A next-generation sequencing–based assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations

Internal tandem duplications in <i>fms-like tyrosine kinase 3</i> (<i>FLT3-</i>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 <i>FLT3-</i>ITD mutated AML could guide therapy decisions. Existing assays for MRD in <i>FLT3-</i>ITD AML have not been particularly useful because of limited sensitivity. We developed a sensitive and specific MRD assay for <i>FLT3-</i>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 <i>FLT3-</i>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 <i>FLT3-</i>ITD AML in remission. Finally, we analyzed bone marrow samples from 80 patients with <i>FLT3-</i>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 <i>FLT3-</i>ITD assays. The assay is being prospectively validated in ongoing randomized clinical trials.