Elevated Allele Frequency of a Common Germline LAG3 Variant Associated with Anemia, Thrombocytopenia and Peripheral Blast Percentage in Acute Myeloid Leukemia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the association between germline variants in LAG3 (rs870849) and CTLA4 (rs231775) with disease risk, clinical characteristics, and outcomes in AML. The study reports a significantly higher minor allele frequency of LAG3 rs870849 in AML patients compared to the European population, along with a dose-dependent increase in disease risk and associations with hematologic parameters. The authors propose that LAG3 rs870849 is a potential genetic predisposition factor in AML.

The topic is timely and relevant, particularly given the increasing interest in immune checkpoint biology and germline predisposition in hematologic malignancies. However, several issues need to be addressed before the manuscript can be considered for publication.

1. Insufficient details are there for reproducibility, for instance, PCR/sequencing conditions are not described. Likewise, software/tools not fully specified. Please add the URL for all the softwares and databases used. Hence, expand methods to ensure reproducibility.
2. Add more recent AML GWAS and immunogenetics studies (2023–2025) for stronger context.
3. Figures 1 and 2 are dense and difficult to interpret. Add clear legends explaining genetic models and improve labelling and readability for better visualization.
4. The study limitations such as small, single-center cohort, lack of matched controls, no functional validation, potential population stratification and limited multivariate significance should be discussed.
5. Briefly discuss the Role of Hardy–Weinberg equilibrium in genetic association studies and corelate it with the meaningful interpretation of the present study.
6. Survival differences (PFS, OS) are either marginally significant or non-significant and not retained in multivariate analysis. Yet conclusions suggest meaningful clinical impact. Authors should reframe conclusions to reflect lack of independent prognostic value and avoid overstating survival associations.
7. Some reported p-values are borderline (e.g., p=0.05–0.06) yet interpreted as biologically meaningful. Clearly distinguish exploratory vs. confirmatory findings.
8. Language improvement and proofreading is essential.

Author Response

Comments and Suggestions for Authors

This manuscript investigates the association between germline variants in LAG3 (rs870849) and CTLA4 (rs231775) with disease risk, clinical characteristics, and outcomes in AML. The study reports a significantly higher minor allele frequency of LAG3 rs870849 in AML patients compared to the European population, along with a dose-dependent increase in disease risk and associations with hematologic parameters. The authors propose that LAG3 rs870849 is a potential genetic predisposition factor in AML.

The topic is timely and relevant, particularly given the increasing interest in immune checkpoint biology and germline predisposition in hematologic malignancies. However, several issues need to be addressed before the manuscript can be considered for publication.

1. Insufficient details are there for reproducibility, for instance, PCR/sequencing conditions are not described. Likewise, software/tools not fully specified. Please add the URL for all the softwares and databases used. Hence, expand methods to ensure reproducibility.

Response: Methods have been expanded. Software and database tools have been specified, URLs have been added.

2. Add more recent AML GWAS and immunogenetics studies (2023–2025) for stronger context.

Response: More recent AML GWAS studies have been added in the discussion.

1. Figures 1 and 2 are dense and difficult to interpret. Add clear legends explaining genetic models and improve labelling and readability for better visualization.

Response: Figures and legends have been improved.

1. The study limitations such as small, single-center cohort, lack of matched controls, no functional validation, potential population stratification and limited multivariate significance should be discussed.

Response: We thank the reviewer for the helpful comments. We have addressed the concerns in the revised discussion. The study was limited by sample size, retrospective single center design and selection bias with a majority of patients with favorable risk disease. Due to the small sample size the investigation is explorative, including the risk of false positive and false negative results. The selection bias with a majority of patients with favorable risk disease may influence allele frequency and gene risk estimates. The comparison of allele frequencies was made against a large European database rather than a locally matched control cohort. Due to the lack of a control cohort, it was not possible to differentiate between prognostic and predictive value. The presented results may reflect genotype-specific characteristics but should be interpreted cautiously and as hypothesis-generating rather than definitive. Adequately powered case-control studies will be required to formally evaluate the role of LAG3 rs870849 in AML susceptibility. This paragraph was added at the end of the discussion. Selection bias in the population stratification has been visualized in the new figure 3.

1. Briefly discuss the Role of Hardy–Weinberg equilibrium in genetic association studies and correlate it with the meaningful interpretation of the present study.

Response: The key inferences from a genetic association study may be compromised if Hardy-Weinberg equilibrium (HWE) is violated (Salanti et al., 2005). With no deviations from HWE in the analyzed data-set, the LAG3 and CTLA4 allele frequencies calculated for the AML groups are validated. This sentence was added to the results section.

1. Survival differences (PFS, OS) are either marginally significant or non-significant and not retained in multivariate analysis. Yet conclusions suggest meaningful clinical impact. Authors should reframe conclusions to reflect lack of independent prognostic value and avoid overstating survival associations.

Response: Conclusions have been revised to avoid overstatements.

1. Some reported p-values are borderline (e.g., p=0.05–0.06) yet interpreted as biologically meaningful. Clearly distinguish exploratory vs. confirmatory findings.

Response: Manuscript has been revised to avoid overstatements.

1. Language improvement and proofreading is essential.

 

Submission Date 23 April 2026

Date of this review 05 May 2026 13:36:58

Date of revision 12 May 2026.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This paper looks at two immune checkpoint germline variants (LAG3 rs870849 and CTLA4 rs231775) in AML. In 140 AML patients, LAG3 rs870849 shows a clearly higher minor allele frequency than the European reference population and is associated with a dose-dependent increase in AML risk, while CTLA4 rs231775 does not show a significant risk signal. The authors also report that baseline blood count profiles differ across LAG3 genotypes, suggesting a link between this germline variant and erythrocyte/leukocyte/platelet imbalance. 

Major Issues:

1.The comparison is made against a large European database frequency rather than a locally matched control cohort. Please discuss more clearly how population structure and selection (e.g., sampling after CR) may influence allele frequency estimates and risk ORs, and frame the findings accordingly.

2. Survival differences by genotype are shown in univariate analyses, but the variant is not significant in multivariable models. Please tone down statements implying a clear survival benefit and label outcome associations as exploratory.

3.The discussion proposes T-cell exhaustion/immune surveillance mechanisms, but no immune profiling is included in this study. Please clearly state this as hypothesis-level interpretation and avoid overstating causality.

Minor Issues:

1. Add a short note on how representative this cohort is compared to an unselected AML population

2. The “predisposition gene” wording in the conclusion could be softened to “associated with increased risk” unless validated in an independent matched cohort.

3. Table 1 reports many genotype–clinical characteristic comparisons with multiple p-values。Please clarify whether multiple-testing correction was applied (and report adjusted p-values if available); otherwise, these associations should be framed as exploratory.

Author Response

Comments and Suggestions for Authors

This paper looks at two immune checkpoint germline variants (LAG3 rs870849 and CTLA4 rs231775) in AML. In 140 AML patients, LAG3 rs870849 shows a clearly higher minor allele frequency than the European reference population and is associated with a dose-dependent increase in AML risk, while CTLA4 rs231775 does not show a significant risk signal. The authors also report that baseline blood count profiles differ across LAG3 genotypes, suggesting a link between this germline variant and erythrocyte/leukocyte/platelet imbalance. 

Major Issues:

1.The comparison is made against a large European database frequency rather than a locally matched control cohort. Please discuss more clearly how population structure and selection (e.g., sampling after CR) may influence allele frequency estimates and risk ORs, and frame the findings accordingly.

Response: We thank the reviewer for the helpful comments. We have addressed the concerns in the revised discussion. The study was limited by sample size, retrospective single center design and selection bias with a majority of patients with favorable risk disease. Due to the small sample size the investigation is explorative, including the risk of false positive and false negative results. The selection bias with a majority of patients with favorable risk disease may influence allele frequency and gene risk estimates. The comparison of allele frequencies was made against a large European database rather than a locally matched control cohort. Due to the lack of a control cohort, it was not possible to differentiate between prognostic and predictive value. The presented results may reflect genotype-specific characteristics but should be interpreted cautiously and as hypothesis-generating rather than definitive. Adequately powered case-control studies will be required to formally evaluate the role of LAG3 rs870849 in AML susceptibility. This paragraph was added at the end of the discussion. Selection bias in the population stratification has been visualized in the new figure 3.

Sampling the peripheral blood to determine germline variants will not affect allele frequency estimates as results of sanger sequencing are unambiguous for homozygosity of an snp (allele dosage equaling signal strength 2:0 or 0:2) and heterozygosity of an snp (allele dosage equaling signal strength 1:1). This sentence was added to the Materials and Methods.

2. Survival differences by genotype are shown in univariate analyses, but the variant is not significant in multivariable models. Please tone down statements implying a clear survival benefit and label outcome associations as exploratory.

Response: Manuscript has been revised to avoid overstatements.

3.The discussion proposes T-cell exhaustion/immune surveillance mechanisms, but no immune profiling is included in this study. Please clearly state this as hypothesis-level interpretation and avoid overstating causality.

Response: The following statements are hypothesis-driven. This sentence was added to the paragraph.

Minor Issues:

1. Add a short note on how representative this cohort is compared to an unselected AML population.

Response:  The ELN risk distribution in the local AML cohort was significantly different (p=0.01) to the ELN distribution in an unbiased AML cohort with 575 (37%) favorable, 410 (26%) intermediate, and 585 (37%) adverse risk cases. This statement is placed in the Results section. A new Figure 3 has been added to visualize the selection bias.

2. The “predisposition gene” wording in the conclusion could be softened to “associated with increased risk” unless validated in an independent matched cohort.

Response: This was a retrospective single center study on the prevalence and impact of two immune checkpoint germline variants, LAG3 rs870849 and CTLA4 rs231775, in AML. While LAG3 rs870849 showed a higher minor allele frequency in AML patients compared to the European reference population and associated with a dose-dependent increase in AML risk, CTLA4 rs231775 did not show a significant risk signal. Baseline blood count profiles differed across LAG3 genotypes, suggesting a link between LAG3 rs870849 and erythrocyte/leukocyte/platelet imbalance. This is the revised conclusion.

3. Table 1 reports many genotype–clinical characteristic comparisons with multiple p-values。Please clarify whether multiple-testing correction was applied (and report adjusted p-values if available); otherwise, these associations should be framed as exploratory.

Response: p values without Bonferroni correction for multiple testing. Associations are exploratory. This sentence was added in the Table footer.

 

Submission Date 23 April 2026

Date of this review 06 May 2026 05:45:28

Date of revision 12 May 2026

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revisions done by the authors is satisfactory and the manuscript is recommended for acceptance.