Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

These authors have collected a lot of information with conventional review process. This reviewer could not clearly feel the uniqueness of their review. However, in most subsection, these authors provide a short summary, which is good. A few specific comments are blow.

 

1. In the 2.3 Fruquintinib subsection, these authors may also add a short summary at the end.

 

2. Section 5 should have a separate short paragraph summary at the end as well – may be, try to revise the part of “treatment selection must ….. ….. continues to evolve” and then have a separate short paragraph summary at the end.

 

3. The manuscript at the end (above Refs, usually should be at the end after refs) listed 4 figures and 3 Tables. However, this reviewer could not see any test description or mention any one of these figures or table throughout the description of the manuscript tetxt. What is the goal of these listed figures and table? This is a very bizarre situation.

Author Response

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments. We appreciate the positive feedback regarding the inclusion of subsection summaries. We have carefully revised the manuscript in response to all comments, as detailed below.

Comment 1
In the 2.3 Fruquintinib subsection, these authors may also add a short summary at the end.

Response 1
Thank you for this helpful suggestion. We agree with the reviewer’s comment.
Accordingly, we have added a concise summary sentence at the end of the Fruquintinib subsection.
This revision has been incorporated in Section 2.4, page 5, lines 197—200, as follows:
" These findings establish fruquintinib as a valuable salvage-line option for mCRC, offering consistent survival benefits across diverse populations regardless of race, biomarker status, or prior treatment history, although careful toxicity monitoring remains essential."
This addition improves the clarity and consistency of the subsection by aligning its structure with that of the other subsections.


Comment 2
Section 5 should have a separate short paragraph summary at the end as well – may be, try to revise the part of “treatment selection must ….. ….. continues to evolve” and then have a separate short paragraph summary at the end.

Response 2
We appreciate this important suggestion and agree with the reviewer.
In response, we have revised Section 5 by adding a separate paragraph immediately before the final paragraph to better highlight the key clinical considerations in treatment selection.
The added text has been incorporated in Section 5, page 15, lines 500–508, as follows:
"Treatment selection should be guided by Eastern Cooperative Oncology Group performance status (ECOG PS), treatment goals, and toxicity profile. In patients with PS 0–1 requiring tumor shrinkage, FTD-TPI plus bevacizumab may be preferred. For patients prioritizing disease control, or those intolerant of hematologic toxicity, regorafenib or fruquintinib may be more appropriate. Prior treatment-related adverse events should also be systematically considered when determining treatment sequencing; for instance, prior proteinuria has been reported to increase the risk of subsequent proteinuria [151], underscoring the importance of a comprehensive toxicity history in clinical practice."
Additionally, the original concluding paragraph has been slightly refined to enhance clarity and flow, while preserving the overall narrative structure.


Comment 3
The manuscript at the end (above Refs, usually should be at the end after refs) listed 4 figures and 3 Tables. However, this reviewer could not see any test description or mention any one of these figures or table throughout the description of the manuscript tetxt. What is the goal of these listed figures and table? This is a very bizarre situation.

Response 3
We sincerely thank the reviewer for pointing out this important issue and apologize for the lack of clarity in the original submission.
In the revised manuscript, we have addressed this concern by integrating all figures into the main text at appropriate locations and explicitly citing them within the relevant sections. These figures present treatment algorithms for molecularly guided therapies based on specific genomic alterations, including RAS wild-type status, HER2 amplification, KRAS G12C mutation, and BRAF V600E mutation, with additional explanations provided to clarify their clinical relevance.
Furthermore, the tables have been reorganized to clearly summarize the key clinical trials and are now structured into the following categories: (1) Conventional Salvage-Line Therapies in mCRC; (2) Contemporary Salvage-Line Therapies in mCRC (Anti-EGFR mAb Rechallenge Trials); (3) Contemporary Salvage-Line Therapies in mCRC (Novel Targeted Agent Trials); and (4) Strategies for Optimal Treatment Sequencing in mCRC.
All figures and tables are now explicitly cited and appropriately contextualized within the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

This review offers a thorough examination of the current salvage-line landscape for mCRC, and suggests combined strategies to enhance therapeutic results. The review is relevant and useful for practicing oncologists.

My comments and suggestions.

1. The title of the MS does not fully correspond to its content. The MS does not give proportional attention to “The Role and Clinical Significance of Circulating Tumor DNA”. Essentially, all information on liquid biopsy is presented in subsection 4.1. Only Figure 1 mentions circulating tumor DNA. It would be desirable to describe in more detail the methodological aspects of determining tumor DNA, its dynamic indicators, and its prognostic significance.

2. It is not entirely clear what is meant by the term 4.“Biomarker-Driven Therapies”: the identification of a mutation, a marker, or the identification of circulating tumor DNA.

3. It would be more logical to place the columns “Treatment regimen and treatment line” on the left side of the tables.

Author Response

We sincerely thank the reviewer for the positive evaluation of our manuscript and for the constructive and insightful comments. We have carefully revised the manuscript in response to all suggestions, as detailed below.

Comment 1
The title of the MS does not fully correspond to its content. The MS does not give proportional attention to “The Role and Clinical Significance of Circulating Tumor DNA.” Essentially, all information on liquid biopsy is presented in subsection 4.1. Only Figure 1 mentions circulating tumor DNA. It would be desirable to describe in more detail the methodological aspects of determining tumor DNA, its dynamic indicators, and its prognostic significance.

Response 1
We sincerely thank the reviewer for this important comment. We agree that the discussion of ctDNA in the original manuscript was not sufficiently comprehensive relative to the scope indicated by the title.
In response, we have substantially expanded the content within Section 4.1 (Anti-EGFR mAb Rechallenge) to provide a more detailed and balanced discussion of ctDNA, encompassing its methodological aspects, the clinical significance of dynamic molecular changes, and its prognostic and predictive value in the management of mCRC.
These revisions have been incorporated on page 7 (lines 254–284) and pages 8–9 (lines 325–337).


Comment 2
It is not entirely clear what is meant by the term 4.“Biomarker-Driven Therapies”: the identification of a mutation, a marker, or the identification of circulating tumor DNA.

Response 2
We thank the reviewer for this important comment highlighting the ambiguity of the term “Biomarker-Driven Therapies.”
In response, we have revised the terminology throughout the manuscript by replacing “Biomarker-Driven Therapies” with “Molecularly Guided Therapies” to improve clarity and precision.
In addition, we have added a clear definition at the beginning of Section 4 to explicitly describe this concept. The revised text reads as follows (Section 4, page 7, lines 241–244):
“In this context, molecularly guided therapies refer to treatment strategies tailored according to specific genomic alterations identified by tissue or ctDNA analysis, including RAS wild-type status, HER2 amplification, KRAS G12C mutation, and BRAF V600E mutation.”
These revisions clarify the intended meaning and ensure terminological consistency throughout the manuscript.


Comment 3
It would be more logical to place the columns “Treatment regimen and treatment line” on the left side of the tables.

Response 3
We thank the reviewer for this helpful suggestion. We agree that placing these columns on the left improves the logical flow and readability of the tables.
Accordingly, we have revised all relevant tables by repositioning the “Treatment Regimen” and “Treatment Line” columns to the leftmost position.

Reviewer 3 Report

Comments and Suggestions for Authors

1. Depth of Discussion on Treatment Sequencing:
While the manuscript provides excellent, detailed algorithms for molecular subtypes (HER2, KRAS G12C, BRAF V600E), the discussion on sequencing for the "all-comers" population (RAS mutant, MSS) is comparatively brief. The mention of the OSERO study and the choice between regorafenib, FTD-TPI/Bev, and fruquintinib is valuable but could be expanded. Consider adding a brief discussion on clinical factors that might influence this choice in the absence of direct comparative data, such as toxicity profiles (e.g., hand-foot skin reaction vs. hypertension vs. hematologic toxicity), patient performance status, and the goal of therapy (disease control vs. tumor shrinkage).

2. Clarification of ctDNA-Guided Rechallenge Criteria:
The manuscript strongly advocates for ctDNA-guided anti-EGFR rechallenge. However, the specific technical and temporal aspects of ctDNA testing are mentioned only briefly in the limitations section. For clinical applicability, it would be helpful to briefly specify the optimal timing of the "pre-rechallenge" liquid biopsy (e.g., immediately before treatment initiation) and mention the challenge of detecting clonal hematopoiesis of indeterminate potential (CHIP) mutations, which can confound results. While CHIP is mentioned, linking it directly to the risk of false positives in RAS testing would strengthen the clinical caution.

3. Figure and Table Presentation:
The figures (Figure 1-4) are conceptually excellent and provide clear clinical algorithms. However, the legends are quite lengthy and contain information that might be better placed in the main text or as a bulleted list within the figure itself. To enhance readability, consider streamlining the figure legends to a concise title and moving the detailed eligibility criteria (e.g., the 4 bullet points for anti-EGFR rechallenge in Figure 1) to the main body of the manuscript where these criteria are discussed. Additionally, ensure that all abbreviations in the figures are fully spelled out in a footnote, as some figures lack a comprehensive abbreviation list (e.g., "T-mab" in Figure 2).

4. Inclusion of Emerging Data on Immunotherapy in MSS Population:
The manuscript focuses heavily on targeted therapy and chemotherapy. While immunotherapy in MSS mCRC remains challenging, there are emerging combination strategies (e.g., with regorafenib or other TKIs) showing modest activity. The current manuscript is largely silent on this area. Given the scope of "salvage-line" treatment, a brief mention of the ongoing research into combining immunotherapy with anti-angiogenic agents or other modulators in the MSS population would provide a more complete picture of the future landscape, even if it is not yet a standard option.

5. Table Consistency and Data Presentation:
Tables 1 and 2 are incredibly comprehensive and a major strength of the review. However, Table 2 is visually dense and contains some inconsistencies in data reporting (e.g., "NA" vs. "NR" for not reached, and some rows with incomplete data). Standardize the notation for "Not Available" and "Not Reached" (e.g., use "NA" and "NR" consistently). For readability, consider splitting Table 2 into two separate tables: one for "ctDNA-guided/Rechallenge Trials" and one for "Novel Targeted Agent Trials (HER2, KRAS, BRAF)." This would make it easier for readers to digest the information.

Author Response

We sincerely thank the reviewer for the thoughtful and constructive comments. We appreciate the positive evaluation of our work and the valuable suggestions to improve the clarity and clinical relevance of the manuscript. We have carefully revised the manuscript in response to all comments, as detailed below.

Comment 1. Depth of Discussion on Treatment Sequencing:
While the manuscript provides excellent, detailed algorithms for molecular subtypes (HER2, KRAS G12C, BRAF V600E), the discussion on sequencing for the "all-comers" population (RAS mutant, MSS) is comparatively brief. The mention of the OSERO study and the choice between regorafenib, FTD-TPI/Bev, and fruquintinib is valuable but could be expanded. Consider adding a brief discussion on clinical factors that might influence this choice in the absence of direct comparative data, such as toxicity profiles (e.g., hand-foot skin reaction vs. hypertension vs. hematologic toxicity), patient performance status, and the goal of therapy (disease control vs. tumor shrinkage).

Response 1
We sincerely thank the reviewer for this insightful suggestion. We agree that expanding the discussion on treatment sequencing for the “all-comers” population would strengthen the manuscript.
In response, we have added a paragraph in Section 5 (page 15, lines 500–508) to incorporate key clinical factors influencing treatment selection, including toxicity profiles, patient performance status, and treatment goals.
The added text reads as follows:
“Treatment selection should be guided by ECOG performance status, treatment goals, and toxicity profile. In patients with PS 0–1 requiring tumor shrinkage, FTD-TPI plus bevacizumab may be preferred. For patients prioritizing disease control or those intolerant of hematologic toxicity, regorafenib or fruquintinib may be more appropriate. Prior treatment-related adverse events should also be systematically considered when determining treatment sequencing; for instance, prior proteinuria has been reported to increase the risk of subsequent proteinuria, underscoring the importance of a comprehensive toxicity history in clinical practice.”


Comment 2. Clarification of ctDNA-Guided Rechallenge Criteria:
The manuscript strongly advocates for ctDNA-guided anti-EGFR rechallenge. However, the specific technical and temporal aspects of ctDNA testing are mentioned only briefly in the limitations section. For clinical applicability, it would be helpful to briefly specify the optimal timing of the "pre-rechallenge" liquid biopsy (e.g., immediately before treatment initiation) and mention the challenge of detecting clonal hematopoiesis of indeterminate potential (CHIP) mutations, which can confound results. While CHIP is mentioned, linking it directly to the risk of false positives in RAS testing would strengthen the clinical caution.

Response 2
We sincerely thank the reviewer for this important and clinically relevant comment. We agree that clarifying the practical considerations of ctDNA testing would enhance clinical applicability.
In response, we have expanded the relevant discussion in Section 4.1 (pages 8–9, lines 325–337).
The added text reads as follows:
“When performing ctDNA assessment, several important considerations warrant attention. First, given the short half-life of ctDNA, testing should ideally be performed immediately prior to rechallenge, as this timing allows for a more accurate reflection of the current tumor biology. Second, when RAS mutations are detected at a low VAF—particularly below 1%—false-positive results attributable to CHIP should be suspected, especially in elderly patients or those with prior chemotherapy exposure; in such cases, matched white blood cell sequencing alongside ctDNA testing is recommended to exclude CHIP-related mutations. Third, no consensus definition currently exists for what constitutes a high ctDNA level, which complicates result interpretation. Fourth, ctDNA shedding may be reduced in patients with low disease burden or specific metastatic sites—such as pulmonary, peritoneal, or brain metastases—potentially leading to false-negative results. Finally, variability across assay platforms may affect the comparability and reproducibility of ctDNA measurements.”


Comment 3. Figure and Table Presentation:
The figures (Figure 1-4) are conceptually excellent and provide clear clinical algorithms. However, the legends are quite lengthy and contain information that might be better placed in the main text or as a bulleted list within the figure itself. To enhance readability, consider streamlining the figure legends to a concise title and moving the detailed eligibility criteria (e.g., the 4 bullet points for anti-EGFR rechallenge in Figure 1) to the main body of the manuscript where these criteria are discussed. Additionally, ensure that all abbreviations in the figures are fully spelled out in a footnote, as some figures lack a comprehensive abbreviation list (e.g., "T-mab" in Figure 2).

Response 3
We thank the reviewer for this helpful suggestion.
In response, we have streamlined all figure legends to concise titles to improve readability. We have also relocated detailed eligibility criteria from the figure legends to the main text in the corresponding sections (Section 4.3 and Section 4.4), where these criteria are more appropriately discussed within their clinical context. In addition, all abbreviations used in the figures have been fully defined in the corresponding figure footnotes to ensure clarity and consistency.
These revisions improve both readability of the figures and their integration with the main text.


Comment 4. Inclusion of Emerging Data on Immunotherapy in MSS Population:
The manuscript focuses heavily on targeted therapy and chemotherapy. While immunotherapy in MSS mCRC remains challenging, there are emerging combination strategies (e.g., with regorafenib or other TKIs) showing modest activity. The current manuscript is largely silent on this area. Given the scope of "salvage-line" treatment, a brief mention of the ongoing research into combining immunotherapy with anti-angiogenic agents or other modulators in the MSS population would provide a more complete picture of the future landscape, even if it is not yet a standard option.

Response 4
We thank the reviewer for this insightful and forward-looking comment. We agree that incorporating emerging data on immunotherapy-based combination strategies would provide a more comprehensive overview of the evolving salvage-line treatment landscape.
Accordingly, we have added a new section entitled “Immune Checkpoint Inhibitor–Based Combination Therapy for MSS mCRC” (Section 6).
In this section, we summarize the key recent clinical trials evaluating combination strategies, including immune checkpoint inhibitors combined with multikinase inhibitors and other immunomodulatory agents. We also discuss both negative phase III trials and studies demonstrating modest efficacy, particularly in selected subgroups such as patients without liver metastases.
This addition provides a more balanced and forward-looking perspective on emerging therapeutic strategies, while clearly distinguishing investigational approaches from current standard treatments.


Comment 5. Table Consistency and Data Presentation:
Tables 1 and 2 are incredibly comprehensive and a major strength of the review. However, Table 2 is visually dense and contains some inconsistencies in data reporting (e.g., "NA" vs. "NR" for not reached, and some rows with incomplete data). Standardize the notation for "Not Available" and "Not Reached" (e.g., use "NA" and "NR" consistently). For readability, consider splitting Table 2 into two separate tables: one for "ctDNA-guided/Rechallenge Trials" and one for "Novel Targeted Agent Trials (HER2, KRAS, BRAF)." This would make it easier for readers to digest the information.

Response 5
We thank the reviewer for this valuable suggestion and for highlighting the importance of clarity in data presentation.

In response, we have standardized the notation by consistently using "NA" for not available data and "NR" for not reached values throughout the tables, thereby resolving the previous inconsistencies. In addition, to improve readability and logical organization, we have divided the original Table 2 into two separate tables: Clinical Trials of Contemporary Salvage-Line Therapies in mCRC (Anti-EGFR mAb Rechallenge Trials) and Clinical Trials of Contemporary Salvage-Line Therapies in mCRC (Novel Targeted Agent Trials).

These revisions improve both consistency and readability, allowing readers to more easily interpret and compare the presented data.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Well done.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have responded thoroughly and thoughtfully to all previous comments. The revised manuscript is significantly improved in terms of clinical depth, practical guidance, data presentation, and coverage of emerging therapeutic areas. It provides a valuable and up-to-date resource for oncologists managing patients with refractory mCRC. This manuscript has been well revised and is recommended for publication.