IMMUNOPHENOTYPIC SIGNATURE OF LEUKEMIA ARREST STAGE IS ASSOCIATED WITH NPM1/FLT3 MUTATIONAL PROFILE AND PROGNOSIS IN ACUTE MYELOID LEUKEMIA

Abstract

Acute Myeloid Leukemia (AML) is a severe hematopoietic disorder marked by uncontrolled proliferation of immature myeloid progenitors due to differentiation arrest, with significant immunophenotypic diversity. FLT3-ITD mutations’ prognostic impact varies with NPM1 mutation presence and the FLT3 allelic ratio. While cytogenetic and molecular features are well-established for risk stratification by European LeukemiaNet (2017 ELN), the role of leukemia arrest stages in prognosis remains unclear. This study classified the maturation arrest stages of leukemic blasts in a Brazilian AML cohort, analyzing its association with genetic risk and clinical data. Bone marrow from 148 de novo AML patients (Sep-2015-Jul-2024) was evaluated for FLT3 and NPM1 mutations, FLT3 allelic ratio, and immunophenotyping. MPO cytochemistry stains and phenotypic markers by flow cytometry defined six stages of leukemia arrest (SLA): Hematopoietic Stem Cells-like (HSC-L:CD34+ CD117±CD13-CD33-HLA-DR+MPO-); Multipotent Progenitors-like (MPP-L:CD34+CD117+CD13±CD33±HLA-DR+MPO-); Common Myeloid Progenitors-like (CMP-L:CD34±CD117+CD13+CD33± HLA-DR+MPO+); Granulocyte-Monocyte Progenitors-like (GMP-L:CD34±CD117±CD13±CD33+HLA-DR+MPO+); Monocyte Progenitors-like (MP-L:CD34-CD117±CD13+CD33+HLA-DR+MPO+); and Granulocyte Progenitors-like (GP-L:CD34-CD117±CD13+ CD33+HLA-DR-MPO+). SLA showed highest frequency in MP-L (n = 57, 38.5%) and GP-L (n = 53, 35.8%), followed by GMP-L (n = 23, 15.5%), CMP-L (n = 7, 4.7%), MPP-L (n = 6, 4.1%), and HSC-L (n = 2, 1.4%). Predominance of FLT3wt/NPM1mut patients (n = 80, 54.1%) was observed, with similar distribution among FLT3mut patients (FLT3mut /NPM1mut:n = 32, 21.6%; FLT3mut/NPM1wt:n = 36, 24.3%). CMP-L and GMP-L frequencies were increased in FLT3low/NPM1wt and FLT3high/NPM1wt vs. FLT3wt/NPM1mut (CMP-L:13.33%, 14.28% vs 1.25%, p < 0.05; GMP-L:40%, 42.85% vs 7.5%, p < 0.05). GP-L frequency was decreased in FLT3low/NPM1wt and FLT3 high/NPM1wt vs. FLT3wt/NPM1mut (0%, 9.52% vs 43.75%, p < 0.05). Response to treatment (CR/CRi, CR/CRiMRD+ and PD–Persistence disease) was evaluated after 1st and 2nd cycles of Induction of Remission (1IR, 2IR). GMP-L was increased in PD after 1IR (OR:6.09, 95% CI:1.22–30.19, p < 0.05). Immature (HSC+MPP+CMP) SLA was higher in MRD+ patients compared to MRD- (OR:6.81, 95% CI:0.86–47.05, p < 0.05). Overall survival (p = 0.81) and relapse-free survival (p = 0.27) analyses showed that GMP-L patients relapsed sooner. In 2017 ELN risk assessment, FLT3-ITD stratification was based on allelic ratio and NPM1 mutation status. A high FLT3-ITD allelic ratio with NPM1 mutation indicated intermediate risk, and with wild-type NPM1, it indicated unfavorable risk. The 2022 ELN guidelines classify FLT3-ITD alone as intermediate risk, regardless of NPM1 status. Mutated NPM1 is favorable only when FLT3-ITD is absent. Supporting these findings, FLT3 mutation, regardless of allelic ratio, in the absence of NPM1, led to immature SLA and increased GMP-L frequency. This SLA was linked to PD after the 1IR. Immature SLA significantly impacts prognosis with lower survival rates, higher relapse risks, and increased post-induction remission failure. Our findings enhance the understanding of the cell of origin in AML, and further investigation of the SLA-dependent mechanisms that contribute to leukemia resistance are warranted.