Immune-Related Adverse Events in Patients with Melanoma Treated with B-RAF/MEK Target Therapy: Occurrence and Circulating Immune Cell Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses an important question regarding whether immune-related adverse events (irAEs) could serve as biomarkers of response in BRAF/MEK–treated melanoma. This is a setting where predictive markers are generally lacking as noted by the authors. The key motivation of this study is that it takes a commonly-assumed but weakly tested hypothesis and directly challenges prior retrospective signals that link irAEs to better outcomes. Their key finding helps clarify that unlike checkpoint inhibitors, irAEs are rare and do not appear to correlate with efficacy of a targeted therapy. Areas of improvement are noted below:

1. Sample size for irAEs is extremely small (n=3), which limits any meaningful efficacy or immune-correlation conclusions. Future pooled analyses would strengthen the confidence
2. Outcome linkage is underdeveloped; deeper time-to-event and longitudinal immune-response modeling could better test whether immune modulation reflects efficacy or progression

Author Response

The manuscript addresses an important question regarding whether immune-related adverse events (irAEs) could serve as biomarkers of response in BRAF/MEK–treated melanoma. This is a setting where predictive markers are generally lacking as noted by the authors. The key motivation of this study is that it takes a commonly assumed but weakly tested hypothesis and directly challenges prior retrospective signals that link irAEs to better outcomes. Their key finding helps clarify that unlike checkpoint inhibitors, irAEs are rare and do not appear to correlate with efficacy of a targeted therapy. Areas of improvement are noted below:

1. Sample size for irAEs is extremely small (n=3), which limits any meaningful efficacy or immune-correlation conclusions. Future pooled analyses would strengthen the confidence.

We thanks the Reviewer for the comments and indications to improve our work. We clearly agree that the sample size is extremely small, and we must plan a multicenter study to amplify the numbers and achieve a statistical significance. We now underline this aspect in the Conclusions.

2. Outcome linkage is underdeveloped; deeper time-to-event and longitudinal immune-response modeling could better test whether immune modulation reflects efficacy or progression.

Regarding point 2, we do have information only on progression-free survival as an outcome measure. To strengthen the paper, we have added the progression-free survival curves estimated with the Kaplan–Meier method to compare patients with occurrence of toxicity vs. patients without toxicity (Figure 1).

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript reports a real‑world cohort of 158 melanoma patients treated with BRAF/MEK inhibitors (iBRAF/iMEK), describing the frequency of immune‑related adverse events (irAEs) and presenting an exploratory peripheral blood immune‑subset analysis (flow cytometry) in patients who developed irAEs. Clinically, quantifying “immune‑mediated toxicity” and potential immune changes under iBRAF/iMEK is of interest, and the presentation of real‑world occurrence rates is valuable. However, there are several concerns as described below.

 

Major comments

1. The definition of irAE/autoimmune AE is unclear. In general, irAE is used for immune checkpoint inhibitors. It is important to specify what constitutes an “immune‑mediated” AE under iBRAF/iMEK in this work.
2. Also, it is helpful for readers showing a concise table summarizing the three immune‑mediated cases, including: CTCAE grade, time to onset, management (e.g., steroids), dose interruption/reduction, outcome (resolution/recurrence), prior autoimmune disease, and prior ICI exposure (and time since last dose). At present, only limited timing information is presented, making it difficult to evaluate clinical significance.
3. Supplementary Table 1 listed frequencies of AE, however, it is unclear whether these numbers represent number of patients experiencing each AE or number of AE events. It should be clarified.
4. The denominator used for incidence is unclear. The manuscript alternates between “irAEs” and “autoimmune AEs.” In Table 3, Autoimmune is reported to have 3 patients with 3% incidence, however, it is unclear what the 3.0% denominator is. It should be clarified.
5. The categorization in Table 3 (“only cutaneous / cutaneous and other / only other”) would benefit from a clear rule for handling overlap and prioritization.
6. They showed that the cohort includes second line treatment in Table 4. Prior therapies, especially ICI, can affect baseline immune profiles and the likelihood of immune‑mediated symptoms. The proportion with prior ICI exposure, the washout interval, and relevant concomitant medications should be shown. The current conclusion that there are “no clinical predictive factors” may be too strong without additional adjustment or clearer analytic framing.
7. The methods of flowcytometry should be shown in more detail, including the gating strategy and subset definitions. The definition of each subset and representative gating plot should be shown.
8. The manuscript acknowledges the rarity of irAEs and limited ability to define predictors. Nonetheless, the title uses “biomarkers,” which may overstate the findings given the very small irAE sample.

 

Minor comments

1. Several grammar issues are present. A full language edit is recommended.
2. Methods mention one case of trametinib monotherapy, whereas Table 2 appears to show a single‑agent category (“C 1”), which could be interpreted as cobimetinib monotherapy.

Author Response

Major comments

1. The definition of irAE/autoimmune AE is unclear. In general, irAE is used for immune checkpoint inhibitors. It is important to specify what constitutes an “immune‑mediated” AE under iBRAF/iMEK in this work.

We thank the Reviewer for the possibility to clarify this aspect. We have added a paragraph in the Introduction with additional references to provide a more detailed explanation (Lanes 101-106).

2. Also, it is helpful for readers showing a concise table summarizing the three immune‑mediated cases, including: CTCAE grade, time to onset, management (e.g., steroids), dose interruption/reduction, outcome (resolution/recurrence), prior autoimmune disease, and prior ICI exposure (and time since last dose). At present, only limited timing information is presented, making it difficult to evaluate clinical significance.

We agree with the Reviewer that these clinical data were lacking, and we have changed Table 5 accordingly in order to include these data.

3. Supplementary Table 1 listed frequencies of AE, however, it is unclear whether these numbers represent number of patients experiencing each AE or number of AE events. It should be clarified.

We agree with the Reviewer that this point was not clear. We have changed the title of the Supplementary Table 1 in “Number of toxicities occurred after iBRAF+iMEK therapy” and we have added this footnote: “*Counts exceed the total number of patients with toxicity because patients may have experienced multiple toxicities”.  In addition, we have also modified the sentence at lane 219 as “In our patient cohort, toxicity occurred in 101 patients (64%), and patients could have experienced multiple toxicities.”

4. The denominator used for incidence is unclear. The manuscript alternates between “irAEs” and “autoimmune AEs.” In Table 3, Autoimmune is reported to have 3 patients with 3% incidence, however, it is unclear what the 3.0% denominator is. It should be clarified.

We have changed the title of Table 3 in “Patients with occurrence of toxicity after iBRAF/iMEK therapy, by toxicity type”. We have carefully revised the text to eliminate the term autoimmune AEs, leaving only the term irAE.

We have also modified the sentence at lane 219 as “Among these patients, approximately half experienced cutaneous toxicity, while irAEs were rare and observed in only three patients (Table 3)”.

5. The categorization in Table 3 (“only cutaneous / cutaneous and other / only other”) would benefit from a clear rule for handling overlap and prioritization.

 For greater clarity, we have specified in the title that these refer to patients (total = 158). We have also revised the category labels to improve prioritization.

6. They showed that the cohort includes second line treatment in Table 4. Prior therapies, especially ICI, can affect baseline immune profiles and the likelihood of immune‑mediated symptoms. The proportion with prior ICI exposure, the washout interval, and relevant concomitant medications should be shown. The current conclusion that there are “no clinical predictive factors” may be too strong without additional adjustment or clearer analytic framing.

As suggested by the Reviewer, we have added some data in Table 4 and Table 5. Of our patients, 67% (18/27) of those who received target therapy as a second line treatment had previously received ICI immunotherapy (ipilimumab, pembrolizumab or nivolumab). The washout interval between the two therapies was 30-40 days. We have added this data in the Results (lanes 231-233).

7. The methods of flow cytometry should be shown in more detail, including the gating strategy and subset definitions. The definition of each subset and representative gating plot should be shown.

Following the Reviewer indications, we have added an additional Supplementary figure showing the flow cytometry gating strategy in detail, including the sequential gating steps and representative plots. We have expanded the Methods section to provide a more detailed description of gating strategy and the definition of each immune cell subset analyzed.

8. The manuscript acknowledges the rarity of irAEs and limited ability to define predictors. Nonetheless, the title uses “biomarkers,” which may overstate the findings given the very small irAE sample. 

As suggested, we changed the title, substituting “biomarkers” with circulating immune cell analysis.

Minor comments

• 1. Several grammar issues are present. A full language edit is recommended.

As indicated, we carefully revised the text.

• 2. Methods mention one case of trametinib monotherapy, whereas Table 2 appears to show a single‑agent category (“C 1”), which could be interpreted as cobimetinib monotherapy.

This was a mistake; we have changed it in the Methods section.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the occurrence of immune-related adverse events (irAEs) in melanoma patients treated with BRAF/MEK inhibitors and explores circulating immune cell modulation as potential biomarkers. The topic is clinically relevant, particularly in the evolving landscape of combination strategies involving targeted therapy and immunotherapy. The study benefits from a real-world cohort and immunophenotyping analysis. However, several methodological and interpretative limitations need clarification before the manuscript can be considered for publication. Substantial revision is recommended to clarify methodology, adjust statistical interpretation, and moderate conclusions.

Comments

1. The reported irAE incidence (3%) is considerably lower than previously published data. While the authors acknowledge this, the manuscript lacks a formal discussion of statistical power. With only three irAE cases, meaningful comparisons are underpowered. The authors should explicitly state whether any power calculation was performed. Conclusions regarding the lack of association between irAEs and clinical outcomes should be more cautiously phrased, tempered and clearly framed as exploratory.
2. The “immune-related” adverse events need to be discussed more in the Introduction section. Discuss how can autoimmune AEs be distinguished from inflammatory AEs?
3. Cytofluorimetric assays for different immune cell subsets is based on only five patients (two patients who developed an irAE and three matched control patients). This extremely small sample size severely limits interpretability. Statistical analysis using ANOVA may not be appropriate given n=5. Please justify the statistical approach or consider descriptive reporting instead.
4. It is unclear whether the observed increase in circulating Thf cells during the first two months of therapy correlates with clinical response, LDH levels, or tumor burden. Please moderate conclusions and emphasize the exploratory nature of this finding.
5. Adding Kaplan–Meier survival analyses curves comparing toxicity vs. no-toxicity groups would strengthen the manuscript.
6. Several typographical issues are present, for instance “TableT3”. kindly correct.

Author Response

Comments

1. The reported irAE incidence (3%) is considerably lower than previously published data. While the authors acknowledge this, the manuscript lacks a formal discussion of statistical power. With only three irAE cases, meaningful comparisons are underpowered. The authors should explicitly state whether any power calculation was performed. Conclusions regarding the lack of association between irAEs and clinical outcomes should be more cautiously phrased, tempered and clearly framed as exploratory.

No a priori sample size estimation was performed, as we enrolled all the patients with unresectable metastatic melanoma treated with iBRAF/iMEK therapy in our Institute during the study period between 2009 and 2020. We have underlined in the Discussion the points raised by the Reviewer.

2. The “immune-related” adverse events need to be discussed more in the Introduction section. Discuss how can autoimmune AEs be distinguished from inflammatory AEs?

We thank the Reviewer for underlining this aspect. We have added a paragraph in the Introduction with additional references to provide a more detailed explanation (Lanes 101-106).

Autoimmune and inflammatory adverse events can overlap clinically, but they differ in their mechanisms of action. Autoimmune events are driven by immune responses against self-antigens and often involve autoantibodies, autoreactive T cells, and tissue-specific pathology, resembling classical autoimmune diseases. Inflammatory events, in contrast, result from non-specific immune activation or cytokine-mediated inflammation without evidence of autoimmunity, and are usually transient and reversible. Distinguishing between them relies on clinical presentation, laboratory markers, histopathology, and disease course, although in many cases these events exist on a continuum of immune-mediated toxicity. Therefore, we have now added a clarification on this point in the Discussion.

3. Cytofluorimetric assays for different immune cell subsets is based on only five patients (two patients who developed an irAE and three matched control patients). This extremely small sample size severely limits interpretability. Statistical analysis using ANOVA may not be appropriate given n=5. Please justify the statistical approach or consider descriptive reporting instead.

After consulting with a statistician, we agree that the number of patients included in the cytofluorimetric assays (n = 5) is very limited and that the study should be considered primarily descriptive from an immunological standpoint. In line with this concern, we have removed the statistical analysis (including ANOVA) from the immunological flow cytometry data and now present these results in a purely descriptive manner.

4. It is unclear whether the observed increase in circulating Thf cells during the first two months of therapy correlates with clinical response, LDH levels, or tumor burden. Please moderate conclusions and emphasize the exploratory nature of this finding.

Following Reviewer indication, we have moderated the Conclusions underlining the explorative nature of our findings.

5. Adding Kaplan–Meier survival analyses curves comparing toxicity vs. no-toxicity groups would strengthen the manuscript.

Unfortunately, we do not have access to the Regional Mortality Registry to obtain vital status data. However, we do have information on progression-free survival. Therefore, to strengthen the paper, we have added the Progression-free survival (PFS) curves estimated with the Kaplan–Meier method to compare patients with occurrence of toxicity vs. patients without toxicity (lanes 247-252, Figure 1). 

6. Several typographical issues are present, for instance “TableT3”. kindly correct.

We are very sorry for these mistakes; we have carefully checked the manuscript to eliminate them.

Reviewer 4 Report

Comments and Suggestions for Authors

This is a comprehensive and interesting publication on a very important topic, but it requires numerous changes to the text to better clarify certain aspects, such as the aim, limitations, and materials and methods. Here are some specific suggestions:

• In the introduction, it would be helpful to add references to the Phase III clinical trials that confirmed the effectiveness of the iBRAF/iMEK combination.
  It would be helpful to clearly define the aim of the study, perhaps with a few more words about the mechanism, and why it is so important in this case.
• The Material and Methods section should be expanded. A description of the exclusion criteria for patients from the study should be added, not just the inclusion criteria. How was the presence of the BRAF mutation verified? How were patients classified and assigned to the irAE/non-irAE group? The Gating Strategy in Flow Cytometry section should also be described.
• The discussion should clearly define the limitations of the study. (3 adverse events (irAE) in 101 patients (3%). It seems that a reliable assessment of the relationship is not statistically possible. Numerous mechanistic explanations should also be added and the tone of some statements should be slightly changed, overinterpretation should not be made (e.g. as above).

Author Response

1. In the introduction, it would be helpful to add references to the Phase III clinical trials that confirmed the effectiveness of the iBRAF/iMEK combination.
It would be helpful to clearly define the aim of the study, perhaps with a few more words about the mechanism, and why it is so important in this case.

We thank the Reviewer for the comment. As for your suggestion, we have added references and explanations about phase III clinical trial in the Introduction (Lanes 70-77).

2. The Material and Methods section should be expanded. A description of the exclusion criteria for patients from the study should be added, not just the inclusion criteria. How was the presence of the BRAF mutation verified? How were patients classified and assigned to the irAE/non-irAE group? The Gating Strategy in Flow Cytometry section should also be described.

We have added an additional Supplementary figure showing the flow cytometry gating strategy in detail, including the sequential gating steps and representative plots.

We have expanded the Methods section to provide a more detailed description of gating strategy and the definition of each immune cell subset analyzed.

 3. The discussion should clearly define the limitations of the study. (3 adverse events (irAE) in 101 patients (3%). It seems that a reliable assessment of the relationship is not statistically possible. Numerous mechanistic explanations should also be added, and the tone of some statements should be slightly changed, overinterpretation should not be made (e.g. as above).

As indicated, we have carefully revised the Discussion and Conclusion in order to avoid any overinterpretation of our data.

 

 

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has largely improved. However, there are still a few concerns exist.

 

1. The new background paragraph in the Introduction is helpful; however, the Methods section still does not clearly state how events were classified as “immune‑related/immune‑mediated” under iBRAF/iMEK in this study.
2. In Table 5, the caption currently reads “Clinical characteristic of two patients who had irAEs…”, but the table includes three irAE cases and three controls (with PBMC availability seemingly limited to two irAE cases). It should be revised.
3. In the Abstract, the sentence stating that patients “present neither clinical features that could predict the onset of an irAE…” reads too definitive given the very small number of irAE cases (n=3). It should be rephrased to a more appropriately cautious wording.

Author Response

1. The new background paragraph in the Introduction is helpful; however, the Methods section still does not clearly state how events were classified as “immune‑related/immune‑mediated” under iBRAF/iMEK in this study.

We understand the Reviewer concern, and we added in the Methods section the indication that we considered as irAE every autoimmune AE (lane 145).

2. In Table 5, the caption currently reads “Clinical characteristic of two patients who had irAEs…”, but the table includes three irAE cases and three controls (with PBMC availability seemingly limited to two irAE cases). It should be revised.

We apology for this mistake and we changed the title accordingly.

3. In the Abstract, the sentence stating that patients “present neither clinical features that could predict the onset of an irAE…” reads too definitive given the very small number of irAE cases (n=3). It should be rephrased to a more appropriately cautious wording.

Following the Reviewer suggestion, we rephrased the sentence in the abstract results and modified the abstract conclusions to emphasize that the small number of patients with irAEs prevented us from proposing any correlation between irAE onset and response to therapy.

Reviewer 4 Report

Comments and Suggestions for Authors

The corrections made by the Authors seem to be satisfactory, especially the expansion of the Methodology.

I have no further comments.

Author Response

We thank the Reviewer for the positive reception of our revision.