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  • Elisa Tarozzi 1,
  • Katja Seipel 1,2,* and
  • Thomas Pabst 1,*
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments to the Authors

1. As autologous stem cell transplantation (ASCT) represents primarily a cytotoxic rather than an immunotherapeutic approach, it would support the rationale of the study if the introduction included a brief discussion of the existing literature addressing the potential immunological effects of ASCT (if any). In particular, outlining prior evidence on immune modulation in this context (even if it is for ASCT in other oncological or autoimmune diseases) would help justify the investigation of CTLA-4-related mechanisms in AML.

2. It would be helpful if you could clarify the clinical context in which ASCT is applied for AML at your center. For instance, is this approach aligned with a specific protocol (e.g., prior, published, HOVON trial protocols)? Is it employed as consolidation therapy preferably in lower-risk cases, or in all cases (probably not)? 

3. In the first paragraph of the Discussion, the suggestion that differences in allele frequency of this CTLA-4 polymorphism between the ALFA registry and your AML ASCT cohort may reflect disease-specific pathogenetic mechanisms appears too speculative (even more so because it refers to the AML cohort you have auto-transplanted, and not to your entire center AML cohort). It would be more appropriate to just state cautiously that such questions require investigation in larger cohorts, ideally including healthy control groups etc, as you briefly note.

4. Have you explored the results specifically within the favorable-risk AML subgroup? Given that this appears to be a relatively larger subgroup in this study, it could be informative to examine whether univariate or multivariate analyses within this cohort yield more statistically robust insights into the potential impact of the CLTA4 genotypes.

Overall assessment:

The scientific and statistical methodology applied in this study appears sound. The presented analysis of this study does not demonstrate statistically significant evidence that the CTLA4 polymorphism of interest influences outcomes following ASCT. Nonetheless, the observed trend toward improved outcomes associated with the polymorphic genotype could, potentially, be meaningful. Even in the absence of statistical significance, such findings could be of interest to researchers in the field of immunotherapy (even if they are to be considered as "negative" results)

At present, therapeutic strategies targeting immune checkpoints (PD-1/PD-L1, CTLA-4) in MDS and AML have not really shown promising results, either in the post-allogeneic setting or in combination with chemotherapy, based on limited studies. Nevertheless, the findings presented here may contribute  to the literature—either as a basis for further, more nuanced investigations with larger, controlled cohorts, or as informative negative results that could guide future research directions.

Author Response

Reviewer 1:

  1. As autologous stem cell transplantation (ASCT) represents primarily a cytotoxic rather than an immunotherapeutic approach, it would support the rationale of the study if the introduction included a brief discussion of the existing literature addressing the potential immunological effects of ASCT (if any). In particular, outlining prior evidence on immune modulation in this context (even if it is for ASCT in other oncological or autoimmune diseases) would help justify the investigation of CTLA-4-related mechanisms in AML.

Authors: We thank the reviewer for this important comment. We have added to the introduction a brief section reviewing the existing literature on the potential immunological effects of ASCT, in order to better justify the investigation of CTLA-4 in this setting (line 75-85).

  1. It would be helpful if you could clarify the clinical context in which ASCT is applied for AML at your center. For instance, is this approach aligned with a specific protocol (e.g., prior, published, HOVON trial protocols)? Is it employed as consolidation therapy preferably in lower-risk cases, or in all cases (probably not)? 

Authors: Autologous stem cell transplantation is the standard of care at our center for patients with intermediate and favorable risk, in accordance with the HOVON protocol. In exceptional cases, autologous transplantation was also performed in patients with adverse risk, either due to the absence of a suitable donor or patient refusal of allogeneic transplantation.

  1. In the first paragraph of the Discussion, the suggestion that differences in allele frequency of this CTLA-4 polymorphism between the ALFA registry and your AML ASCT cohort may reflect disease-specific pathogenetic mechanisms appears too speculative (even more so because it refers to the AML cohort you have auto-transplanted, and not to your entire center AML cohort). It would be more appropriate to just state cautiously that such questions require investigation in larger cohorts, ideally including healthy control groups etc, as you briefly note.

Authors: We thank the reviewer for this important comment. We have revised the opening paragraph of the Discussion section, further emphasizing that any potential effect of the CTLA-4 rs231775 polymorphism on AML susceptibility is purely speculative, and that adequately powered case-control studies would be required to draw meaningful conclusions.

 

  1. Have you explored the results specifically within the favorable-risk AML subgroup? Given that this appears to be a relatively larger subgroup in this study, it could be informative to examine whether univariate or multivariate analyses within this cohort yield more statistically robust insights into the potential impact of the CLTA4 genotypes.

 

Authors: We thank the reviewer for this comment. We have already explored this possibility; however, univariate and multivariate analyses restricted to the favorable-risk subgroup did not show any statistically significant differences among the three genotypes. The same result was observed when comparing patients within the intermediate- and adverse-risk groups.

Overall assessment:

The scientific and statistical methodology applied in this study appears sound. The presented analysis of this study does not demonstrate statistically significant evidence that the CTLA4 polymorphism of interest influences outcomes following ASCT. Nonetheless, the observed trend toward improved outcomes associated with the polymorphic genotype could, potentially, be meaningful. Even in the absence of statistical significance, such findings could be of interest to researchers in the field of immunotherapy (even if they are to be considered as "negative" results).

At present, therapeutic strategies targeting immune checkpoints (PD-1/PD-L1, CTLA-4) in MDS and AML have not really shown promising results, either in the post-allogeneic setting or in combination with chemotherapy, based on limited studies. Nevertheless, the findings presented here may contribute to the literature—either as a basis for further, more nuanced investigations with larger, controlled cohorts, or as informative negative results that could guide future research directions.

Reviewer 2 Report

Comments and Suggestions for Authors
  1. Explain the prevelance of AML among European POpulation.
  2. How you have calculated the sample size for this study.
  3. What are AML inclusion exclusion criteria is M3 is excluded.
  4. Is samples are newly diagnosis cases and what are treatment plan adopted, is it 7+3 in the starting phase, venetoclax is used or not.
  5. CTLA Gene Analysis method must be included only given reference is not sufficient.
  6. One latest paper published in Cancers they did exome sequencing in relapse and Denovo cases.  "Integrated Genomic Profiling of Newly Diagnosed and Relapsed Acute Myeloid Leukemia Identifies Driver Genes, Mutational Signatures, and Therapeutic Targets" this can be included and discuss if relevant.

Author Response

Reviewer 2

Comments and Suggestions for Authors

  1. Explain the prevalence of AML among European Population.

Authors. We are grateful for this important suggestion. We have added epidemiological data on the incidence and prevalence of AML in Europe in order to better highlight the clinical relevance of this malignancy and its aggressive disease course.

  1. How you have calculated the sample size for this study.

Authors. We thank the Reviewer for this important question. The sample size was not determined through a formal statistical calculation. Instead, it was defined by the number of patients with AML who underwent ASCT at the University Hospital of Bern and met the study inclusion criteria during the study period.

  1. What are AML inclusion exclusion criteria is M3 is excluded.

Authors. We thank the reviewer for this important comment. In the “Study Design and Patient Cohort” section, we have added a final paragraph to further clarify the inclusion and exclusion criteria of our study.

  1. Is samples are newly diagnosis cases and what are treatment plan adopted, is it 7+3 in the starting phase, venetoclax is used or not.

Authors: We agree with the reviewer’s comment. In the Study Design and Patient Cohort section, we have specified the type of induction therapy (7+3) and the high-dose chemotherapy regimen used. Venetoclax is not part of the standard treatment protocol at our center.

  1. CTLA Gene Analysis method must be included only given reference is not sufficient.

Authors. We agree with the reviewer and we have included the CTLA4 Gene Analysis methods.

  1. One latest paper published in Cancers they did exome sequencing in relapse and Denovo cases.  "Integrated Genomic Profiling of Newly Diagnosed and Relapsed Acute Myeloid Leukemia Identifies Driver Genes, Mutational Signatures, and Therapeutic Targets" this can be included and discuss if relevant.

 Authors: We thank the Reviewer for this thoughtful suggestion. The referenced article, which highlights the complex polygenic architecture underlying AML oncogenesis, is indeed relevant to the broader context of our work. We have incorporated this citation into the Introduction (line 54-56), where it reinforces our framing of AML as a genetically heterogeneous and multifactorial disease. We respectfully opted not to expand on this topic within the Discussion, as doing so would risk shifting the focus away from the central aim of the manuscript — namely, the immunogenetic stratification of AML patients based on CTLA4 rs231775 genotype.

Reviewer 3 Report

Comments and Suggestions for Authors

In this article “Clinical Impact of the CTLA4 rs231775 Polymorphism in Acute Myeloid Leukemia treated with Autologous Stem Cell Transplantation” by Elisa Tarozzi et al., the authors have performed a retrospective analysis of CTLA4 gene polymorphism with relation to the risk of developing acute myeloid leukemia (AML). The authors have analysed 140 AML patients with a comparable number of male and female patients treated for AML during the period 2004-2025. The main focus of the study was to investigate the rs231775 single nucleotide polymorphism (SNP) in immune modulatory molecule CTLA-4 and its correlation with the risk of developing AML following autologous stem cell transplantation (ASCT) in AML patients. The authors have reported three genetic subgroups, 62 patients (44%) homozygous for the major allele encoding the CTLA4-T17 isoform (T17hom; AA genotype), 64 patients (46%) heterozygous carriers of the rs231775 SNP (T17Ahet; AG genotype), and 14 patients (10%) homozygous for the minor allele (A17hom; GG genotype) within the 140 study participants. They have reported the similarities and differences in the baseline clinical characteristics within these three genetic subgroups, which suggest a clear trend in the risk of AML pathogenesis associated with each genotype, and interestingly a favourable response for the A17hom subgroup. Using univariate and multivariate analyses, they report a favourable one-year and four-year progression free survival (PFS) and overall survival (OS) against AML pathogenesis following ASCT in the A17hom subgroup. Interestingly, they have reported a graded response to the risk of developing AML with CTLA4 T17hom subgroup having the highest risk, T17het intermediate and A17hom the lowest risk of developing the disease. This is potentially of interest and could be helpful in devising strategies to treat AML patients carrying a distinct CTLA4 rs231775 SNP. The authors conclude that analysis of rs231775 polymorphism may serve as a candidate biomarker for post-transplant risk stratification, which is of interest for the treatment management of AML patients.

            The article is written and described well with the content easily comprehensible and clinically relevant. However, there are some points which need attention and should be clarified to make the study robust.

Comments

  1. Concerning the data on leukocytes, leukopenia, leukocytosis, blasts in PB and blasts in BM in Table 1, is there any information on how at molecular level CTLA4 with A17hom polymorphism confers these beneficial effects?
  2. What could be the mechanism of this graded response (section 3.3. Treatment Outcomes-Univariate Analysis) in the progression-free survival (PFS) and overall survival (OS) among the three genetic subgroups with A17hom and T17hom groups at the opposite extremes?
  3. For the data shown in Fig. 1A and B, there is not much difference among the three groups for both PFS and OS in the long term at 120 months if looked at % survived. The authors need to discuss if their conclusion is still valid in this situation.
  4. For the CR and PR data in Table 2, response is comparable in all three groups. With the sample size 4.5 times smaller than the T17hom and T17Ahet groups, the CR and PR might change if compared with similar number of patients in the A17hom group. The small sample size analysed in the study, especially in the A17hom group, is a problem to accept the conclusions convincingly.
  5. For the rest of the data in Table 2, although, there seems to be a trend with T17hom the least, T17Ahet in between and A17hom the highest one- and five-year PFS, again the smaller sample size in the A17hom group could influence the outcome in favour of this group. Therefore, it is difficult to agree with the conclusion made here.
  6. The observations for A17hom SNP as reported in the study, if true, it is of interest and significance for the AML patients with this particular CTLA4 polymorphism. Do the authors have any idea about how A17hom SNP confers this better survival to AML patients considering the mechanism of action of CTLA-4?
  7. Concerning lines 253-257, the graded response with the three CTLA4 genetic subgroups with regard to PFS and OS is very interesting. But how does that happen at the molecular and cellular level? Need to discuss.
  8. Concerning lines 288-292, what is the molecular basis for it? It will be of interest to understand this adverse response (the AA genotype), as well as the favourable response imparted by the A17hom SNP (GG genotype) as this has been observed in multiple blood cancers.
  9. Abbreviations such as MDS and BPDCN etc., should be extended in full at the point of first use.

Author Response

Reviewer 3

Comments and Suggestions for Authors

In this article “Clinical Impact of the CTLA4 rs231775 Polymorphism in Acute Myeloid Leukemia treated with Autologous Stem Cell Transplantation” by Tarozzi et al., the authors have performed a retrospective analysis of CTLA4 gene polymorphism with relation to the risk of developing acute myeloid leukemia (AML). The authors have analyzed 140 AML patients with a comparable number of male and female patients treated for AML during the period 2004-2025. The main focus of the study was to investigate the rs231775 single nucleotide polymorphism (SNP) in immune modulatory molecule CTLA-4 and its correlation with the risk of developing AML following autologous stem cell transplantation (ASCT) in AML patients. The authors have reported three genetic subgroups, 62 patients (44%) homozygous for the major allele encoding the CTLA4-T17 isoform (T17hom; AA genotype), 64 patients (46%) heterozygous carriers of the rs231775 SNP (T17Ahet; AG genotype), and 14 patients (10%) homozygous for the minor allele (A17hom; GG genotype) within the 140 study participants. They have reported the similarities and differences in the baseline clinical characteristics within these three genetic subgroups, which suggest a clear trend in the risk of AML pathogenesis associated with each genotype, and interestingly a favorable response for the A17hom subgroup. Using univariate and multivariate analyses, they report a favorable one-year and four-year progression free survival (PFS) and overall survival (OS) against AML pathogenesis following ASCT in the A17hom subgroup. Interestingly, they have reported a graded response to the risk of developing AML with CTLA4 T17hom subgroup having the highest risk, T17het intermediate and A17hom the lowest risk of developing the disease. This is potentially of interest and could be helpful in devising strategies to treat AML patients carrying a distinct CTLA4 rs231775 SNP. The authors conclude that analysis of rs231775 polymorphism may serve as a candidate biomarker for post-transplant risk stratification, which is of interest for the treatment management of AML patients.

The article is written and described well with the content easily comprehensible and clinically relevant. However, there are some points which need attention and should be clarified to make the study robust.

Comments

  1. Concerning the data on leukocytes, leukopenia, leukocytosis, blasts in PB and blasts in BM in Table 1, is there any information on how at molecular level CTLA4 with A17hom polymorphism confers these beneficial effects?

Authors: We thank the Reviewer for this insightful question. At present, no direct mechanistic evidence specifically links the CTLA4 A17 homozygous polymorphism to leukocyte counts, peripheral blood or bone marrow blast levels in acute myeloid leukemia. However, CTLA4 is a key immune checkpoint receptor involved in the negative regulation of T-cell activation, and genetic variation in CTLA4 may influence immune surveillance and anti-tumor immune responses. Therefore, it is conceivable that CTLA4 polymorphisms may indirectly modulate the host immune environment and tumor–immune interactions, which could in turn contribute to differences in disease burden and hematologic parameters. Nevertheless, these associations remain speculative and require further functional validation.

  1. What could be the mechanism of this graded response (section 3.3. Treatment Outcomes-Univariate Analysis) in the progression-free survival (PFS) and overall survival (OS) among the three genetic subgroups with A17hom and T17hom groups at the opposite extremes?

Authors: We thank the Reviewer for this insightful comment. The observed graded pattern in progression-free survival (PFS) and overall survival (OS) across rs231775 genotypes may be biologically explained by a dose-dependent effect of CTLA4-mediated immune regulation. CTLA-4 is a key immune checkpoint receptor that negatively regulates T-cell activation; therefore, genetic variation affecting its expression or functional activity may result in different levels of immune surveillance against residual leukemic cells after ASCT. In this context, homozygous genotypes at opposite ends of the spectrum (A17hom and T17hom) may correspond to differential CTLA-4 expression levels and thus distinct degrees of T-cell inhibition, whereas heterozygous carriers (T17Ahet) may exhibit intermediate functional effects, consistent with an additive genetic model. This could potentially translate into a graded antitumor immune response and, consequently, differences in post-transplant outcomes. We acknowledge that this mechanism remains speculative, as functional data directly linking rs231775 genotypes to post-ASCT immune reconstitution and clinical outcomes in AML are limited, and further mechanistic studies are required to validate this hypothesis.

  1. For the data shown in Fig. 1A and B, there is not much difference among the three groups for both PFS and OS in the long term at 120 months if looked at % survived. The authors need to discuss if their conclusion is still valid in this situation.

Authors: We agree with the reviewer’s observation. In lines 315–319, we clarified that the benefit associated with the A17hom genotype is mainly restricted to the immediate post-transplant period and is no longer evident in the long term. This finding further highlights the importance of initiating consolidation therapy shortly after transplantation in order to maximize clinical benefit.

  1. For the CR and PR data in Table 2, response is comparable in all three groups. With the sample size 4.5 times smaller than the T17hom and T17Ahet groups, the CR and PR might change if compared with similar number of patients in the A17hom group. The small sample size analyzed in the study, especially in the A17hom group, is a problem to accept the conclusions convincingly.

Authors: We fully agree with the reviewer and acknowledge this major limitation of our study. In lines 327–329, we explicitly state that the small size of this cohort reduces the statistical power of our analyses, and we emphasize the need for validation in larger studies.

We would like to draw the reviewer’s attention to the fact that Table 2 has been updated with respect to the “best outcomes” after transplantation. During manuscript revision, we identified an error and corrected it by properly reporting patients who achieved complete remission and those who did not. The resulting values remain comparable across the three genotypes, likely reflecting the limited sample size of the cohort.

  1. For the rest of the data in Table 2, although, there seems to be a trend with T17hom the least, T17Ahet in between and A17hom the highest one- and five-year PFS, again the smaller sample size in the A17hom group could influence the outcome in favor of this group. Therefore, it is difficult to agree with the conclusion made here.

Authors: We thank the reviewer for the comment. As mentioned above, the small number of patients limits the strength of our conclusions; however, we believe that validation in larger studies could confirm the trends observed in our cohort.

  1. The observations for A17hom SNP as reported in the study, if true, it is of interest and significance for the AML patients with this particular CTLA4 polymorphism. Do the authors have any idea about how A17hom SNP confers this better survival to AML patients considering the mechanism of action of CTLA-4?

Authors: We thank the Reviewer for this insightful question. We hypothesize that the improved survival observed in A17hom carriers may be related to lower CTLA-4 surface expression. This could result in reduced inhibition of T-cell activation and consequently a stronger antitumor immune response against leukemic cells.

  1. Concerning lines 253-257, the graded response with the three CTLA4 genetic subgroups with regard to PFS and OS is very interesting. But how does that happen at the molecular and cellular level? Need to discuss.

Authors: We thank the Reviewer for this valuable point of discussion. We hypothesize that the graded response observed across the three genotypes may reflect a dose-dependent effect of CTLA-4 surface expression, with A17hom showing the lowest expression levels, T17hom the highest, and T17Ahet intermediate levels. We kindly refer the Reviewer to point 2 for further discussion of this aspect.

  1. Concerning lines 288-292, what is the molecular basis for it? It will be of interest to understand this adverse response (the AA genotype), as well as the favorable response imparted by the A17hom SNP (GG genotype) as this has been observed in multiple blood cancers.

Authors: We thank the Reviewer for this valuable input. We hypothesize that a plausible molecular explanation may be related to the differential expression of CTLA-4 among the three genotypes. Several studies have reported higher CTLA-4 expression levels in the AA genotype, resulting in stronger inhibition of the antitumor immune response, which may be consistent with the poorer post-transplant survival observed in these patients. Conversely, lower CTLA-4 expression levels have been described in the GG genotype, potentially leading to a more robust anti-leukemic immune response. A paragraph discussing this hypothetical molecular mechanism has been added in lines 299–308.

  1. Abbreviations such as MDS and BPDCN etc., should be extended in full at the point of first use.

Authors: We agree with the Reviewer’s comment and have defined these abbreviations in full at their first mention in the manuscript.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have responded satisfactorily to the comments made on the original submission. They agree that detailed study to understand the mechanistic details, with large sample size in all three groups is essential to make any solid conclusion.