PONE-D-25-07416Identification of the immune-associated enhancer RNA SATB1-AS1 as a novel biomarker for thymic cancer prognosisPLOS ONE

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Additional Editor Comments:

Very nice study, reviewers have made specific comments please address them. I am looking forward to your reply and revised version of the manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: 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: The study analyzes enhancer RNAs in thymoma using data from the TCGA database and explores their potential prognostic and therapeutic roles.

Comments:

1. The identification of a novel thymoma biomarker could be highly valuable. However, a major weakness of this study is that the authors rely solely on database analysis without any supporting in vitro or in vivo experiments. The manuscript only establishes a correlation between eRNA SATB1-AS1 and thymoma disease stage. Therefore, the claim in the title that SATB1-AS1 is "a novel biomarker for thymic cancer prognosis" appears to be an overstatement.

2. The manuscript lacks fluency and contains some errors. For example, in section 2.2, the first sentence is repeated twice. Careful proofreading and editing are needed to improve readability.

3. The placement of figures could be improved. I recommend positioning them after the relevant result descriptions rather than grouping them between the results and discussion sections. Additionally, the figures have low resolution, making it difficult to read the text, particularly in Figure 1.

Conclusion:

While the discovery of a new thymoma biomarker could have significant clinical implications, additional in vitro, in vivo, or epidemiological studies are necessary to validate its prognostic potential.

Reviewer #2: In their manuscript titled "Identification of the immune-associated enhancer RNA SATB1-AS1 as a novel

biomarker for thymic cancer prognosis" Wang et al. have performed bioinformatic and statistical analyses on publicly available TCGA data to determine the roles of enhancer RNAs, specifically SATB1-AS1, in thymomas and other tumors. I have several comments that must be addressed prior to the publication of this manuscript.

GENERAL

The symbols for genes and RNAs should be written in the italic font style (proteins should be written in regular style).

ABSTRACT

Methods: “Validation of the prognostic potential of target eRNAs across cancers in other cancer types.” This sentence has no verb; perhaps “was performed” is missing at the end?

Results: “SATB1-AS1 and the target gene SATB1 were associated with poor patient prognosis” This statement is misleading as high SATB1-AS1 expression wasa positive prognostic marker!

“…SATB1-AS1 expression was correlated with patient stage…” You should add “negatively” in front of correlated.

INTRODUCTION

-“Surgery is the treatment of choice for tumor management. Surgery, as the treatment of choice for tumor therapy…” No need to write the same information twice.

-“It is also involved in driving the transcription of target genes in various ways, such as three-dimensional chromatin remodeling and enhancer‒promoter loops.” Again, this was already stated in the previous sentence and should be removed.

-lncRNA, miRNA, SCCHN, and ESCA should be written in full form the first time they are mentioned.

-”A recent study also revealed that eRNA transcripts are not associated with oncogenic genes.” No reference is provided.

MATERIALS AND METHODS

2.1 The exact numbers of tumor patients and controls for which expression data was obtained should be stated.

2.2 “The R package "limma" was used to screen for differentially expressed eRNAs…” This is stated twice.

2.5 A closing bracket is missing after “stimulatory (36)”

2.7 Which test was used to test the normality of data distribution? This information should be added.

RESULTS

3.2 SATB1-AS1 or SATB1-as1? You need to be consistent with terminology.

3.2 “higher SATB1-AS1 expression was more strongly associated with tumor grade and lymphatic infiltration than lower SATB1-AS1 expression was, and lower SATB1-AS1 expression was more strongly associated with tumor grade and lymphatic infiltration.” This statement contradicts itself, so it is not clear what the authors meant to convey.

3.2 “We found that high SATB1-AS1 expression was associated with a favorable prognosis in OV patients.” Why is this mentioned in this section?

3.2 “we also found that SATB1-AS1 expression levels were independent of age, sex, and tumor grade.” Figure 2B clearly demonstrates that expression levels are significantly lower in stage III and IV tumors compared to stage I and II. Also, the text mentions tumor grade but the graph is annotated as tumor stage. These terms are not identical, so you should correct whichever one is wrong.

3.3 The categories of GO enrichment should be written in full form since they are mentioned the first time here.

3.5 Again, full forms of the tumors should be written since they are mentioned the first time here.

FIGURES

Figure 1: The resolution is too small and I cannot tell what is written on the axes or the R and p values on the graphs. The figure should be larger and of a higher resolution.

Figure 2B-E: The description states SATB1-AS1, but SATB1 is written on the y-axis of the graphs. This should be corrected.

Figure 3: The text should be larger, especially on 3B, and everything should be of a higher resolution.

Figure 4: The same as Figures 1 and 3, the text is too small, low resolution, everything is smushed together. In my opinion, this Figure, even when corrected, does not provide any valuable information and the data contained inside would be better suited for a supplementary table.

Figures 5 and 6: Same comment as Figures 1 and 3.

DISCUSSION

-“The optimal strategy for prolonging long-term survival and minimizing the side effects of treatment should be developed by a multidisciplinary team of thoracic surgeons” No need to write the same information twice.

-“However, cancer treatment is limited by both intratumor and intertumor factors. However,

cancer treatment is limited by intra- and intertumor heterogeneity” Same as above.

Reviewer #3: Review of manuscript by Wang et al. “Identification of the immune-associated enhancer RNA SATB1-AS1 as a novel biomarker for thymic cancer prognosis”

In the study by Wang et al., the authors performed a bioinformatics analysis using publicly available databases to explore the role of enhancer RNAs (eRNAs) as prognostic biomarkers in thymoma. The study successfully identified the immune-associated enhancer RNA SATB1-AS1 as a novel biomarker for thymic cancer prognosis.

This manuscript is well-written and easily understood. The introduction provides a clear and concise background, giving sufficient context for the study. The materials and methods section is well-structured and described in adequate detail. The manuscript can be accepted for publication after addressing the following comments.

Specific Comments:

1. Data Extraction:

o Please specify the date when the data was extracted from the TCGA databases and Xena.

2. Figure Quality and Assembly:

o Please provide details on how the figures were assembled.

o The quality of the figures is low, making it difficult to distinguish details. Please improve the resolution of Figures 1, 3, and 4. If the issue is due to the PDF format of the manuscript, this comment can be disregarded.

3. Discussion:

o There appears to be redundancy in the following sentence; please revise it for clarity: "However, cancer treatment is limited by both intratumor and intertumor factors. However, cancer treatment is limited by intra- and intertumor heterogeneity, which requires the study of mechanisms that work across patient characteristics to develop innovative therapies."

o Please include references and elaborate on the known information about SATB1-AS1 and its connection to other known tumors.

4. Conclusion:

o The following sentence appears incomplete; please revise for clarity and coherence: (4) Differential expression of SATB1-AS1 across cancers and its impact on survival.

After addressing these comments, the manuscript will be suitable for publication.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Identification of the immune-associated enhancer RNA SATB1-AS1 as a novel biomarker for thymic cancer prognosis

PONE-D-25-07416R1

Dear Dr. jiang,

We’re 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,

Benjamin Benzon, Ph.D., M.D.

Academic Editor

PLOS ONE

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