Emerging Role of BTK Inhibitors in Multiple Sclerosis: From Immunobiology to Clinical Translation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The study you presented is fine with clearly stated aims and conclusions. However I have some suggestion of corrections and improvements including substantial and editorial ones.

Substantial corrections:

1. In Abstract it is stated “Serious hepatotoxicity was reported in 11.0% of BTKi-treated patients compared with 13.7% of control patients (pooled RR 0.80; 95% CI 0.66–0.96), although increased transaminase elevations were reported in the placebo-controlled trials.” (line 40-42) followed by information “Hepatotoxicity is an issue for the class.” (line 44). These two parts of the manuscript makes some confusion. Please make them more compatible with each other.
2. In Data extraction section Authors indicates that they extract data on adverse events (lines 296-297) but later on safety data are limited to hepatotoxicity. I think that information about focusing on hepatotoxicity should be clearly introduced in methodology, not only in results and discussion.
3. In Results section (disability outcomes), in lines 375-376 the statement “Post-treatment EDSS scores showed minimal changes in both BTK inhibitor and control groups.” is too general. Please make it more informative and put the evaluation in the discussion.
4. In study limitation section the part “The majority of participants had relapsing-remitting MS, which may limit the applicability of findings to progressive disease subtypes.” (lines 445-446) in my opinion should be more definite e.g. “The majority of participants had relapsing-remitting MS, which limits the applicability…”. In the next line Authors indicate “Follow-up durations were relatively short across studies…” but the duration of the studies was not clearly indicated and possibly it may be done in Table 1. Please, consider such opportunity. In my opinion these limitations are crucial for conclusions about the role of BTK in progression of MS and should be more clearly expressed as majority of studies were conducted in RMS and RRMS.

Editorial corrections:

1. There are repeating parts in the introduction. These are: “Current high-efficacy disease-modifying therapies (HE-DMTs) for multiple sclerosis 73 include Ocrelizumab, Ofatumumab, Natalizumab, Alemtuzumab, Rituximab, and 74 Cladribine. Early initiation of HE-DMTs in relapsing-remitting multiple sclerosis patients 75 has a significant clinical impact on disease activity, relapse, and disability [5]. Despite their 76 effectiveness in reducing relapses, HE-DMTs have failed to address microglial activation, 77 smouldering inflammation, and progressive disability [6].” (lines 73-77) and “Current high-efficacy disease-modifying therapies (HE-DMTs) for multiple sclerosis 79 (MS), including Ocrelizumab, Ofatumumab, Natalizumab, Alemtuzumab, Rituximab, 80 and Cladribine, have improved clinical outcomes in patients with relapsing-remitting MS 81 (RRMS). Early initiation of these therapies is associated with reductions in disease activity 82 and relapse rates [5]. However, despite their efficacy in controlling acute inflammatory 83 activity, these agents have limited impact on microglial activation, smoldering inflammation, and the progression of disability, particularly in progressive forms of the disease [6].” (lines 79-85). Please compare and choose the preferred one. I would recommend to leave the latter one. Similarly compare “These limitations have driven interest in therapies capable of modulating both adaptive and innate immune responses, including those operating within the CNS. Recent pharmacotherapy for MS focuses on Bruton’s tyrosine kinase (BTK) receptor-expressing activated immune cells (e.g., microglia).” (lines 89-92) and “These limitations have prompted growing interest in therapeutic strategies that can target both adaptive and innate immune pathways, including those operating within the CNS compartment. In this context, recent advances in MS pharmacotherapy have focused on targeting Bruton’s tyrosine kinase (BTK), a key signaling molecule expressed in activated B cells and myeloid-lineage cells such as microglia.” (lines 92-97).

Author Response

Comment 1: In Abstract it is stated “Serious hepatotoxicity was reported in 11.0% of BTKi-treated patients compared with 13.7% of control patients (pooled RR 0.80; 95% CI 0.66–0.96), although increased transaminase elevations were reported in the placebo-controlled trials.” (line 40-42) followed by information “Hepatotoxicity is an issue for the class.” (line 44). These two parts of the manuscript makes some confusion. Please make them more compatible with each other.
Response 1: We agree. These 2 lines can cause confusion without further context. Accordingly, the section has been updated to make it more compatible: "Serious hepatotoxicity was reported in 11.0% of BTKi-treated patients compared with 13.7% of control patients (pooled RR 0.80; 95% CI 0.66–0.96). However, increased transaminase elevations were reported in the placebo-controlled trials, which indicates that hepatotoxicity remains a clinically relevant safety concern for the class. Conclusion: BTK inhibitors reduce inflammatory disease activity in relapsing MS and have emerging efficacy in progressive MS phenotypes; however, continued monitoring for hepatotoxicity is warranted."

Comment 2: In Data extraction section Authors indicates that they extract data on adverse events (lines 296-297) but later on safety data are limited to hepatotoxicity. I think that information about focusing on hepatotoxicity should be clearly introduced in methodology, not only in results and discussion.
Response 2: Agreed. We have modified the part accordingly: "Outcomes: changes in EDSS scores (e.g., at 3 and 6 months), number of new T1 gadolinium-enhancing and T2 lesions on MRI, percentage brain volume change, and safety outcomes showing hepatotoxicity."

Comment 3: In Results section (disability outcomes), in lines 375-376 the statement “Post-treatment EDSS scores showed minimal changes in both BTK inhibitor and control groups.” is too general. Please make it more informative and put the evaluation in the discussion.
Response 3: Certainly. We have revised the results section to make it more elaborate: "Among the included BTK inhibitor trials, the Montalban 2019 study reported no significant difference in EDSS change from baseline, while the FENopta extension study reported stable EDSS scores over follow-up; however, numerical post-treatment EDSS values were not provided." (lines 389-392). Also, the study limitations section has been changed in the discussion to interpret this: "Because most studies did not publish post-treatment EDSS scores or mean changes in EDSS from baseline, it is difficult to interpret disability results across BTK inhibitor trials. The lack of EDSS data limits direct quantitative comparison of disability trajectories among studies. Instead, few studies evaluated disability through confirmed disability worsening (CDW) or confirmed disability progression (CDP) endpoints." (lines 466-471)

Comment 4: In study limitation section the part “The majority of participants had relapsing-remitting MS, which may limit the applicability of findings to progressive disease subtypes.” (lines 445-446) in my opinion should be more definite e.g. “The majority of participants had relapsing-remitting MS, which limits the applicability…”. In the next line Authors indicate “Follow-up durations were relatively short across studies…” but the duration of the studies was not clearly indicated and possibly it may be done in Table 1. Please, consider such opportunity. In my opinion these limitations are crucial for conclusions about the role of BTK in progression of MS and should be more clearly expressed as majority of studies were conducted in RMS and RRMS.
Response 4: We understand and have have revised the "may limit the applicability..." to: "The majority of participants were diagnosed with relapsing MS (RMS), which limits the applicability of our findings to progressive disease subtypes." For the duration of the studies section, the lines have been revised to make them elaborate, consistent with the articles, and an extension to the response 3: "The reporting of CDW and CDP outcomes was largely confined to trials with longer follow-up durations, whereas shorter phase 2 studies primarily focused on MRI and relapse-related endpoints. Given the slow accumulation of disability in multiple sclerosis, shorter follow-up periods may be insufficient to detect meaningful changes in disability status, which may explain the limited reporting of disability outcomes in these studies." (lines 471-476)

Editorial Comment 1: There are repeating parts in the introduction. These are: “Current high-efficacy disease-modifying therapies (HE-DMTs) for multiple sclerosis 73 include Ocrelizumab, Ofatumumab, Natalizumab, Alemtuzumab, Rituximab, and 74 Cladribine. Early initiation of HE-DMTs in relapsing-remitting multiple sclerosis patients 75 has a significant clinical impact on disease activity, relapse, and disability [5]. Despite their 76 effectiveness in reducing relapses, HE-DMTs have failed to address microglial activation, 77 smouldering inflammation, and progressive disability [6].” (lines 73-77) and “Current high-efficacy disease-modifying therapies (HE-DMTs) for multiple sclerosis 79 (MS), including Ocrelizumab, Ofatumumab, Natalizumab, Alemtuzumab, Rituximab, 80 and Cladribine, have improved clinical outcomes in patients with relapsing-remitting MS 81 (RRMS). Early initiation of these therapies is associated with reductions in disease activity 82 and relapse rates [5]. However, despite their efficacy in controlling acute inflammatory 83 activity, these agents have limited impact on microglial activation, smoldering inflammation, and the progression of disability, particularly in progressive forms of the disease [6].” (lines 79-85). Please compare and choose the preferred one. I would recommend to leave the latter one. Similarly compare “These limitations have driven interest in therapies capable of modulating both adaptive and innate immune responses, including those operating within the CNS. Recent pharmacotherapy for MS focuses on Bruton’s tyrosine kinase (BTK) receptor-expressing activated immune cells (e.g., microglia).” (lines 89-92) and “These limitations have prompted growing interest in therapeutic strategies that can target both adaptive and innate immune pathways, including those operating within the CNS compartment. In this context, recent advances in MS pharmacotherapy have focused on targeting Bruton’s tyrosine kinase (BTK), a key signaling molecule expressed in activated B cells and myeloid-lineage cells such as microglia.” (lines 92-97).
Response 1: Thank you for pointing this out. We have removed the first part of both the queries and retained the second part. For the first, we have kept: "Current high-efficacy disease-modifying therapies (HE-DMTs) for multiple sclerosis (MS), including Ocrelizumab, Ofatumumab, Natalizumab, Alemtuzumab, Rituximab, and Cladribine, have improved clinical outcomes in patients with relapsing-remitting MS (RRMS). Early initiation of these therapies is associated with reductions in disease activity and relapse rates [5]. However, despite their efficacy in controlling acute inflammatory activity, these agents have limited impact on microglial activation, smoldering inflammation, and the progression of disability, particularly in progressive forms of the disease [6]." For the second, we have kept: "These limitations have prompted growing interest in therapeutic strategies that can target both adaptive and innate immune pathways, including those operating within the CNS compartment. In this context, recent advances in MS pharmacotherapy have focused on targeting Bruton’s tyrosine kinase (BTK), a key signaling molecule expressed in activated B cells and myeloid-lineage cells such as microglia."

Reviewer 2 Report

Comments and Suggestions for Authors

Your paper provides a comprehensive discussion of the role of BTK inhibitors in MS. This reviewer agrees with the conclusions of your systemic analysis. However, (1) the discussion does not address the fact that a reported pooled relative risk of relapse rate of 0.24, indicates this class of medication is indeed a high-efficacy DMT, even though the effect on disability exhibits discrete numbers perhaps, as the authors adduced, this factor may be influenced by the number of subjects with progressive forms as well as the limitations of time of study.   

(2) The analysis did not include mortality or number of subjects abandoning the trials due to hepatotoxicity. I personally believe this information is important, and that was retrievable through the analysis. An explanation or comment on this point would be very informative. 

(3) This study is authored by researchers from India, Nepal and the USA, and while the absence of commercial or financial relationships, hence no COI, are clearly stated, the source(s) of support of the study and publication, are not mentioned. 

Author Response

Comment 1: the discussion does not address the fact that a reported pooled relative risk of relapse rate of 0.24, indicates this class of medication is indeed a high-efficacy DMT, even though the effect on disability exhibits discrete numbers perhaps, as the authors adduced, this factor may be influenced by the number of subjects with progressive forms as well as the limitations of time of study.  
Response 1: We thank the reviewer for this insightful comment. We agree that our pooled analysis of the relapse rate (RR 0.24) provides a strong clinical signal that was perhaps insufficiently highlighted in our initial draft. We have amended the discussion to elevate this finding by adding: "The pooled relative risk of 0.24 for relapse observed in this meta-analysis highlights the potent clinical efficacy of BTK inhibitors, establishing this class as a high-efficacy disease-modifying therapy. While individual Phase 3 trials have presented challenges in demonstrating superiority against active comparators, these aggregate data show that the class possesses strong intrinsic therapeutic activity. This clinical signal suggests that the initial trial results may have been constrained by trial design rather than a deficiency in efficacy, providing a robust rationale for the continued clinical advancement of BTK inhibitors. Further studies might cement the position of BTK inhibitors as an effective class by establishing success in progressive MS subtypes and refining the pharmacological features required to treat relapsing MS." (lines 457-466)  We have also elaborated about the disability outcomes in the study limitation section to make it more consistent and informative: "Because most studies did not publish post-treatment EDSS scores or mean changes in EDSS from baseline, it is difficult to interpret disability results across BTK inhibitor trials. The lack of EDSS data limits direct quantitative comparison of disability trajectories among studies. Instead, few studies evaluated disability through confirmed disability worsening (CDW) or confirmed disability progression (CDP) endpoints. The reporting of CDW and CDP outcomes was largely confined to trials with longer follow-up durations, whereas shorter phase 2 studies primarily focused on MRI and relapse-related endpoints. Given the slow accumulation of disability in multiple sclerosis, shorter follow-up periods may be insufficient to detect meaningful changes in disability status. Consequently, disability outcomes were less reported in these studies." (lines 472-482)

Comment 2: The analysis did not include mortality or number of subjects abandoning the trials due to hepatotoxicity. I personally believe this information is important, and that was retrievable through the analysis. An explanation or comment on this point would be very informative.
Response 2: Certainly. Based on the analysis of all the articles for treatment discontinuations or mortalities due to hepatotoxicity, we have added a new paragraph in discussion showing significant findings: "While hepatotoxicity-related mortality and treatment discontinuation were rare across the included studies, severe hepatic events were clinically significant when they occurred. For instance, in the evolutionRMS trials, three participants met Hy's law criteria and recovered following treatment discontinuation. However, the 2 deaths did occur during the trial, which were not considered treatment-related [24]. In the phase 3 tolebrutinib study, one participant underwent liver transplantation and subsequently died from postoperative complications following tolebrutinib-related severe liver injury [26]. This highlights the importance of liver-function monitoring during BTK inhibitor therapy." There are other points, such as the FDA's hold on tolebrutinib due to cases of drug-induced liver injury, which has already been mentioned.

Comment 3: This study is authored by researchers from India, Nepal and the USA, and while the absence of commercial or financial relationships, hence no COI, are clearly stated, the source(s) of support of the study and publication, are not mentioned. 
Response 3: We thank the reviewer for pointing out the omission of a funding statement. We have now included a 'Funding' section in the manuscript to clarify that this study received no specific external funding.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

thank you for your corrections and explanations. I do not have any further.

Kind regards

Reviewer

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing the questions. observations, posed by this reviewer.