Optimizing FLT3 Inhibitor Use in Adult Acute Myeloid Leukemia with FLT3 Mutations Using Proteomics

FLT3 mutations are the most frequent genetic alteration in adult Acute Myeloid Leukemia (AML). The use of FLT3 inhibitors (FLT3I) has improved outcome among FLT3 mutant patients, but not all benefit. We developed a proteomic-based approach to stratify patients according to their response to FLT3I therapy. The expression of 429 proteins (339 total, 90 post-translational modified) was measured by Reverse Phase Protein Arrays (RPPA) in fresh samples from 805 newly diagnosed AML patients, with levels normalized to normal CD34+ cells. Of 137 with FLT3 ITD or D835 mutations, 54 who were treated with FLT3I (Sorafenib=22, Quizartinib=11, Midostaurine=6, Gilteritinib=4 and Crenolanib=1) combined with other agents (Hypomethylating agents (HMA)=23, HMA+Venetoclax (VTX)=5, Idarrubicin+Cytarabine (AraC)+Purine Analogs (IA+Pur)=19, IA+Pur+VTX=2, AraC=3, Fludarabine+AraC (FA)=1, none=1) were selected for analyses. Proteins prognostic for Overall Survival (OS) and Complete Remission Duration (CRD) were identified using pairwise LogRank tests (p < 0.05) adjusted for False Discovery Rate, followed by unbiased hierarchical clustering. Continuous and categorial variables were compared with Wilcoxon, Kruskal-Wallis or Fisher's Exact test with simulated p-values (10000 replicates). Cox proportional hazards models (CoxPH) were developed for Uni-(UV) and Multi-variate (MV) analysis. Deep neural network analysis using random forest models identified proteins most predictive of cluster assignment. 28 proteins were individually prognostic for both OS and CRD and divided patients into 3 clusters (Fig. 1: C1=red, C2=blue, C3=green). The distribution of nearly all other clinical and molecular features did not differ between clusters, including: age, gender, race, cytogenetic risk and AML group. Co-occurrence with other mutations was not biased, except for FLT3+TET2 and FLT3+TET2+NPM1 mutants, more frequent in C1. OS was significantly longer (Fig. 2), in C1 (Median: C1>84mo, C2=14.8mo, C3=11.4mo; p84mo, C2=8.6mo, C3=5.7mo; p70yo), primary, but not secondary AML, diploid karyotype and intermediary cytogenetic risk, and with Sorafenib or IA+PR therapies. In the UV CoxPH model for OS, C1, C2 and C3 were predictive (HR=1, 4.1, 7.5; p=r