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.