A TFPI missense variant provides protection against severe febrile neutropenia in pediatric Acute Myeloid Leukemia

Abstract Background: Toxicity due to antileukemic agents is common in acute myeloid leukemia (AML) patients and significantly impacts morbidity and mortality. Intensive chemotherapy regimens, such as the standard “7+3” induction protocol with cytarabine and daunorubicin, frequently result in severe neutropenia, markedly increasing susceptibility to infections (PMID: 38961525). Here we evaluated the impact of genetic variants on risk of severe febrile neutropenia post-induction 1 therapy. Methods: Febrile Neutropenia (FN) severity was graded based on the Common Terminology Criteria for Adverse Events (CTCAE), version 3.0. Grade 3 FN was defined as an absolute neutrophil count <1.0 × 10⁹/L with fever ≥38.5°C, without clinically or microbiologically documented infection. Grade 4 FN represented life-threatening complications. Patients enrolled in multisite trials AML02 and AML08 (N=400) with toxicity data were included in the study. Patients were stratified into two groups: those with severe FN (Grade ≥3) and those with low-grade (Grade 1–2) or no FN. Genome wide association study used the Illumina 2.5 Omni array, established quality control steps were used for data preparation. Logistic regression model, adjusted for ancestry, was used to test SNP associations with severe FN. Genome-wide significance was defined as P < 5 × 10⁻⁸, with a suggestive threshold at P < 1 × 10⁻⁴. Transcriptomic data from diagnostic specimen were available in 137 patients from AML02 trial and was used for evaluation with toxicity. Results: One of the top SNPs, rs8176592, (A>G) significantly associated with severe FN mapped to chromosome 2 in the regions with genes TFPI (tissue factor pathway inhibitor), CALCRL (calcitonin receptor-like receptor) and CALCRL-AS. Presence of G allele for the tag SNP rs8176592 associated with a significantly reduced risk of developing grade ≥3 FN (odds ratio [OR] = 0.46; 95% CI: 0.30–0.70; p = 9.79×10⁻⁵). rs8176592 is in intronic region of TFPI, CALCRL-AS1 and in promoter of CALCRL, it occurs in LD with several SNPs (~49 SNPs in LD at r2>0.95) spanning across the 3 genes including a mis-sense variant rs7586970 (T>C) in TFPI resulting in Asparagine to Serine change. The protective effect was dose-dependent: 83% of patients with homozygous genotype for the reference allele developed grade 3 or more FN, compared to 73% of heterozygous and 54% of patients with homozygous genotype for the variant allele (p <0.001). Further evaluation of using the GTEx database showed that for rs8176592, G allele was associated with significantly low levels of CALCRL and TFPI in several tissues and as a splice QTL for TFPI. Further evaluation of these genes showed CALCRL expression in AML02 cohort (N=137 with available transcriptomics data) to be associated with greater risk of grade 3 or more FN (p = 0.0004), though such an association with TFPI expression was not observed. These findings suggest that lower CALCRL and TFPI levels may protect against FN. TFPI is a known regulator of coagulation and has been implicated in inflammation through signaling mediated via activation of protease activated receptors, due to its dual anticoagulant and anti-inflammatory role it has been closely linked to the pathophysiology of sepsis; CALCRL which encodes a G-protein-coupled receptor has emerged as biologically relevant gene due to its role in vascular stability and inflammation, it is involved in adrenomedullin signaling which maintains vascular integrity and regulates immune responses during infection and sepsis. The presence of associated variants in both CALCRL and TFPI suggests that this genomic region may influence FN risk through coordinated modulation of vascular, inflammatory, and coagulative pathways. Conclusion: Our study reveals a common genetic variant in CALCRL and TFPI to be associated with substantially reduced risk of severe FN during induction therapy in pediatric AML. Future directions include independent validation in larger and more diverse AML cohorts, and functional validation studies to uncover the mechanistic role of CALCRL and TFPI in observed toxicity. Ultimately, these insights could support the development of personalized supportive care strategies in pediatric AML.