Mutation Spectrum of FLT3 and Significance of Non-Canonical FLT3 Mutations in Hematological Malignancies

Background FLT3 mutations are detected in 15%-35% of newly diagnosed AML patients. The canonical FLT3 mutation types are internal tandem repeats (ITD) in the JMD and TKD1 regions and point mutations at codons D835/I836 (TKD). Conventional sequencing for FLT3 mutations only covers the above-mentioned hotspot regions. With the advent of next generation sequencing which spans the full-length FLT3, a series of non-canonical mutations have also been reported. However, the kinase activities and clinical characteristics of these mutations remain unclear.

Methods To profile the spectrum of FLT3 mutations, we retrospectively analyzed 869 consecutive newly diagnosed AML, ALL, and MDS patients with FLT3 mutation by NGS-seq from June 2016 to December 2020 at Jiangsu Institute of Hematology. The clinical data of 316 AML (excluding APL) patients were retrospectively collected and analyzed after informed consent. Since previous studies have elaborated the FLT3 functional characteristics of FLT3-ITD and FLT3 point mutations, we focus on other FLT3 mutation types in this study. Seven representative FLT3 mutations, including four deletion mutations in JMD, one frameshift mutation in JMD, one deletion mutation in TKD1 and one ITD in TKD2, were constructed to study the kinase activities and sensitivities to FLT3 inhibitors in vitro.

Results FLT3 mutational spectrums of 869 patients showed that the NGS method could detect numerous novel non-canonical FLT3 mutations, accounting for 21.2% of FLT3 mutation events compared with conventional tests (Figure 1). According to the changes in gene structures resulted by different mutations, we classified the non-canonical mutations into four types: non-canonical point mutations (NCPM) (19.2%), deletion (0.7%), frameshift (0.8%), and ITD outside the JMD and TKD1 regions (0.5%). Given the enormous differences in frequency, NCPM was further divided into FLT3 recurrent point mutations of NPM (FLT3-NCPM-RPM) and FLT3 non-recurrent point mutations (FLT3-NRPM). Kaplan-Meier analyses of the RFS and OS showed that there is no difference between canonical FLT3-TKD and FLT3-NCPM-RPM while statistical differences were observed not only between canonical FLT3-TKD and FLT3-NRPM but also between FLT3-NCPM-RPM and FLT3-NRPM. Due to the similarities in prognosis and kinase activity, classical FLT3-TKD and FLT3-NCPM-RPM were grouped into a class called FLT3 recurrent point mutations (FLT3-RPM). Further prognosis analyses indicated that patients with FLT3-ITD, FLT3-RPM and FLT3-NRPM had significant differences in RFS (P=0.005) and OS (P=0.01). Interestingly, the presence of FLT3-NRPM may confer a favorable prognosis compared with FLT3-ITD and FLT3-RPM. Multivariable analysis using Cox models also confirmed that FLT3-NRPM had a survival benefit over FLT3-ITD and FLT3-RPM. The significant difference in the prognosis of patients with three FLT3 mutation subtypes and Cox models proved the rationality of this strategy. Besides the non-canonical point mutation, a series of FLT3 mutations with small deletions and frameshift alterations were also detected. In addition, we also discovered several completely random insertion mutations in the non-ITD region. According to our previous study, these mutageneses were considered a subtype of FLT3-ITD. Based on the results, FLT3 alterations could be divided into FLT3-ITD, FLT3-RPM, FLT3-NRPM, FLT3-deletion, and FLT3-frameshift groups.

In vitro analysis showed that the deletion mutants in TKD1 (p.K614_G617del) and the FLT3-ITD mutant in TKD2 (p.S941_F942insRVS) had significantly higher kinase activity than wild-type FLT3. Whereas the deletion mutants in JMD have comparable phosphorylation levels to wild-type FLT3 (Figure 2). All tested deletion mutations and ITD in the TKD2 region were sensitive to both AC220 and sorafenib.

Conclusion Conventional sequencing that only covers hotspot regions omitted part of FLT3 mutation and full-length sequencing of the FLT3 gene may be indispensable. FLT3 alterations detected by NGS-seq could be reclassified into 5 types: FLT3-ITD, FLT3-RPM, FLT3-NRPM, FLT3-deletion, and FLT3-frameshift. In vitro studies indicated patients with FLT3 deletion mutations and FLT3-ITD outside the JMD and TKD1 regions might be ought to be treated with FLT3 inhibitors. Our new classification of FLT3 mutation was expected to promote more precise management of patients with FLT3 non-canonical mutations.

No relevant conflicts of interest to declare.