Molecular Landscape and Prognostic Impact of FLT3 Internal Tandem Duplication Insertion Site in Acute Myeloid Leukemia (AML): Results from the Ratify Study (Alliance 10603)

Introduction: Internal tandem duplications of the FLT3 gene (FLT3-ITD), present in ~25% of newly diagnosed adult acute myeloid leukemia (AML), are associated with poor prognosis, in particular in cases of high mutant to wild-type allelic ratio (AR) and/or insertion site (IS) in the beta1-sheet.
Aims: To investigate the relationship between ITD IS and patient (pt) outcome, Roche 454 next generation sequencing (NGS) was performed in 452/555 (81.4%) FLT3-ITD positive pts enrolled into the RATIFY trial (NCT00651261).
Results: NGS identified 908 ITDs with up to 9 ITDs/pt; 242 (53.5%) pts exhibited ≥2 ITD clones (2 ITDs, 29%; 3 ITDs, 12.8%; 4 ITDs, 5.3%; 5 ITDs, 4%; 6 ITDs, 0.7%; 7 ITDs, 1.5%; 9 ITDs, 0.2%). Median ITD-size was 45 nucleotides (range, 6-246); all ITDs were in-frame with direct head-to-tail orientation. Molecular characterization revealed the majority (70.8%, n=643) of IS within JMD, whereas 29.2% inserted 3´ of JMD, predominantly in the beta1-sheet of TKD1 (27.5%, n=250). In the 242 pts featuring multiple ITD clones, 698 concurrent IS were delineated with coexistent IS within JMD-Z being the most frequent interaction (41.5%) followed by JMD-Z and beta1-sheet (20.2%), within beta1-sheet (12.2%), by JMD-Z and hinge region (11.5%), by beta1-sheet and hinge region (5.5%), and within the hinge region (4.8%). ITD size strongly correlated with IS, in that the more C-terminal the IS, the longer the inserted fragment (P<0.001).
FLT3-ITD IS, categorized as (i) JMDsole (n=251, 55.5%), (ii) JMD/TKD1 (n=117, 25.9%), and (iii) TKD1sole (n=84, 18.6%) were not associated with differences in clinical characteristics, but with genetic features: JMDsole was positively correlated with NPM1^mut (P=.029), inversely with the NPM1^wt/FLT3-ITD^low genotype (P<.001), and showed higher AR, determined by both Genescan and NGS (P<.001, each). Pts with JMD/TKD1 more frequently were FLT3-ITD^low (AR ≤0.5) (P<.001) and had more ITD subclones (P<.001).
Complete remission (CR) was achieved in 274/452 (60.6%) pts. Regression model including the 3 categorized IS groups revealed NPM1^mut as a favorable marker for achievement of CR (OR, 2.16; P=.001), while WBC (OR, 0.83; P=.009) and number of ITDs (OR, 0.80; P=.035) predicted for a lower CR rate. The 3 IS groups had no impact on CR achievement.
The estimated median follow-up of the 452 pts was 60.6 months (95% CI, 57.7-63.6). Survival analysis according to categorized IS groups showed that pts exhibiting insertion exclusively in TKD1 had significantly inferior OS (P=.032) compared to the other 2 groups. In multivariate Cox models for OS and CIR, including allogeneic hematopoietic stem-cell transplantation in CR1 (HCT) as time-dependent covariate, FLT3-ITD IS in TKD1sole (compared to JMDsole: HR, 1.61; P=.014, and compared to JMD/TKD1: HR, 2.17; P=.004), WBC (HR, 1.12; P=.010), and age (HR, 1.02; P=.029) were unfavorable factors for OS, whereas NPM1^mut (HR, 0.58; P<.001) and HCT (HR, 0.45; P<.001) were both favorable. For CIR, FLT3-ITD IS in TKD1sole (compared to JMDsole: HR, 2.14; P=.002), WBC (HR, 1.18; P=.002), and NGS-based AR (HR, 1.16; P=.007) were associated with higher risk of relapse, whereas HCT (HR, 0.51; P=.007) reduced risk of relapse significantly; treatment with midostaurin had no impact on OS and CIR in the whole cohort. Cox regression models on OS and CIR according to the 3 IS subgroups revealed for the TKD1sole group HCT (HR, 0.29; P=.005 and HR, 0.24; P=.015) as the only significant factor for improved OS and CIR, whereas WBC (HR, 1.38; P=.008 and HR, 1.32; P=.026) were of adverse impact for both endpoints. Within the JMDsole group, NGS-based AR (HR, 1.18; P=.006 and HR, 1.43; P<.001) and multiclonality (HR, 1.29; P=.022 and HR, 1.43; P=.047) were negative on both OS and CIR, and age (HR, 1.02; P=.024) on OS only, whereas treatment with midostaurin (HR, 0.56; P=.005 and HR, 0.56; P=.034) and NPM1^mut (HR, 0.47; P<.001 and HR, 0.47; P=.006) were of beneficial impact on both endpoints. A significant beneficial effect for HCT was also observed for CIR (HR, 0.48; P=.040). The only significant factor for the JMD/TKD1 group was HCT (HR, 0.18; P=.018) associated with improved OS.
Conclusions: In this cohort of 452 FLT3-ITD mutated AML pts treated within the RATIFY trial the negative prognostic impact of TKD1 IS was confirmed. A beneficial effect of midostaurin was only found for patients with JMDsole IS. In this subset, NPM1^mut also exerted a strong beneficial effect.