Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript provides a thorough and comprehensive review of malignant transformation in low-grade gliomas (LGGs). It primarily focuses on IDH-mutant subtypes and presents a detailed synthesis of multidimensional datasets to support the argument that transformation into more malignant and treatment-refractory phenotypes is a continuous process driven by environmental factors and selective pressures.

 

While the topic is timely and of interest, a major concern relates to the potential use of artificial intelligence (AI) in the preparation of this manuscript. Figures 1 and 2 appear to be generated using Google Gemini, and the overall writing style is consistent with large language model (LLM)-generated text. However, there is no disclosure of AI usage in the acknowledgments or elsewhere in the manuscript.

 

The authors should explicitly state whether AI tools were used in generating text, figures, or other content, and clearly delineate the extent of such use. Transparency in this regard is essential. Until this information is provided, the manuscript should not be considered for publication in Biomedicines.

Author Response

We thank the reviewer for raising this important point. We agree that transparency regarding the use of AI-assisted tools is essential.

Comments 1: This manuscript provides a thorough and comprehensive review of malignant transformation in low-grade gliomas (LGGs). It primarily focuses on IDH-mutant subtypes and presents a detailed synthesis of multidimensional datasets to support the argument that transformation into more malignant and treatment-refractory phenotypes is a continuous process driven by environmental factors and selective pressures.

While the topic is timely and of interest, a major concern relates to the potential use of artificial intelligence (AI) in the preparation of this manuscript. Figures 1 and 2 appear to be generated using Google Gemini, and the overall writing style is consistent with large language model (LLM)-generated text. However, there is no disclosure of AI usage in the acknowledgments or elsewhere in the manuscript.

The authors should explicitly state whether AI tools were used in generating text, figures, or other content, and clearly delineate the extent of such use. Transparency in this regard is essential. Until this information is provided, the manuscript should not be considered for publication in Biomedicines.

Response 1:  In the revised manuscript, we have now added an explicit Declaration on the Use of Generative AI. Specifically, generative AI tools were used in a limited and transparent manner during manuscript preparation. ChatGPT (OpenAI) was used for language editing, wording refinement, structural polishing, and drafting prompts for schematic figures. Gemini was used to generate draft schematic figures based on author-defined scientific content and layout. No AI tool was used to generate scientific conclusions, select or interpret the literature, analyze data, or make editorial decisions about the manuscript. All content was reviewed, revised, and verified by the authors, who take full responsibility for the accuracy and integrity of the submitted work.

Changes made in the manuscript: A Declaration on the Use of Generative AI has been added to the manuscript. We have added this statement to the revised manuscript to ensure full transparency in Line 656-665 in Page 16 before References.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this work, Wu et al. reviewed recent advances of the malignant transformation of lower-grade gliomas, focusing on the link between epigenetic constraints and evolutionary escape. The manuscript is interesting, but there are several issues that need to be addressed before the paper can be considered for publication. I recommend a major revision. My specific comments are as follows:

1. Important concepts such as epigenetic constraint and evolutionary escape are not defined clearly. I suggest the authors to add one more section to clearly describe these concepts, like how a "constraint" is measured, how to determine when "escape", the specific biomarkers/thresholds/experimental criteria to define these, etc, so that readers can have a clear idea of what epigenetic constraint and evolutionary escape mean. The same is for terms like "adaptive landscape" and "selection". What measurable parameters are used to define these concepts?

2. The different components of the framework are presented in a largely additive manner without integration. For example, Section 3 lists therapy, hypoxia, immune remodeling, and genomic instability as selective pressures, but it is unclear how these interact. Are these independent processes, or do they act sequentially or synergistically? A more integrated model would strengthen the manuscript.

3. The review heavily extrapolates findings from glioblastoma and other contexts to lower-grade gliomas. However, IDH-mutant LGGs are biologically distinct, and mechanisms derived from glioblastoma may not be directly applicable. The authors should be more cautious in generalizing these findings and clearly indicate when evidence is indirect.

4. The manuscript lacks critical appraisal of the cited literature. Most studies are presented as supporting the framework, but there is little discussion of limitations, conflicting findings, or alternative models. For example, the single-cell studies cited are treated as definitive, but these approaches have known biases (e.g. sampling bias, state annotation uncertainty, etc). A more balanced discussion is needed.

 

Author Response

We appreciate the reviewer’s recognition of the interest of this topic and agree that the manuscript would benefit from a clearer conceptual framework, stronger integration across sections, more cautious interpretation of indirect evidence, and a more balanced discussion of the limitations of the current literature. We have revised the manuscript accordingly.

Comments 1: Important concepts such as epigenetic constraint and evolutionary escape are not defined clearly. I suggest the authors to add one more section to clearly describe these concepts, like how a "constraint" is measured, how to determine when "escape", the specific biomarkers/thresholds/experimental criteria to define these, etc, so that readers can have a clear idea of what epigenetic constraint and evolutionary escape mean. The same is for terms like "adaptive landscape" and "selection". What measurable parameters are used to define these concepts?

Response 1: We thank the reviewer for this important suggestion. In the revised manuscript, we have added a new Section 2 (“Defining the core conceptual terms”), to clarify how the terms epigenetic constraint, evolutionary escape, selection, and adaptive landscape are used in this review. We now explicitly state that these are integrative conceptual frameworks rather than single experimentally measurable variables, while also linking them to observable biological correlates wherever possible. We further clarify that these concepts are best approximated through convergent evidence from histopathology, longitudinal molecular profiling, and single-cell/spatial analyses, rather than through fixed universal thresholds.

Changes made: Lines 147–176 in Page 4 and 5 as a new Section 2.

Comments 2: The different components of the framework are presented in a largely additive manner without integration. For example, Section 3 lists therapy, hypoxia, immune remodeling, and genomic instability as selective pressures, but it is unclear how these interact. Are these independent processes, or do they act sequentially or synergistically? A more integrated model would strengthen the manuscript.

Response: We agree with this point and have revised the manuscript to make the framework more integrated. In particular, we added a new integrative paragraph at the end of the selective-pressure section to explain that therapy, hypoxia/vascular stress, immune remodeling, and genomic or epigenomic instability are not isolated processes, but can act sequentially and synergistically through iterative feedback between tumor-intrinsic adaptation and ecosystem-level remodeling. This revised paragraph clarifies how these pressures interact across time and biological scale, rather than functioning as a simple additive list.
Changes made: Lines 309–320 in Page 7 at the end of Section 4.3.

Comments 3: The review heavily extrapolates findings from glioblastoma and other contexts to lower-grade gliomas. However, IDH-mutant LGGs are biologically distinct, and mechanisms derived from glioblastoma may not be directly applicable. The authors should be more cautious in generalizing these findings and clearly indicate when evidence is indirect.

Response: We fully agree and have revised the manuscript accordingly. First, we clarified in the Introduction that this review focuses primarily on adult-type IDH-mutant lower-grade gliomas, while selected evidence from glioblastoma is used only where it provides indirect mechanistic insight. Second, we revised the wording in the hypoxia/vascular stress section to avoid implying direct transferability of glioblastoma-derived mechanisms, and now explicitly describe such evidence as indirect mechanistic insight rather than direct evidence for LGG malignant transformation. Third, we added a cautionary statement noting that glioblastoma-derived vascular mechanisms should not be assumed to operate identically in lower-grade gliomas, and that direct validation in LGGs remains limited.
Changes made: Lines 250–253 in Page 6 in Section 4.2 and Lines 408–412 in Page 10 in Section 6.1.

Comments 4: The manuscript lacks critical appraisal of the cited literature. Most studies are presented as supporting the framework, but there is little discussion of limitations, conflicting findings, or alternative models. For example, the single-cell studies cited are treated as definitive, but these approaches have known biases (e.g. sampling bias, state annotation uncertainty, etc). A more balanced discussion is needed.

Response: We appreciate this important comment and agree that the original version did not sufficiently discuss methodological limitations and alternative interpretations. In response, we added a new subsection, Section 5.4 (“Methodological limitations and alternative interpretations”), in which we now discuss sampling bias, underrepresentation of spatially diffuse or treatment-altered regions, uncertainty in state annotation, and the dependence of inferred trajectories on computational frameworks. We also note that apparent state transitions may in some cases reflect shifts in the relative abundance of pre-existing populations rather than true lineage conversion. In addition, we now explicitly acknowledge that alternative models remain possible, including the selective expansion of pre-existing aggressive clones. The proposed framework is therefore presented as a working model rather than a definitive account of LGG malignant transformation.
Changes made: Lines 372–392 in Page 9 in Section 5.4 and Lines 632–634 in Page 15 in Section 9.

Reviewer 3 Report

Comments and Suggestions for Authors

The review article sounds good, but some modifications are recommended as follows:

1. At the beginning of the introduction, clarify how much does LGGs represent from overall gliomas, the incidence, or any mortality rate.
2. In introduction, add a short paragraph about any risk factors associated with the development of LGGs.
3. Check the abbreviation throughout the review. The full term must be clearly stated at their first mention. for example, in line 48, IDH mutation should clarify that is isocitrate dehydrogenase.
4. I suggest adding a subheading or paragraph how LGG differs from other gliomas.
5. In line 164, "The mutagenic consequences of TMZ are now well recognized in subsets of IDH-mutant glioma.". What do the authors mean by mutagenic consequences of TMZ. It should be clearer or more explanatory with at least one clinical evidence.
6. in line 173, 3.2 Hypoxia and vascular stress, this all subheading involves only one reference (9). The authors should cite more work relevant to this topic.
7. Overall in the heading of "Selective Pressures That Destabilize the Constrained State", the authors wrote a general speech without any specific biomarkers or signalling pathways for each discussed topic. For example, in Hypoxia and vascular stress, the authors could add some details about HIF, VEGF, ...etc. 
8. In line 221-224, recent single-cell studies have demonstrated that transformation is accompanied by shifts away from relatively differentiated glial-like states toward more proliferative, stem-like, developmentally plastic, and stress-adapted states [3], The authors stated that "single-cell studies", but there is only one reference (3)!.
9. Again like comment 6, only one reference -(3)- is not enough for such large paragraph in the subheading From lineage restriction to developmental plasticity.
10. In general, much more references and updated ones must be included since it is not logic that a review article cited only 19 references!!!
11. The authors should add a heading about which types exactly of epigenetic modifications happen in LGGs i.e. clarify whether that modifications are DNA methylation, histone modification, ...etc.
12. For table 1, add a suitable reference for each row.
13. The authors should provide a subheading about the current available treatment modalities for LGGs and discuss their limitations and also clarify hoe knowing of epigenetic modifications could improve the treatment and the response of the patients.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted in Red in the re-submitted files.

Comments 1: At the beginning of the introduction, clarify how much does LGGs represent from overall gliomas, the incidence, or any mortality rate.

Response 1: We thank the reviewer for this helpful suggestion. We have revised the opening part of the Introduction to include a brief epidemiologic background on lower-grade gliomas, including their place within the broader spectrum of diffuse gliomas and their clinical burden. This addition is intended to provide a clearer context for the relevance of malignant transformation in LGGs.

Changes made: Lines 59–68 in Page 2 in Introduction.

Comments 2: In introduction, add a short paragraph about any risk factors associated with the development of LGGs.

Response 2: We agree and have added a short paragraph in the Introduction summarizing currently recognized risk factors and epidemiologic considerations relevant to glioma development, while noting that well-established risk factors specific to LGGs remain limited.

Changes made: Lines 69–77 in Page 2 in Introduction.

Comments 3: Check the abbreviation throughout the review. The full term must be clearly stated at their first mention. for example, in line 48, IDH mutation should clarify that is isocitrate dehydrogenase.

Response 3:Thank you for pointing this out. We have carefully revised the manuscript to ensure that abbreviations are defined at first mention throughout the text, including clarification of IDH as isocitrate dehydrogenase.

Comments 4: I suggest adding a subheading or paragraph how LGG differs from other gliomas.

Response 4: We agree that this distinction should be made more explicit. In the revised manuscript, we added clarifying text in the Introduction to distinguish LGGs, particularly adult-type IDH-mutant tumors, from other glioma subtypes in terms of biology, clinical behavior, and progression patterns.

Changes made: Lines 78–87 in Page 2 in Introduction.

Comments 5: In line 164, "The mutagenic consequences of TMZ are now well recognized in subsets of IDH-mutant glioma." What do the authors mean by mutagenic consequences of TMZ. It should be clearer or more explanatory with at least one clinical evidence.

Response 5: We thank the reviewer for this important comment. We have revised this sentence to clarify that the “mutagenic consequences” of TMZ refer to treatment-associated mutational burden, mismatch repair-deficient hypermutation, and the emergence of recurrence patterns associated with poorer outcomes after high-grade transformation. We have also added clinical evidence to support this statement.

Changes made: Lines 238–244 in Page 6 in Section 4.1.

Comments 6: In line 173, 3.2 Hypoxia and vascular stress, this all subheading involves only one reference (9). The authors should cite more work relevant to this topic.

Response 6: We agree and have expanded this section by adding additional relevant references and by broadening the discussion of hypoxia- and vascular stress-related mechanisms in glioma progression in Page 7 in Section 4.2.

Comments 7: Overall in the heading of "Selective Pressures That Destabilize the Constrained State", the authors wrote a general speech without any specific biomarkers or signaling pathways for each discussed topic. For example, in Hypoxia and vascular stress, the authors could add some details about HIF, VEGF, ...etc.

Response 7: We appreciate this suggestion. In the revised manuscript, we have added more specific mechanistic detail to the section on hypoxia and vascular stress, including discussion of representative signaling pathways and mediators such as HIF-related adaptation and pro-angiogenic signaling. We have also revised other parts of Section 4 to make the discussion more mechanistically informative.

Changes made: Lines 261–271 in Page 7 in Section 4.2 and 238-244 in Page 6 in Section 4.1.

Comments 8: In line 221-224, recent single-cell studies have demonstrated that transformation is accompanied by shifts away from relatively differentiated glial-like states toward more proliferative, stem-like, developmentally plastic, and stress-adapted states [3], The authors stated that "single-cell studies", but there is only one reference (3)!.

Response 8: We agree. We have revised the wording and added additional relevant references to support this statement more appropriately.

Changes made: Lines 316-322 in Page 8 in Section 5.1.

Comments 9: Again like comment 6, only one reference -(3)- is not enough for such large paragraph in the subheading From lineage restriction to developmental plasticity.

Response 9: We appreciate this point and have added further references in this section to better support the discussion and to avoid overreliance on a single study.

Changes made: Lines 316-322 in Page 8 in Section 5.1.

Comments 10: In general, much more references and updated ones must be included since it is not logic that a review article cited only 19 references!!!

Response 10: We agree that the original reference list was too limited for the scope of the review. In the revised version, we have substantially expanded the reference list to 33 and incorporated additional recent and relevant literature throughout the manuscript.

Comments 11: The authors should add a heading about which types exactly of epigenetic modifications happen in LGGs i.e. clarify whether that modifications are DNA methylation, histone modification, ...etc.

Response 11: We thank the reviewer for this suggestion. In the revised manuscript, we added text clarifying the major types of epigenetic dysregulation relevant to LGGs, including DNA methylation, histone-related regulation, and broader chromatin-associated changes.

Changes made: Lines 178-188 in Page 5 in Section 3.1.

Comments 12: For table 1, add a suitable reference for each row.

Response 12: We agree and have revised Table 1 accordingly by adding appropriate supporting references for each row.

Comments 13: The authors should provide a subheading about the current available treatment modalities for LGGs and discuss their limitations and also clarify hoe knowing of epigenetic modifications could improve the treatment and the response of the patients.

Response 13: We thank the reviewer for this important suggestion. In the revised manuscript, we added a dedicated discussion of current treatment modalities for LGGs and their limitations, and further clarified how understanding epigenetic dysregulation may inform treatment strategies, patient stratification, and therapeutic response.

Changes made: Lines 532-546 in Page 13-14 in Section 8.1.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I do not have further comments.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed my comments. I recommend the paper for acceptance.

Reviewer 3 Report

Comments and Suggestions for Authors

All comments have been covered and addressed as requested.