Diagnostic evaluation methods and prognostic impact of FLT3-ITD microclones in Acute Myeloid Leukemia (AML): A retrospective multicenter study on behalf of the EHA AML-specialized working group (SWG)

Abstract

Abstract ELN recommendations for AML diagnosis, genetic risk stratification and treatment have been updated in 2022. Accordingly, FLT3-ITD+ AML is now included in the intermediate-risk category, regardless of the allelic ratio (AR), and capillary electrophoresis (CE) is the recommended detection method. In two large trials combining intensive chemotherapy with the FLT3 inhibitors midostaurin (RATIFY) and quizartinib (QuANTUM-First), the AR threshold to define FLT3-ITD+ was conventionally set at 0.05 and 0.03, respectively. CE analysis requires high level of expertise specifically to detect FLT3-ITD microclones (FLT3-ITDm, AR: >0.01,<0.05), whose clinical role is not yet well established. Here, we leveraged the EHA AML-SWG framework to explore diagnostic evaluation methods and prognostic impact of FLT3-ITDmin AML. In a first methodological part, results of CE from 63 FLT3-ITD+patient samples were blindly analyzed by 7 European labs, and reproducibility of results was assessed. The intraclass correlation coefficient (ICC) was computed as an index of interrater reliability of AR data. Overall, we observed a good ICC for ITD length determination (95%CI=0.52–0.92), but a less satisfactory ICC for AR estimation (95%CI=0.06–0.63). Next, we compared NGS and CE for FLT3-ITD status determination, by running in parallel an additional set of 48 FLT3-ITD+ AML cases. Results showed a significant correlation of AR/VAF (r=0.923,p<0.001), and a 94% concordance overlap, with only 3/48 cases with AR<0.02 detected by CE, but not by NGS. Next, we enrolled AML patients diagnosed between 2017 and 2022 with FLT3-ITD AR<0.05 by CE, and a comparator cohort of cases with AR>0.05 treated with standard chemotherapy without FLT3-inhibitors, or with less-intensive strategies. A total of658 patients with FLT3-ITD+ AML were included in our study. Of these, 212 (32%) had FLT3-ITDm (AR range:0.01-0.04), whereas 446 (68%) had an AR>0.05 (0.05-15.26). Median age at AML onset was 59 years (range 18-94). Patients with microclones were older (62 vs 58 years, p<0.001), and had a less proliferative phenotype. AML with FLT3-ITDm were also more frequently classified as “secondary” with regards to disease ontogeny (14% vs 8%, p=0.012), and had a higher frequency of myelodysplasia-related (MR) genes mutations (60% vs 30%, p<0.001), whereas no difference was noticed for NPM1. A total of 161 patients (24%, median age 76 years) were deemed unfit and were managed with less-intensive approaches, while 497 patients (76%) received intensive chemotherapy. Among the latter, complete response (CR) was achieved in 88% of cases with AR>0.05 vs. 74% of cases with FLT3-ITDm (p<0.001). CR achievement was associated with younger age (p<0.001), presence of NPM1 (p=0.018), absence of DNMT3A (p=0.035) and MR-genes (p=0.011) mutations, FLT3-ITD AR >0.05 (p<0.001), and ELN 2017/22 favorable/intermediate vs adverse categories (p=0.015/0.045). With a median follow-up of 30 months (18-41), 3-year overall survival (OS) was 58% in intensively treated patients, censoring for transplant. The multivariable analysis (MVA) for OS, including clinical and genetic variables, and ELN 2022 categories, showed that increasing age and WBC, together with NPM1 mutation had an independent prognostic role (HR=1.03, 1.03 and 0.63 respectively, p<0.001, 0.021 and 0.012, respectively). Overall, 136 intensively treated patients (27%) relapsed at a median of 8 months (range 1-56) from AML diagnosis, leading to a 3-year disease-free survival (DFS) of 47%, when censoring for transplant, with no impact of FLT3-ITD AR (HR=0.9, p=0.6). The MVA identified increased WBC and presence of NPM1mutation as independent predictors of DFS (HR=1 and 0.61, p=0.007 and 0.017, respectively). Of cases with available FLT3-ITD status at relapse (n=33), 45% remained positive, of which 64% were originally FLT3-ITDm. Dissecting their FLT3-ITD clonal make-up, the majority (91%) recapitulated the same clone harbored at onset (18% also acquiring additional clones, and 27% losing prior clones), whereas a new clone was detected in 9% of cases. Finally, the clonal burden expanded in all cases (median 0.027 vs 0.686, p<0.001), with 89% of FLT3-ITDm patients relapsing with AR >0.05. Our data show that FLT3-ITDmare associated with older age, secondary-type AML and higher frequency of MR-gene mutations vs AML with FLT3-ITD >0.05. The potential benefit of FLT3 inhibitors in patients with FLT3-ITDm needs to be evaluated in randomized clinical trials.