The Development of Novel Treatment Strategies for Rhabdomyosarcoma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review provides an extensive overview of current and emerging risk-classification systems and therapeutic strategies for rhabdomyosarcoma. It outlines traditional risk factors such as histologic subtype, age, site, and metastasis, summarizes the evolution of standard treatments based on risk stratification, and discusses the potential of novel molecular targets such as MYOD1 mutations, RAS/MAPK, ALK, NTRK, FGFR, and MSI-high status. The paper emphasizes the need for personalized, genomically informed treatment approaches, especially for refractory or high-risk RMS. 

This is a well-researched and informative review that effectively summarizes recent progress in RMS classification and treatment. It would serve as a valuable reference for oncologists and translational scientists.

However, the manuscript would benefit from the following: 

• Including a table or scheme comparing traditional versus molecular risk classification approaches would help readers in gaining information

• immunotherapy section focuses solely on MSI-high RMS. A broader consideration ( checkpoint inhibitors, CAR-T developments) would strengthen the review.

• It would be relevant to further highlight that gene-fusion events beyond the classic PAX3/FOXO1 or MYOD1 mutations are relevant. For instance, it has been recently reported a novel RAB3IP–HMGA2 fusion in adult head-and-neck RMS, illustrating that gene-fusion diversity extends beyond the pediatric setting (10.1111/odi.14036) and suggesting the importance of anatomical site and age in the underlying molecular biology. Morover, reports of additional rare fusion events such as EP300–VGLL3, NCOA2–MEIS1, CAV1–MET and HMGA2–NEGR1 in spindle cell rhabdomyosarcoma further illustrate the expanding spectrum of fusion-driven RMS subtypes and the need for broad molecular testing (10.1002/gcc.22978).

Author Response

Including a table or scheme comparing traditional versus molecular risk classification approaches would help readers in gaining information
immunotherapy section focuses solely on MSI-high RMS. A broader consideration ( checkpoint inhibitors, CAR-T developments) would strengthen the review.

→ Regarding MSI-H tumors, it was noted that aside from immune checkpoint inhibitors, no other novel immunotherapies have yet been established as specific treatments, and the development of other immunotherapies is desired.

It would be relevant to further highlight that gene-fusion events beyond the classic PAX3/FOXO1 or MYOD1 mutations are relevant. For instance, it has been recently reported a novel RAB3IP–HMGA2 fusion in adult head-and-neck RMS, illustrating that gene-fusion diversity extends beyond the pediatric setting (10.1111/odi.14036) and suggesting the importance of anatomical site and age in the underlying molecular biology. Morover, reports of additional rare fusion events such as EP300–VGLL3, NCOA2–MEIS1, CAV1–MET and HMGA2–NEGR1 in spindle cell rhabdomyosarcoma further illustrate the expanding spectrum of fusion-driven RMS subtypes and the need for broad molecular testing (10.1002/gcc.22978).

→ We have added references to the literature you introduced and included mention of new fusion genes that may serve as candidate prognostic factors.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript offers a clear, well-structured overview of current and emerging approaches to risk stratification and treatment optimization in rhabdomyosarcoma (RMS). The topic is timely and relevant to Cancers, and the author effectively synthesizes a large body of clinical literature. The discussion of risk factors, staging systems, and established chemotherapy regimens is strong, and the manuscript provides valuable updates on targeted therapies currently under investigation.

 

However, several areas require deeper mechanistic discussion, improved integration of molecular insights, and more critical analysis to meet the expected scholarly standard. With substantial revision and expansion in key sections, this review has the potential to serve as an authoritative resource for clinicians and translational researchers in pediatric and AYA sarcomas.

 

Major Comments

 

1. Strengthen mechanistic and conceptual synthesis

While the manuscript is descriptive and comprehensive, several sections should be expanded to provide deeper insight into why certain molecular alterations shape RMS biology and risk. Examples include:

• The mechanistic consequences of FOXO1 fusion–driven transcriptional reprogramming, including enhancer hijacking, super-enhancer activation, and 3D chromatin remodeling.
• The distinct developmental and transcriptional programs underlying embryonal versus alveolar RMS.
• The biological rationale for MYOD1-mutant RMS being exceptionally aggressive.

To support these themes, the authors may consider incorporating literature on chromatin boundaries and 3D genome regulation, such as PMID: 41151055 , which provide relevant mechanistic frameworks for how boundary elements and chromatin architecture influence oncogene activation and promoter insulation.

2. Expand molecular and epigenetic landscape of RMS

The review would benefit from a more detailed synthesis of:

• Epigenetic dysregulation (PRC2, histone modification patterns) in RMS.
• The role of Polycomb-mediated repression in maintaining differentiation arrest.
• How chromatin accessibility differs between fusion-positive and fusion-negative RMS.

3. The authors should Expand discussion of transcriptomic heterogeneity, immune biology, and emerging therapies. Gene-expression variability across RMS subtypes (fusion-positive vs fusion-negative, MYOD1-mutant, FGFR4-mutant) warrants further discussion. Methodological work such as PMID: 26732976 which compares RNA-seq normalization and differential-expression approaches may help contextualize these differences.

 

4. Pleomorphic RMS is biologically distinct from pediatric RMS and follows different treatment paradigms. The authors should clearly delineate key molecular features unique to pleomorphic RMS.

 

5. The authors should expand the discussion on survivorship, long-term toxicities, and late effects.

Author Response

Major Comments

1 Strengthen mechanistic and conceptual synthesis
While the manuscript is descriptive and comprehensive, several sections should be expanded to provide deeper insight into why certain molecular alterations shape RMS biology and risk. Examples include:

The mechanistic consequences of FOXO1 fusion–driven transcriptional reprogramming, including enhancer hijacking, super-enhancer activation, and 3D chromatin remodeling.
The distinct developmental and transcriptional programs underlying embryonal versus alveolar RMS.

→ I added information on the function of the FOXO1 fusion gene and its role in cancer development.

The biological rationale for MYOD1-mutant RMS being exceptionally aggressive.

To support these themes, the authors may consider incorporating literature on chromatin boundaries and 3D genome regulation, such as PMID: 41151055 , which provide relevant mechanistic frameworks for how boundary elements and chromatin architecture influence oncogene activation and promoter insulation.

→ Regarding the poor prognosis associated with MYOD1 mutations, I have added a discussion based on pathological findings.

2 Expand molecular and epigenetic landscape of RMS
The review would benefit from a more detailed synthesis of:

Epigenetic dysregulation (PRC2, histone modification patterns) in RMS.
The role of Polycomb-mediated repression in maintaining differentiation arrest.
How chromatin accessibility differs between fusion-positive and fusion-negative RMS.

→ A section specifically addressing EZH2 was included to explain it as an epigenetic growth factor for RMS (section 4.3.7).

3 The authors should Expand discussion of transcriptomic heterogeneity, immune biology, and emerging therapies. Gene-expression variability across RMS subtypes (fusion-positive vs fusion-negative, MYOD1-mutant, FGFR4-mutant) warrants further discussion. Methodological work such as PMID: 26732976 which compares RNA-seq normalization and differential-expression approaches may help contextualize these differences.

→ I added the escription About transcriptomic heterogeneity in the section 4.2.
 
4 Pleomorphic RMS is biologically distinct from pediatric RMS and follows different treatment paradigms. The authors should clearly delineate key molecular features unique to pleomorphic RMS.
 
→ Section 2.1 has been updated to note that pleomorphic RMS is molecularly similar to UPS.

5 The authors should expand the discussion on survivorship, long-term toxicities, and late effects.

→ The description about importance of survivorship has been added to the conclusion section.

Reviewer 3 Report

Comments and Suggestions for Authors

General Comment

Overall, the manuscript is well structured and clearly written. It provides a comprehensive overview of RMS, its risk classification, and new therapeutic strategies. The logical flow from epidemiology to molecular targets is consistent, and the inclusion of recent literature is commendable.

However, several sections are rather descriptive and could benefit from more in-depth scientific analysis and discussion to elevate the review from a summary to a critical synthesis. In particular, the sections dealing with molecular subtypes, new biomarkers, and targeted therapies would benefit from additional context and interpretation.

Major Comments

1. Section 2.1 – Pathological subtypes
   The authors mention embryonal, alveolar, spindle cell/sclerosing, and pleomorphic RMS. However, the genomic discussion focuses only on eRMS and aRMS.
   Please include a brief description of the genomic landscape of pleomorphic RMS and spindle cell/sclerosing RMS, as these have distinct molecular features and prognostic implications.
2. Section 2.2 – Patient age
   While age-related prognosis is discussed, spindle cell/sclerosing RMS is not mentioned.
   Clarify whether spindle cell/sclerosing RMS shows any age-specific presentation or outcome differences, as this subtype may occur in both pediatric and adult populations.
3. Sections 2.3–2.4 – Primary lesion and risk stratification
   It would be helpful to clarify whether prognosis differs when lesions arise at the same anatomical site but belong to different subtypes (e.g., alveolar vs. embryonal in the head and neck region).
   Adding a comparative table or short commentary on this point would enhance the clinical relevance of the section.
4. Section 3.2 – Intermediate-risk patients
   The section could be strengthened by explaining why vinorelbine plus low-dose cyclophosphamide improved prognosis in the RMS2005 study.
   A short explanation on the rationale would help readers understand the biological basis of this therapeutic improvement.
5. Section 3.3 – High-risk/metastatic, recurrent cases
   This section is concise but underdeveloped.
   Consider expanding on current investigational strategies for relapsed RMS, such as high-dose chemotherapy with stem cell rescue, targeted agents, or immunotherapeutic approaches. Including recent clinical trial identifiers would add depth.
6. Section 4.1 – Novel risk factors: MYOD1 mutation
   The paragraph correctly highlights MYOD1 as a poor-prognosis factor but lacks an explanation of its biological function.
   Briefly describe the role of MYOD1 as a myogenic transcription factor and how the L122R mutation alters its transcriptional activity and promotes aggressiveness.
7. Section 4.3.2 – ALK
   It is well known that ALK overexpression, copy gain, and mutation occur frequently in fusion-positive aRMS.
   Emphasize this correlation, as it may identify a biologically distinct subpopulation relevant for future clinical trials. It should also be clarified that ALK does not cause PAX3/7–FOXO1 fusions but may be transcriptionally upregulated downstream of them.
8. Section 4.3.5 – MSI-high status and pleomorphic RMS
   This is an excellent and original addition, but its impact would be greater if the pleomorphic subtype were described earlier.
   Expand the early sections (2.1–2.2) to better define the clinical and molecular features of pleomorphic RMS, providing continuity with the discussion of MSI-high tumors and Lynch syndrome.
9. Finally, I would strongly encourage the authors to consider adding a short dedicated paragraph on two emerging biological dimensions that are increasingly relevant in RMS.

First, microRNAs are rapidly gaining attention as regulatory drivers of tumor progression, treatment resistance, and metastatic potential in RMS, and several studies have begun to highlight their possible translational value.

Second, growing evidence points to the relevance of the PRC2 epigenetic complex—particularly EZH2—in sustaining oncogenic programs in RMS (10.1186/s13148-021-01147-w).

Integrating these aspects would further strengthen the biological and translational completeness of the review.

 

Minor Comment

1. Introduction – Terminology consistency:

The frequent shift between “soft tissue sarcomas (STS)” and “rhabdomyosarcoma (RMS)” may confuse readers.

2. Figure 2

The treatment schedule figure is clear and very effective. Consider maintaining this schematic style for other visual summaries.

3. Line 210

…ing survivors' quality of life of [41]….

There’s an extra “of” before citation [41]

4. Line 293

…RAS mutations, and is is thus possible that…

There’s an extra “is” between “and” – “thus”

 

 

Author Response

Major Comments

Section 2.1 – Pathological subtypes
The authors mention embryonal, alveolar, spindle cell/sclerosing, and pleomorphic RMS. However, the genomic discussion focuses only on eRMS and aRMS.
Please include a brief description of the genomic landscape of pleomorphic RMS and spindle cell/sclerosing RMS, as these have distinct molecular features and prognostic implications.

→ Characteristics of the pleomorphic RMS and spindle cell/sclerosing RMS based on the WHO classification are described.

Section 2.2 – Patient age
While age-related prognosis is discussed, spindle cell/sclerosing RMS is not mentioned.
Clarify whether spindle cell/sclerosing RMS shows any age-specific presentation or outcome differences, as this subtype may occur in both pediatric and adult populations.

→ It was added that in spindle cell/sclerosing RMS, the prognosis is favorable in pediatric-onset cases but poor in adult-onset cases.

Sections 2.3–2.4 – Primary lesion and risk stratification
It would be helpful to clarify whether prognosis differs when lesions arise at the same anatomical site but belong to different subtypes (e.g., alveolar vs. embryonal in the head and neck region).
Adding a comparative table or short commentary on this point would enhance the clinical relevance of the section.

→ I reviewed the literature cited in this paper and other pooled data analyses. However, I found no studies that quantitatively assessed how much differences in histological type affect prognosis differences when the primary site is the same. Therefore, I noted that no established information is available regarding this matter.

Section 3.2 – Intermediate-risk patients
The section could be strengthened by explaining why vinorelbine plus low-dose cyclophosphamide improved prognosis in the RMS2005 study.
A short explanation on the rationale would help readers understand the biological basis of this therapeutic improvement.

→ It is speculated that this improvement in prognosis may have resulted from controlling “tumor cells in a state close to dormancy” following standard treatment through the continuous administration of low-dose anticancer drugs. 以上の考察を追記した。

Section 3.3 – High-risk/metastatic, recurrent cases
This section is concise but underdeveloped.
Consider expanding on current investigational strategies for relapsed RMS, such as high-dose chemotherapy with stem cell rescue, targeted agents, or immunotherapeutic approaches. Including recent clinical trial identifiers would add depth.

→ An update has been added regarding the current status of high-dose chemotherapy. An update has been added stating that molecularly targeted therapy will be discussed in Section 4.

Section 4.1 – Novel risk factors: MYOD1 mutation
The paragraph correctly highlights MYOD1 as a poor-prognosis factor but lacks an explanation of its biological function.
Briefly describe the role of MYOD1 as a myogenic transcription factor and how the L122R mutation alters its transcriptional activity and promotes aggressiveness.

→ Additional considerations based on pathological findings have been added regarding the fundamental background for the poor prognosis associated with MYOD1 mutations.

Section 4.3.2 – ALK
It is well known that ALK overexpression, copy gain, and mutation occur frequently in fusion-positive aRMS.
Emphasize this correlation, as it may identify a biologically distinct subpopulation relevant for future clinical trials. It should also be clarified that ALK does not cause PAX3/7–FOXO1 fusions but may be transcriptionally upregulated downstream of them.

→ Added description regarding the background for ALK positivity in PAX3/7-FOXO1 fusion gene-positive cases.

Section 4.3.5 – MSI-high status and pleomorphic RMS
This is an excellent and original addition, but its impact would be greater if the pleomorphic subtype were described earlier.
Expand the early sections (2.1–2.2) to better define the clinical and molecular features of pleomorphic RMS, providing continuity with the discussion of MSI-high tumors and Lynch syndrome.

→ In sections 2.1-2.2, we noted that pleomorphic RMS is pathologically similar to UPS. Based on this, we stated that pleomorphic RMS can be observed in Lynch Syndrome in a form similar to UPS.

Finally, I would strongly encourage the authors to consider adding a short dedicated paragraph on two emerging biological dimensions that are increasingly relevant in RMS.

First, microRNAs are rapidly gaining attention as regulatory drivers of tumor progression, treatment resistance, and metastatic potential in RMS, and several studies have begun to highlight their possible translational value.

Second, growing evidence points to the relevance of the PRC2 epigenetic complex—particularly EZH2—in sustaining oncogenic programs in RMS (10.1186/s13148-021-01147-w).

→ A new section 4.3.7 has been added to explain the significance of EZH2 in RMS. It also mentions the possibility that microRNA may be a driver of RMS.

Minor Comment

Introduction – Terminology consistency:
The frequent shift between “soft tissue sarcomas (STS)” and “rhabdomyosarcoma (RMS)” may confuse readers.

→ Following the feedback, the notation has been revised to minimize the need to switch between STS and RMS.

Figure 2
The treatment schedule figure is clear and very effective. Consider maintaining this schematic style for other visual summaries.

→ I am honored by your kind words. In addition to the figures, I made a conscious effort to create clear and easy-to-read tables for the tables added in this revision, aiming to make the intended meaning easier to grasp.

Line 210
…ing survivors' quality of life of [41]….

There’s an extra “of” before citation [41]

→ I omitted the extra "of".

Line 293
…RAS mutations, and is is thus possible that…

There’s an extra “is” between “and” – “thus”

→ I omitted the extra "is".

Reviewer 4 Report

Comments and Suggestions for Authors

This is a relatively well put-together overview of the current landscape of rhabdomyosarcoma, but there are several issues which need to be addressed:

1. The title is misleading as the author does not develop a novel risk-classification; the article is more a review and future targeted therapies
2. The author should also describe current alternative risk classification eg in the European FaR-RMS study
3. The EpSSG studies do not use VAC, but use ifosfamide rather than cyclophosphamide as the alkylating agent in the intensive cycles
4. The author should describe what current questions are being evaluated in COG and European trials eg integration of new agents/targeted therapy, extending duration of maintenance, radiotherapy dosing
5. MSI-High is extremely uncommon in RMS, and this section should be remved. A sentence describing determining MSI/TMB as part of CGP would suffice;
6. The author should describe whether there are any targeted therapies to be introduced to all patients at diagnosis/relapse versus personalised targeted therapy following CGP
7. There are other mutations seen more frequently than NTRK in RMS eg CDK4/CCND2A so the author should consider including a discussion of these

Author Response

1 The title is misleading as the author does not develop a novel risk-classification; the article is more a review and future targeted therapies

Under your advice, I deleted the word "risk-classification" from the title.

2 The author should also describe current alternative risk classification eg in the European FaR-RMS study

→ Added the risk classification from the FaR-RMS study and included the table.

3 The EpSSG studies do not use VAC, but use ifosfamide rather than cyclophosphamide as the alkylating agent in the intensive cycles

→ The presence of treatment protocol using IFM as an alkylating agent has been documented.

4 The author should describe what current questions are being evaluated in COG and European trials eg integration of new agents/targeted therapy, extending duration of maintenance, radiotherapy dosing

→ Information regarding ongoing clinical trials and new drug candidates has been added to Section 3-4.

5 MSI-High is extremely uncommon in RMS, and this section should be remved. A sentence describing determining MSI/TMB as part of CGP would suffice;

→ Other reviewers instructed me to add additional information regarding MSI-High, so I decided to retain this section without deletion.

6 The author should describe whether there are any targeted therapies to be introduced to all patients at diagnosis/relapse versus personalised targeted therapy following CGP

→ In addition to adding challenges in current treatment development to Section 3, Section 4.2 mentions the necessity of developing new treatments based on CGP.

7 There are other mutations seen more frequently than NTRK in RMS eg CDK4/CCND2A so the author should consider including a discussion of These

→ A section explaining CDK4 and cyclin-dependent kinase pathways has been added to 4.3.6.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The author has addressed the major comments by expanding the relevant sections of the manuscript and adding discussion on FOXO1 fusion biology, MYOD1-mutant rhabdomyosarcoma, epigenetic regulation, transcriptomic heterogeneity, pleomorphic RMS, and survivorship. While the specific references suggested by the reviewer were not incorporated, the conceptual issues raised have been acknowledged and partially addressed through additional explanatory text. Overall, the revisions improve the scope and completeness of the review, though further integration of mechanistic literature could strengthen the synthesis.

Author Response

Thank you for your comments. Your feedback helped me improve the paper. I apologize that I was unable to add the suggested references to the citations due to the balance with the original text's arguments.

Reviewer 3 Report

Comments and Suggestions for Authors

The author has responded exhaustively to the comments. As far as I am concerned, it is ready for publication. 

Author Response

Thank you for checking the revised manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review this evised manuscript. On the whole, it reads better and is more comprehensive. 

One comment:

Line 261 - EpSSG added doxorubicin to backbone of VAI not VAC

 

Author Response

Thank you for pointing out the error. I corrected the error.