Limited Efficacy of Anti-EGFR Monoclonal Antibodies in Colorectal Cancer Patients with Rare RAS Variants: Analysis of the C-CAT Database

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This research manuscript has scientific merit that might benefit readers, but some minor revisions still need to be included. Therefore, we recommend that the authors make the following modifications:

1.      Line 27-29
This study provides real-world evidence supporting the clinical relevance of rare RAS variants in treatment decision-making for colorectal cancer.
The authors are suggested to add one line on future perspectives at the end of the abstract to point out the future directions regarding this issue.
As the authors explained well about the rare variants, a few words about the future perspectives will make the abstract more comprehensive and well-defined.

2.      Line 40-41
Current standard testing methods in many healthcare systems, including Japan, are limited to detecting common mutations.
The authors are suggested to give examples of common mutations associated with colorectal cancer, especially in RAS wild-type cases. It will make the statement more demonstrative.

3.      Line 43-44
The molecular landscape of colorectal cancer is complex, with RAS mutations representing the most frequent alterations, occurring in approximately 40% of cases.
The authors are suggested to add details about the most frequent alterations (RAS mutations). The current statement lacks clarity, and the revised statement will make it more understandable.

4.      Line 101
Genomic analysis included evaluation of gene calls, tumor mutational burden, microsatellite instability status, and specific genetic alterations.
The authors mistakenly wrote "gene calls" instead of "gene cells" in the above sentence. The authors are suggested to review this miscommunication.

5.      Line 261-263
From a biological perspective, our results support the hypothesis that rare RAS variants can have functional consequences similar to common mutations in terms of pathway activation and treatment resistance.
The authors are suggested to add the mechanism of rare RAS variants in the introduction part. This will provide readers with a more comprehensive overview of the issue and improve understanding.

Author Response

Dear Reviewer 1,

We sincerely appreciate your thoughtful review and constructive suggestions that will help improve our manuscript. We have carefully addressed each of your points as detailed below:

Comments 1:Line 27-29 This study provides real-world evidence supporting the clinical relevance of rare RAS variants in treatment decision-making for colorectal cancer.The authors are suggested to add one line on future perspectives at the end of the abstract to point out the future directions regarding this issue.As the authors explained well about the rare variants, a few words about the future perspectives will make the abstract more comprehensive and well-defined.
Response 1:Thank you for this excellent suggestion. We have added the following sentence to the abstract's conclusion:
"Future studies should focus on developing cost-effective comprehensive testing strategies and evaluating alternative treatment approaches for patients with rare RAS variants."

Comments 2:Line 40-41 Current standard testing methods in many healthcare systems, including Japan, are limited to detecting common mutations.
The authors are suggested to give examples of common mutations associated with colorectal cancer, especially in RAS wild-type cases. It will make the statement more demonstrative.
Response 2:We appreciate this suggestion for improved clarity. We have added examples in the introduction:
"Common mutations include KRAS G12D, G12V, and G13D mutations in exon 2, which together account for approximately two-thirds of all RAS mutations in colorectal cancer."

Comments 3: Line 43-44 The molecular landscape of colorectal cancer is complex, with RAS mutations representing the most frequent alterations, occurring in approximately 40% of cases.The authors are suggested to add details about the most frequent alterations (RAS mutations). The current statement lacks clarity, and the revised statement will make it more understandable.
Response 3:Thank you for highlighting this lack of clarity. We have expanded this section to read:
"The molecular landscape of colorectal cancer is complex, with RAS mutations representing the most frequent alterations, occurring in approximately 40% of cases. These mutations primarily occur in KRAS (30-35%) and NRAS (3-5%), with specific hotspots in codons 12, 13, and 61 being most common."

Comments 4: Line 101
Genomic analysis included evaluation of gene calls, tumor mutational burden, microsatellite instability status, and specific genetic alterations.
The authors mistakenly wrote "gene calls" instead of "gene cells" in the above sentence. The authors are suggested to review this miscommunication.
Response 4:We respectfully maintain that "gene calls" is the correct terminology in this context, as it refers to the process of determining genetic variants from sequencing data. This is standard terminology in genomic analysis.

Comments 5:Line 261-263 From a biological perspective, our results support the hypothesis that rare RAS variants can have functional consequences similar to common mutations in terms of pathway activation and treatment resistance. The authors are suggested to add the mechanism of rare RAS variants in the introduction part. This will provide readers with a more comprehensive overview of the issue and improve understanding.
Response 5:Thank you for this valuable suggestion. We have added the following text to the introduction:
"Rare RAS variants can affect RAS protein function through various mechanisms, including altered GTP binding, impaired GTPase activity, or modified interactions with downstream effectors. These alterations can lead to constitutive pathway activation similar to common mutations, potentially conferring resistance to anti-EGFR therapy."

We believe these revisions have significantly strengthened the manuscript and hope they meet with your approval.

Reviewer 2 Report

Comments and Suggestions for Authors

Could you clarify how CGP significantly impacts treatment decisions for rare RAS variants if the therapeutic options remain limited after identification?

Why were potential alternative therapeutic approaches, such as combinational therapies or non-EGFR-targeting regimens, not evaluated or discussed as a follow-up to CGP findings?

Could you include a cost-benefit analysis to justify recommending CGP for first-line therapy selection in colorectal cancer?

Why was MSI not further investigated as a potential confounding factor in rare RAS variant cases?

How did you account for the statistical limitations of analyzing rare subgroups, and were confidence intervals calculated to validate the findings?

Author Response

Dear Reviewer 2,

We deeply appreciate your insightful questions that have helped us identify areas requiring additional clarity in our manuscript. 

Comments 1:Could you clarify how CGP significantly impacts treatment decisions for rare RAS variants if the therapeutic options remain limited after identification?
Response 1:Regarding your question about how CGP impacts treatment decisions for rare RAS variants, we acknowledge that therapeutic options may be limited after identifying these variants. However, CGP significantly influences clinical decision-making by preventing the use of potentially ineffective anti-EGFR therapy, thereby avoiding unnecessary toxicity and costs. Furthermore, early identification of rare RAS variants enables timely transition to alternative treatment strategies, such as bevacizumab-based regimens. Additionally, CGP often identifies other actionable alterations that may guide clinical trial enrollment. We have clarified these points in the discussion section.

Comments 2:Why were potential alternative therapeutic approaches, such as combinational therapies or non-EGFR-targeting regimens, not evaluated or discussed as a follow-up to CGP findings?
Response 2:Your question about alternative therapeutic approaches is particularly pertinent. We have expanded our discussion to address the role of bevacizumab-based combinations, emerging targeted therapy approaches, and the potential for novel combination strategies. We have also included information about ongoing clinical trials investigating new treatment options for RAS-mutant colorectal cancer.

Comments 3:Could you include a cost-benefit analysis to justify recommending CGP for first-line therapy selection in colorectal cancer?
Response 3:Concerning the cost-benefit analysis of CGP implementation, you raise an excellent point. While a comprehensive cost-benefit analysis was beyond the scope of our current study, we have enhanced our discussion to address the economic implications, drawing from available literature on anti-EGFR therapy costs. We have also outlined key considerations for future cost-effectiveness analyses and acknowledged this as an important area for future research.

Comments 4:Why was MSI not further investigated as a potential confounding factor in rare RAS variant cases?
Response 4:Regarding MSI status, we appreciate you bringing attention to this potential confounding factor. In our cohort, we found that 88.9% of cases were MSS while 11.1% had unknown status, with no MSI-H cases identified among rare RAS variant cases. We have added this information to the results section and discussed its implications in detail.

Comments 5:How did you account for the statistical limitations of analyzing rare subgroups, and were confidence intervals calculated to validate the findings?
Response 5:Finally, we acknowledge your crucial point about statistical limitations in analyzing rare subgroups. We have strengthened our statistical methodology by adding confidence intervals for all key outcome measures and including a more detailed statistical methods section. We have also explicitly acknowledged the limitations of small subgroup analyses and added appropriate caveats to our conclusions.

We believe these revisions have substantially improved the manuscript's methodological rigor and clinical relevance. We thank you again for your thoughtful review that has helped enhance the quality of our work.