Subtype-Dependent Expression Patterns of Core Hippo Pathway Components in Thymic Epithelial Tumors (TETs): An RT-qPCR Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a transcript-level analysis of key Hippo signaling pathway components across thymic epithelial tumor subtypes using RT-qPCR on FFPE samples and relates these findings to previously reported immunohistochemical observations. The topic is relevant given the limited molecular characterization of thymic epithelial tumors and the need for pathway-based biomarkers. The experimental workflow is technically sound and adheres to reporting standards. However, the study is largely descriptive, limited by small subtype-specific cohorts, and occasionally overinterprets mRNA-level findings as pathway deregulation. Stronger contextualization within the existing molecular literature, clearer discussion of biological limitations, and a more cautious framing of translational implications are required.

1. The subtype-specific sample sizes are small, which substantially limits statistical power. While this is understandable for rare tumors, the manuscript should more clearly emphasize the exploratory nature of subtype-level conclusions and avoid overgeneralization.
2. The exclusive reliance on bulk RT-qPCR from FFPE tissue raises concerns about cellular heterogeneity, particularly in thymic tumors where epithelial cells are intermingled with thymocytes and stromal components. This limitation should be more explicitly discussed when interpreting differential expression.
3. The manuscript frequently refers to “Hippo pathway deregulation” based solely on transcript-level changes. Given that Hippo signaling is predominantly regulated post-translationally, these statements should be tempered, and conclusions reframed as transcriptional associations rather than functional pathway activation or suppression.
4. Discordance between transcript expression and previously reported protein-level findings is acknowledged but not sufficiently explored. A more critical discussion of possible biological and technical explanations would strengthen the interpretation.
5. The integration of the present findings with known genomic features of thymic epithelial tumors (e.g., established molecular subgroups and driver alterations) is limited. Explicitly positioning the Hippo pathway data within this broader molecular framework would enhance relevance.
6. Claims regarding biomarker or therapeutic potential are premature based on the current data. The authors should clarify whether diagnostic, prognostic, or predictive utility is being proposed and what additional validation would be required.
7. The Discussion section is comprehensive but somewhat repetitive and could be streamlined to focus more clearly on the novel contributions of this study rather than reiterating general Hippo pathway biology.
8. The manuscript would benefit from citing additional integrative and pathway-focused molecular studies in thymic epithelial tumors and related epithelial malignancies to better contextualize the findings. In particular, recent comprehensive molecular and systems-level analyses of thymic tumors and Hippo/YAP signaling—by independent groups active in this area—would strengthen the Discussion and support balanced interpretation.
9. Figures are generally clear and legible, but some multi-panel figures are dense. Simplifying legends and reducing redundancy between text and figures would improve clarity.
10. Minor issues include typographical inconsistencies and overly detailed figure captions that repeat information already provided in the Methods section.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The present work is based in a very exhaustive analysis and validation of several different parameters in order to evaluate the expression profile of various transcripts asocciated with the Hippo pathway in thymic epithelial tumors (TETs). Authors analyzed a modest number of tissues by RT-PCR, but considering the low frequency of this type of cancer is accepted.

The manuscript show interesting data that can help to understand the interplay between different components of this pathways.

At present manuscript requires minor revision.

1) Introduction, a general explanation of Hippo pathway and its role in normal cells is required in order that reader can associate the several proteins that participate in this pathway.

2) M&M. The protocol for IHC assay is required, antigen retrieval, antibodies dilution and were the different tissues validated by one pathologist or at least two independent pathologist? The same for the case of IHC score.

3) Results. Add a graph (pie chart) that resume the sub cellular localization of the different proteins analyzed, in order to get a global view of the distribution.

4) Discussion: Add a preliminary image emphasizing the low and high expression levels of the mRNAs and/or proteins studied in this work adding a brief paragraph resuming these findings

After these minor changes are made, manuscript is ready for a new final revision

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors The Authors concluded that TETs exhibit subtype-dependent Hippo pathway alterations, characterized by enhanced YAP1–TEAD4 transcriptional output and progressive reduction of the MST1/LATS1 kinase module, most pronounced in TC. These transcript-level patterns support the potential of Hippo-based biomarkers and may guide future therapeutic strategies. While the aim of the study is valid and clinically important, the extend of experimental data is limited.   Specific comments: 1. The number of samples is low considering the aim and the extend of the study. The Authors should discuss the limiations of their study in this respect. 2. Gene names should be consistently written in italics. 3. To substantiate the conclusions, the Authors should add the gene expression levels from publicly available datasets. If necessary, including the subtype of tumor.   Discussion is valid and sound, the references are up to date. The conclusions are partially supported by the results, and the enrichment of the result section as described above is expected to improve the quality of the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors!

 

Currently, the study of epithelial thymic tumors is an incredibly relevant area due to their low incidence and aggressive nature. However, the complexity of diagnosis limits treatment options for this tumor. The authors have accurately noted that the Hippo signaling pathway plays a key role in the regulation of cell growth, apoptosis, and tissue autoregulation. Disruptions in the activity of this pathway are associated with carcinogenesis and metastasis, making it a potential therapeutic target. Undoubtedly, studying the expression profile (as one of the simple yet reliable methods of analysis) allows us to identify molecular markers for prognosis and evaluation of therapy effectiveness.

 

In this paper, the authors provide a detailed analysis of the expression of Hippo signaling pathway genes depending on tumor subtypes.

 

The text is well structured, the terminology is clear, and the conclusions follow logically from the presented results. Given that the authors have a similar publication planned for 2025 (Elm, L., Gerlitz, N., Hochholzer, A., Papadopoulos, T., & Levidou, G. (2025). Hippo Pathway Dysregulation in Thymic Epithelial Tumors (TETs): Associations with Clinicopathological Features and Patients' Prognosis. International Journal of Molecular Sciences, 26(13), 5938), this work is a logical continuation of the study. Overall, the manuscript leaves a positive impression; however, the authors should nevertheless note the following comments.

 

Section "Materials and Methods":

Overall, the chapter should be shortened and made shorter.

 

In particular, it is recommended to retain the heading "2.4. One-Step RT-qPCR Workflow" (line 142) and remove the remaining subheadings: 2.4.1, 2.4.2, etc. Since these sections describe the same PCR reaction process, such a detailed description is unnecessary. Reducing the text size will not affect the essence and results of the article, but will make the material easier to understand.

 

Line 246 - The author should explain: why were these specific reference genes chosen? Are they specific for thymus tumors? β-actin, GAPDH, rpl11, and others can be used as standard reference genes in RT-PCR.

 

Section "Results":

The authors describe the obtained results well and in a structured manner, allowing for easy comprehension of the data.

 

Line 305 - section "3.1 qPCR assay performance and normalization strategy" pertains more to materials and methods. It is recommended to remove it from the results and begin the description of the obtained data with section "3.2 Relative Expression of Hippo Pathway Genes."

 

Line 488—item 3.3. Consolidated Overview of the Relative Expression Results of Hippo Pathway Genes—is recommended to be moved to the appendix, as it contains extensive tabular data. However, this is at the discretion of the authors.

 

The "Discussion" section is written quite comprehensively and logically. The technical limitations of this work are also described. There are no comments.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have provided a thorough, well-structured, and largely satisfactory point-by-point response to all reviewer comments. The revisions appropriately address the key concerns raised, particularly regarding the limited subtype-specific sample sizes, the exploratory nature of subtype-level analyses, and the need to temper conclusions related to Hippo pathway deregulation. Authors have strengthened the manuscript by (i) clearly emphasizing the hypothesis-generating character of the study, (ii) expanding the discussion of limitations related to bulk RT-qPCR from FFPE tissue and cellular heterogeneity, (iii) reframing pathway-level interpretations as transcript-level associations rather than functional activation, and (iv) integrating the findings more explicitly within the broader molecular framework of thymic epithelial tumors, including TCGA-based contextualization. Claims regarding biomarker and therapeutic relevance have been appropriately moderated and clarified, and the Discussion has been streamlined to focus on the novel contributions of the study.