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Abstract: Internal tandem duplications in fms-like tyrosine kinase 3 (FLT3-ITDs) are common in acute myeloid leukemia (AML) and confer a poor prognosis. A sensitive and specific assay for the detection of minimal residual disease (MRD) in FLT3-ITD mutated AML could guide therapy decisions. Existing assays for MRD in FLT3-ITD AML have not been particularly useful because of limited sensitivity. We developed a sensitive and specific MRD assay for FLT3-ITD mutations using next-generation sequencing. The initial validation of this assay was performed by spiking fixed amounts of mutant DNA into wild-type DNA to establish a sensitivity of detection equivalent to ≥1 FLT3-ITD–containing cell in 10 000, with a minimum input of 100 000 cell equivalents of DNA. We subsequently validated the assay in bone marrow samples from patients with FLT3-ITD AML in remission. Finally, we analyzed bone marrow samples from 80 patients with FLT3-ITD relapsed/refractory AML participating in a trial of a novel FLT3 inhibitor, gilteritinib, and demonstrated a relationship between the mutation burden, as detected by the assay, and overall survival. This novel MRD assay is specific and 2 orders of magnitude more sensitive than currently available polymerase chain reaction– or next-generation sequencing–based FLT3-ITD assays. The assay is being prospectively validated in ongoing randomized clinical trials.