Prognostic Impact of FLT3-ITD Microclones in AML: An EHA AML-Specialized Working Group Study

The 2022 ELN recommendations reclassified fms-like tyrosine kinase-3 (FLT3)-ITD mutated acute myeloid leukemia (AML) as intermediate-risk, irrespective of allelic ratio (AR), and designated capillary electrophoresis (CE) as the preferred detection method. However, CE requires substantial expertise, particularly to identify FLT3-ITD microclones (FLT3-ITD^m; AR < 0.05), whose clinical significance remains uncertain. Herein, we investigated diagnostic aspects and the prognostic impact of FLT3-ITD^m. An initial methodological study performed on 48 FLT3-ITD+ samples, with AR 0.001-6.00, showed overall concordance between CE and next-generation sequencing (NGS) results (p < 0.001), with only three microclones (AR: 0.002, 0.001, and 0.008) identified by CE and not by NGS. We next analyzed 658 AML patients diagnosed with a FLT3-ITD+ AML (n = 446 with AR ≥ 0.05, and n = 212 with a microclone) between 2017 and 2022. Patients with FLT3-ITD^m, as compared to patients with AR ≥ 0.05, were older, more often had secondary-type AML, and displayed a higher prevalence of myelodysplasia-related gene mutations, while NPM1-mutation frequency was similar in the two groups. Complete remission rates were lower in FLT3-ITD^m (74%) than in patients with AR ≥ 0.05 (88%) treated with intensive chemotherapy without FLT3-inhibitors (FLT3i). In the intensively treated subgroup, multivariate analyses demonstrated that age, NPM1 mutation, and allogeneic stem cell transplantation, rather than FLT3-ITD AR, were independent predictors of disease-free survival. At relapse, 15 of 33 (45%) evaluable patients remained FLT3-ITD+, with 10 expanding the original microclone. In conclusion, FLT3-ITD^m are associated with older age, secondary AML, and with expansion of the FLT3-ITD clone upon relapse, thereby providing a rationale for the evaluation of FLT3i in randomized clinical trials.