C083 | CD99 EXPRESSION IN ACUTE MYELOID LEUKEMIA WITH FLT3 INTERNAL TANDEM DUPLICATION (FLT3-ITD^MUT-AML): A FLOW-CYTOMETRIC MARKER FOR AN ACCURATE DIAGNOSTIC WORKUP

Background: FLT3 internal tandem duplication (FLT3-ITD) in adult acute myeloid leukemia (AML) is associated with poor outcome compared to FLT3 wild-type. Multiparameter flow cytometry (MFC) is pivotal in AML diagnosis. CD99 is frequently overexpressed in AML and has emerged as a potential diagnostic marker and a novel therapeutic target. Methods: In this retrospective study, we aimed to develop an optimized cytofluorimetric diagnostic panel for predicting FLT3-ITD mutation in AML cases by integrating CD99 into our MFC panel. Data from 90 consecutive newly diagnosed AML patients in the period between 2021 and 2024 at our center were analyzed. Immunophenotypic profile obtained by routine MFC panel were correlated with molecular assessment. Patients were categorized based on FLT3 mutation status into FLT3-ITD^mut (n = 26; 28.9%) and FLT3^wt (n = 64; 71.1%). Results: CD99 was expressed in 100% of FLT3-ITD^mut-AML compared to 48% in FLT3^wtcases. Moreover, FLT3-ITD^mut-AML exhibited a significantly higher median CD99 expression percentage [100% (range, 38–100) vs. 1% (range, 0–100); p<0.001] and a higher CD99 MFI [59 (range, 20–203) vs. 20 (range, 2–392); p<0.001]. Conversely, CD34 expression was markedly lower in FLT3-ITD^mut-AML [median 3% (range, 0–100) vs. 100% (range, 0–100); p<0.001], with a positivity rate of 31% versus 83% in FLT3^wtcases (p<0.001). Univariate logistic regression revealed significant associations with FLT3-ITD mutation for CD99 positivity [OR 56.40; p<0.001], CD34 negativity [OR 11.11; p<0.001], HLA-DR negativity [OR 3.70; p=0.0019], and CD133 negativity [OR 9.09; p=0.001]. Multivariate analysis confirmed that CD99 positivity [OR 17.67 (95%CI, 1.77–251.06); p=0.010] and CD34 negativity [OR 10.00 (95%CI, 1.12–100.00); p=0.039] were independently associated with FLT3-ITD^mut-AML. CD99 and CD34 displayed negative predictive value (NPV) of 100% and 86.9%, respectively, with moderate positive predictive value (PPV) (45.6% and 62.1%) for the diagnosis of FLT3-ITD^mut-AML. Conclusions: Our data suggest a distinctive immunophenotypic pattern useful for rapid suspicion of this subtype of AML, to confirm with molecular assay, which benefits of a FLT3-targeted therapy. In fact, the integration of CD99 into routinely specific AML-MFC panel significantly enhances the early identification of FLT3-ITD^mut-AML. The consistent overexpression of CD99 in FLT3-ITD^mut-AML makes it a highly reliable marker, while its absence virtually excludes the presence of a FLT3-ITD mutation.