All abbreviations should have definitions in the text; some do not, such as KEGG,.... ", which stands for what?

5.       The authors should avoid using abbreviated letters at the beginning of the paragraph. For example in 2.3. section, they started a paragraph as given “MLH1 exhibited a significant impact on various aspects of cellular activities, includ- 180 ing the mismatch repair, somatic hypermutation of immunoglobulin genes”.

6.      Figures should be easily understandable without additional explanation. Therefore, any abbreviations used should be well clarified in the footnotes, allowing readers to understand them without needing to refer back to the main text.

7.      The authors created beautiful figures, but the figures in the manuscript are not clear enough. The original or high-definition figures should be provided

8.       The manuscript missing Tables 1 and 2.

Author Response

Dear reviewer,

We appreciate your thorough review of our manuscript, "Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An in silico Approach." Your constructive comments are invaluable, and we have carefully considered each point raised. Here are our responses and planned actions:

1. Proofreading for Minor Errors:

We acknowledge the need for careful proofreading to address minor spacing, syntax, and language corrections. We have conducted a comprehensive proofread to ensure overall clarity and correctness of the revised manuscript.

2. Expansion on Pathophysiology:

We recognize the importance of providing a more detailed explanation of the pathophysiology of Uterine Corpus Endometrial Cancer in the introduction. We have expanded this section to enhance the reader's understanding of the disease in lines 24 to 63.

3. Inclusion of Updated Statistical Data:

Your suggestion to include updated statistical data on the occurrence and death rate in metastatic endometrial cancer patients worldwide is well-taken. We have incorporated the latest available data in lines 24 to 63 to provide a more current perspective.

4. Abbreviation Definitions:

We will ensure that all abbreviations, including KEGG, are defined in the text for clarity. Definitions will be provided upon first mention in the manuscript.

5. Avoidance of Abbreviated Letters at Paragraph Beginnings:

We appreciate your feedback on avoiding abbreviated letters at the beginning of paragraphs. We will revise the text in accordance with this suggestion for improved readability.

6. Clarification of Abbreviations in Figures:

Abbreviations used in figures have been clarified in the footnotes to ensure that the figures are easily understandable without the need for additional explanation from the main text in lines 189 to 191 and SI figures 7-8.

7. High-Definition Figures:

We understand the importance of clarity in figures. High-definition versions of the figures will be provided to enhance visual clarity and aid in comprehension.

8. Missing Tables 1 and 2:

We apologize for the oversight regarding Tables 1 and 2. These tables have been labeled well in the revised manuscript for completeness.

We appreciate your meticulous review and constructive feedback, which will undoubtedly improve the quality and comprehensibility of our manuscript. If you have any further suggestions or concerns, please do not hesitate to communicate, and we will address them promptly in the revised version.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is interesting and provide valuable data but some modifications are needed.

1. I suggest to start the introduction with a paragraph about UCEC; its prevalence and statistics.

2. You have do add some information in the introduction about the role of MLH1 in cancer in general 

3.  Results of Multi-Omics Approach Identified MLH1 as a Druggable Target in UCEC, you should add some statistics in your text.

4. Mention the used statistical test under each figure to be mole clear.

 5. Did you get an ethical permission for data collection, specially the 33 malignancies?

6. I can't find a relationship between reference 65 and investigation of MLH1 in Predicting Drug Sensitivity. Can you clarify your opinion in citation.

7. English revision is required.

Comments on the Quality of English Language

requires revision

Author Response

Dear reviewer，

We appreciate your thorough review of our manuscript, "Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An in silico Approach." Your insights are valuable, and we are committed to addressing your suggestions to enhance the manuscript. Here are our responses and planned actions:

1. Introduction Paragraph about UCEC:

We acknowledge your suggestion to include a paragraph about Uterine Corpus Endometrial Cancer (UCEC) prevalence and statistics at the beginning of the introduction. We have incorporated in lines 32 to 63 this information to provide a comprehensive introduction to the topic.

2. Inclusion of MLH1 Role in Cancer in General:

We recognize the importance of providing information about the role of MLH1 in cancer in general. We have incorporated the relevant information in the introduction section.

3. Statistics in "Results of Multi-Omics Approach" Section:

Your suggestion to add statistics in the "Results of Multi-Omics Approach Identified MLH1 as a Druggable Target in UCEC" section is noted. We have incorporated relevant statistical data to support and enhance the findings presented in this section.

4. Mention of Statistical Test under Each Figure:

We agree that clarity is essential. We have specified the statistical tests used under each figure to provide a transparent and comprehensive presentation of the data.

5. Ethical Permission for Data Collection:

In response to your comment about the potential analysis and use of the open database data twice or any ethical permission-related issue, we would like to emphasize that while the data from open public databases, such as TCGA, can be accessed and analyzed multiple times, each analysis is conducted with distinct research questions and objectives. The richness and complexity of the available data allow for multifaceted investigations, enabling researchers to explore various aspects of the biological and clinical landscape.

In our study, we utilized the open public database data to investigate the association of MLH1 expression with prognosis, immunotherapeutic efficacy, and drug sensitivity in Uterine Corpus Endometrial Cancer (UCEC). The data served as a valuable resource for our comprehensive analysis, contributing to the scientific understanding of MLH1's role in UCEC.

We want to assure you that, in adherence to ethical and scientific principles, the data analyses conducted were distinct and tailored to address specific research aims. Reusing the open database data thoughtfully and purposefully is a common practice in bioinformatics and computational biology research, allowing researchers to derive new insights and validate findings across diverse aspects of the investigated phenomenon.

We appreciate your diligence in reviewing our work and hope this clarification addresses any concerns regarding the reuse of open database data. If you have any further questions or comments, please feel free to communicate, and we will gladly address them in our revision.

6. Clarification of Reference 65:

We appreciate your attention to detail. We will clarify the relationship between reference 65 and the investigation of MLH1 in predicting drug sensitivity to ensure coherence and relevance in the citation.

7. English Revision:

We acknowledge the reviewer’s comment. We comprehensively proofread the revised manuscript.

We appreciate your meticulous review and constructive feedback. These suggested modifications will undoubtedly strengthen the manuscript, and we are committed to delivering a revised version that addresses each of your valuable comments.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

I am impressed by your manuscript, "Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An in silico Approach." Your study addresses a critical gap in cancer research, focusing on MLH1 in UCEC and its unexplored prognostic value and treatment implications. Your multi-omics approach, using data from TCGA, GEPIA, and GeneMANIA, provides valuable insights into MLH1's association with adverse outcomes and potential as a biomarker. This work is a commendable contribution to oncological research and offers significant scientific advancement.

Author Response

Subject: Response to Appreciation of Manuscript "Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An in silico Approach"

Dear reviewer,

Thank you for your positive overview and insightful remarks on our manuscript, "Depleted-MLH1 Expression Predicts Prognosis and Immunotherapeutic Efficacy in Uterine Corpus Endometrial Cancer: An in silico Approach." We appreciate the time and effort you took to review our work, and we are pleased to see that our research has resonated positively within the scientific community.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

·         All comments have been addressed

·         The manuscript presented in an intelligible fashion and written in standard English