Prognostic relevance of multiparameter flow cytometry-based MRD assessment in patients with FLT3-ITD mutated AML considering concomitant mutations including DNMT3A/NPM1/WT1 mutation Free

Introduction Measurable residual disease (MRD) monitoring is informative for long-term prognosis in patients with acute myeloid leukemia (AML) at the time of remission assessment. While next-generation sequencing-based MRD monitoring in FLT3-ITD AML is being explored for clinical use, its adoption is slow due to technological and cost limitations. Multiparameter flow cytometry (MFC)-based MRD is widely used for MRD assessment across all AML subtypes. In our previous report, patients with lower FLT3-ITD allele frequency and negative MFC-MRD showed 12-month OS of 86% (vs 16.7%, p=0.023; ASH 2024).

Concurrent mutations (e.g., NPM1, DNMT3A, WT1) in FLT3-ITD AML has diverse prognostic outcomes. DNMT3A and WT1 predict adverse outcomes, while NPM1 improves relapse-free survival (RFS). We hypothesized that MFC-MRD provides prognostic value in FLT3-ITD AML independently of these mutations and explored whether it stratifies prognosis for RFS and OS regardless of molecular subtype.

Patients and method The present study evaluated the outcomes of 111 AML patients with FLT3-ITD diagnosed and treated from 2018 to 2022 at Princess Margaret Cancer Centre. We compared outcomes by MFC-MRD status at remission and molecular subtype (i.e. NPM1, DNMT3A, or WT1 mutation) using a 0.1% MRD cutoff. Primary endpoints were OS and RFS. Secondary endpoints included cumulative incidence of relapse (CIR) and non-relapse mortality (NRM). Kaplan-Meier method with log-rank test and Cox proportional hazard models were used for OS/RFS analyses, while Gray test and Fine-Grey models were used for CIR and NRM.

Results A total of 111 patients were included with a mean age of 63.5 years. According to the European LeukemiaNet (ELN) 2022 classification, patients were stratified as favorable (2, 1.8%), intermediate (67, 60.4%), and adverse (42, 37.8%). NPM1 co-mutations were found in 56 patients (50.5%), DNMT3A or WT1 in 39 (35.1%), and combined NPM1 with DNMT3A or WT1 in 28 (25.2%). Ninety patients received treatment. Seventy-two patients (80%) received intensive chemotherapy with midostaurin used in combination for 60 patients (83.3%).

Overall response was achieved in 79 patients (87.8%), including 69 (76.7%) with complete remission (CR) or CR with incomplete recovery (CRi). MFC-MRD data was available for 61 patients, with 44 (56.4%) negative and 17 (21.8%) positive. Fifty-four patients achieved CR1 after one induction cycle, highest in those with NPM1 co-mutations but no other founder or MDS-related mutations (92%, p=0.017).

With a median follow-up of 437 days among treated patients, 50 (45%) were alive. Two-year OS and RFS rates were 55.6% and 57.1%, respectively. Among FLT3-ITD AML patients achieving CR1, MFC-MRD negativity was associated with improved RFS (2-year RFS 71.2% vs 35.2%, p=0.04), though no OS differences were observed in the overall cohort. Forty-six patients (51.1%) received an allogeneic transplant.

Patients were categorized into three groups based on co-mutation profiles: FLT3-ITD and NPM1 mutation; FLT3-ITD and DNMT3A/WT1 without NPM1; and combined FLT3-ITD, NPM1, and DNMT3A/WT1. No differences in OS, RFS, CIR, or NRM were observed among these groups. When evaluating the impact of MFC-MRD status at CR1 in each subgroup according to their co-mutation profiles, we could not observe any significant differences of its impact on OS probably due to small subject numbers in each subgroup.

In co-mutation subgroups, FLT3-ITD and NPM1 had a 2-year OS of 68.6% (vs 51.4%, p=0.37) and RFS of 69.2% (vs 53.2%, p=0.34). FLT3-ITD and DNMT3A/WT1 without NPM1 showed 2-year OS of 50% (vs 56%, p=0.98) and RFS of 51.9% (vs 57.8%, p=0.63). Combined FLT3-ITD, NPM1, and DNMT3A/WT1 had 2-year OS of 72.7% (vs 49.6%, p=0.63) and RFS of 48.3% (vs 60.5%, p=0.44). No differences were noted for CIR or NRM.

Multivariate analysis for OS, incorporating age at diagnosis, ELN 2022 risk stratification, MFC-MRD status post-induction, and co-mutation profiles, identified MFC-MRD status as the only independent prognostic factor for OS (HR 3.07 [1.24-7.60], p=0.015). No independent factors were identified for RFS, CIR, or NRM.

ConclusionThe present study suggests that MFC-based MRD assessment is feasible in AML with FLT3-ITD regardless of the co-mutations in NPM1, DNMT3A, or WT1. MFC-MRD negativity showed better RFS compared to MFC-MRD positivity. However, larger studies are needed to clarify the prognostic impact of MFC-MRD within specific co-mutational subgroups.