Patients with Colorectal Cancer and BRAF^V600E-Mutation in Argentina: A Real-World Study—The EMOGI-CRC01 Study

Comments 1:  Please explain why stage I–III (potentially curable patients) would require BRAF-targeted therapy. Did these patients undergo curative or palliative surgery? (Table 2 mentions surgery, but Table 1 does not.) Additionally, information on adjuvant therapy is crucial but missing in the report.

Response 1: Nowadays, target therapy in earlier-stage is in Curative surgery was undergone in 47.8% (n=77) of the total patients included (n=161). Correction in table 1 (add information about surgery) and 3th paragraph in the result, we add information about adjuvant treatment. In the second paragraph of the discussion, we add information about target therapy trial in the earlier-setting.

**In our cohort, 28% of the included patients were diagnosed at an early-stage (stage I-III) but experienced metastatic progression during the course of the disease. For this reason, several ongoing clinical trials are investigating the use of BRAF-targeted therapies in this setting. These studies aim to determine whether incorporating targeted treatments can improve outcomes in this potentially curable population. The Alliance A022004 trial (NCT05710406), a phase II/III study evaluates whether the combination of encorafenib and cetuximab after surgery and chemotherapy; AIO-KRK-0420 (NCT05510895), a phase II trial investigates the efficacy of a neoadjuvant regimen combining encorafenib, binimetinib and cetuximab; and the NEORAF study (NCT05706779), a phase II evaluates the effectiveness of neoadjuvant treatment with encorafenib plus cetuximab in patients with operable BRAFV600E-mutant adenocarcinoma of the colon or upper rectum. The re-sults of these three trials are not publish yet but reflect a growing interest in integrating BRAF-targeted therapies into the treatment paradigm for earlier-stage BRAF-mutant CRC.**

Comments 2: Analyzing survival outcomes (PFS and OS) for all stages together seems inappropriate. A stage-specific survival analysis is recommended. Moreover, the starting point for survival analysis is indicated as first-line therapy (lines 97–100, pages 2–3), but BRAFi is not always used as a first-line treatment. It would make more sense to calculate survival time from the initiation of BRAFi use no matter of the lines of BRAFi. Otherwise, the reported PFS and OS have no clear association with BRAFi usage, rendering subsequent analyses meaningless.

Response 2: If it seems appropriate, I can remove the overall survival (OS) and only put on the manuscript progression-free survival (PFS) analysis for the entire population.

All patients included in the study are metastatic (either de novo or as part of disease progression), which may limit the generalizability of the results across all stages. I am open to revising the analysis to better align with the patient population and treatment lines. We agree that a more appropriate approach would be to calculate survival time from the initiation of BRAFi therapy, regardless of the treatment line. Due to the limited number of patients who received BRAF inhibitors (BRAFi), performing a stage-specific survival analysis would have been challenging. Given the small sample size, it is difficult to draw robust conclusions based solely on the use of BRAFi in different treatment lines.

Comments 3: Details about immunotherapy monotherapy or combination therapy are lacking. Were all these cases dMMR? Please explain why the longest survival times were observed in this group per the regimens they received or the molecular patterns they have.

Response 3:  We describe better the explanation about the ICI treatment.

3.4 Immunotherapy and BRAF inhibitors

Considering the total number of patients included in the treatment analysis (n=151), 15.2% (n=23) received ICI: 43.5% (n=10) in the first-line, 30.4% (n=7) in the second-line, and 26.1% (n=6) in the third-line setting. Among them, 93.1% (n=21) patients received pembrolizumab, while only 6.9% (n=2) received nivolumab/ipilimumab.

In the context of BRAF-mutant colorectal cancer, the activation of the MAPK signaling pathway, induced by the BRAF mutation, may contribute to increased expression of immune evasion-related proteins, such as PD-L1, thereby enhancing the tumor’s vulnerability to immunotherapy. Thus, the combination of a higher mutational burden in dMMR tumors and alterations in the MAPK signaling pathway facilitates the efficacy of immunotherapeutic agents, which, by interfering with immune checkpoints, allow for a more robust and effective immune response against the tumor.

Comments 4: In the first paragraph of the Discussion, the authors mention two types of BRAF but fail to clearly explain BRAF mutation subtypes. It is unclear how this relates to the study's findings.

Response 4: Thank you for the correction. We have modified this paragraph.

BRAFV600E-mutation mCRC is a highly aggressive and biologically heterogeneous disease, associated with poor prognosis and limited therapeutic options. In this re-al-world multicenter study, we analyzed the clinical characteristics, treatment strate-gies, and outcomes of 161 patients with BRAFV600E-mutation mCRC in Argentina. Our findings highlight significant variability in treatment approaches, with limited access to BRAF-targeted therapies in second- and third-line settings. Despite a mOS 20.6 months, disease progression remained a major challenge. Notably, while 93.8% of patients re-ceived first-line treatment, only 15.5% were able to undergo third-line therapy, reflect-ing the aggressive nature of the disease and the rapid clinical deterioration that limits the feasibility of subsequent treatment lines.

Comments 5: Lines 269–270 (page 9) state: "Notably, the median OS and PFS were higher in our cohort (20.6 and 9 months, respectively) in contrast to findings from previous reports." The authors do not provide reasons for this phenomenon. Please add a detailed explanation and expand on this in the Discussion section.

Comments 6: In the Discussion, the authors frequently propose that the survival differences in this report compared with previous studies are owing to the status of dMMR. However, when analyzing survival outcomes or conducting multivariate analyses, no significant statistical differences were observed. This weakens their argument. Please provide stronger and more convincing explanations in the section of Discussion.

Response 5-6: The higher median overall survival (OS) and progression-free survival (PFS) observed in our cohort compared to previous reports may be attributed to several statistical and methodological factors. First, our cohort may have included a more favorable patient population, such as those with better baseline performance status or more localized disease at diagnosis, which could have led to better outcomes. Additionally, the size of our cohort, while sufficient for meaningful analysis, may have resulted in a more homogeneous group in terms of treatment adherence and follow-up, which can influence survival outcomes. Also, the majority of the patients included in our cohort were followed up in the private healthcare sector, where patient monitoring tends to be more stringent. Unfortunately, in our country, there is a significant gap between the public and private healthcare systems, particularly regarding the availability of human resources and physical resources, such as medications, hospital infrastructure, medical equipment, and facilities, which can directly impact patient care and survival outcomes.

Comments 7: In the Discussion, the authors frequently propose that the survival differences in this report compared with previous studies are owing to the status of dMMR. However, when analyzing survival outcomes or conducting multivariate analyses, no significant statistical differences were observed. This weakens their argument. Please provide stronger and more convincing explanations in the section of Discussion.

Response 7: Thank you for your valuable comment. I agree with your observation regarding the survival differences and the role of dMMR status. We acknowledge that the lack of statistically significant differences in the survival analysis, is likely due to the small follow up of dMMR patients in our cohort, which limits the statistical power to detect a significant effect.

Comments 8: The approval number of the IRBs is missing. Also, as the authors declared, this is an observational study, did you really get the written ICFs from the subjects? Please confirm.

Response 8: Although this is a retrospective study, we confirm that all patients and/or their families signed informed consent forms (ICFs) to participate in the study and to allow the use of their data. This study involved the participation of several centers across Argentina and began several years ago. During the initial phase of the project, informed consent was collected from all participants, ensuring that their participation was voluntary and in accordance with ethical guidelines.