Background: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML), occurring in approximately 25–30% of adult cases and conferring an adverse prognosis through constitutive activation of FLT3 signaling, enhanced proliferation, and impaired differentiation of leukemic blasts. FLT3-ITD mutational burden, allelic ratio, insertion site, and co-mutations have been widely studied as prognostic markers. However, the impact of the number of distinct FLT3-ITD mutated clones—reflecting clonal heterogeneity—remains underexplored. Clonal diversity at diagnosis has been linked to therapeutic resistance and relapse in AML, yet limited data exist on whether multiple FLT3-ITD clones influence treatment outcomes. This study aimed to investigate the prognostic significance of FLT3-ITD clone numbers in a real-world cohort of FLT3-ITD–positive AML patients.

Materials & Methods: We retrospectively analyzed consecutive adult AML patients harboring FLT3-ITD mutations diagnosed between April 2017 and June 2024. FLT3-ITD mutations were detected and characterized by PCR followed by capillary electrophoresis–based fragment analysis. Patients were classified into monoclone (one FLT3-ITD clone) and multiclone (two or more distinct FLT3-ITD clones) groups based on diagnostic molecular testing. Clinical characteristics, cytogenetics, treatment modalities, chemotherapy responses (per European LeukemiaNet 2022 criteria), and survival outcomes were compared. Survival was estimated using the Kaplan–Meier method, with group comparisons by the log-rank test. Multivariable Cox regression analysis adjusted for age, white blood cell count, and treatment type.

Results: A total of 221 FLT3-ITD–positive AML patients were analyzed. Of these, 155 patients received intensive chemotherapy; 52 received midostaurin in combination with daunorubicin plus cytarabine, while the remainder received standard anthracycline/cytarabine (7+3) induction without FLT3 inhibitor. Low-intensity regimens consisted of hypomethylating agent plus venetoclax or low-dose cytarabine with etoposide. In total, 168 patients (76.0%) were in the monoclone group and 43 patients (19.5%) were in the multiclone group. Among the multiclone group, 16.6% harbored two clones and 3.5% had three or more clones. Multiclone patients exhibited significantly higher bone marrow blast percentages at diagnosis (median 90% vs. 84%, p = 0.032). Median OS was shorter in the multiclone group compared with the monoclone group (1.6 years vs. 1.8 years, p = 0.046). Multivariable analysis confirmed multiclone status as an independent adverse factor for OS (HR 1.78, 95% CI 1.05–3.01, p = 0.032). Among patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT, n = 130), the negative prognostic impact of multiclone status persisted, particularly among those achieving morphologic CR prior to transplant (HR 2.05, 95% CI 1.10–3.82, p = 0.024). Initial induction response rates (composite complete remission [CR], CR with incomplete platelet recovery, and CR with incomplete hematologic recovery) were similar between the monoclone and multiclone groups (53.9% vs. 51.9%, p = 1.0). However, the final response rate, including responses achieved after salvage therapy, was significantly lower in the multiclone group (54.8% vs. 73.1%, p = 0.026). The 2-year cumulative incidence of relapse was higher in multiclone patients (48.2% vs. 31.6%, p = 0.041), with correspondingly shorter relapse-free survival (median 9.8 vs. 15.4 months, p = 0.038).

Conclusion: The presence of multiple FLT3-ITD mutated clones at diagnosis is associated with adverse clinical features, lower CR rates, and inferior OS in AML patients. The negative prognostic association persisted in post-HSCT outcomes. These findings highlight the potential of FLT3-ITD clonal heterogeneity as a biomarker for risk stratification and therapeutic decision-making in FLT3-ITD–positive AML. Although capillary electrophoresis–based fragment analysis has limitations in detecting minor subclones with very low allelic burden, it remains a valuable and practical method for assessing clonal heterogeneity in routine clinical practice. Future prospective studies integrating molecular co-mutation profiling and FLT3 inhibitor exposure are warranted to validate these observations and to develop targeted strategies for this high-risk subgroup.

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2025
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