Comprehensive profiling of AML and T-cell states across disease stages in acute myeloid leukemia using spectral flow cytometry

1. Immune profiling reveals dynamic shifts in leukemic and non-leukemic cell populations across AML stages. 2. Mutation-specific alterations in immune subsets provide insights into AML progression and therapy resistance. Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal expansion of myeloid precursors yet the interplay between leukemic and immune cells across disease stages remains poorly understood. Here, we used spectral flow cytometry and high-dimensional computational analyses to profile peripheral blood mononuclear cells from 72 AML patients across three disease stages: newly diagnosed, remission, and relapsed/refractory. Clustering analyses identified stage-specific enrichment patterns in myeloid and lymphoid populations, with leukemic CD34+ and CD123+ cells dominating in relapsed/refractory patients and monocytic and CD45low clusters enriched in remission. T-cell profiling revealed terminal effector and senescent subsets in relapsed/refractory patients, suggesting immune exhaustion as a contributor to disease progression. Mutation-specific analyses linked TP53 , DNMT3A , and NPM1 mutations to distinct enrichment patterns in both leukemic and immune populations, including increased CD71+ AML cells and altered T-cell distributions. These findings provide insights into the dynamic cellular ecosystem of AML, highlighting mutation-driven immune dysregulation and potential therapeutic targets to improve outcomes. This comprehensive profiling underscores the critical role of immune modulation in AML progression and relapse, paving the way for novel immune-targeted therapies.