Novel deep targeted sequencing method for minimal residual disease monitoring in acute myeloid leukemia

Abstract

A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease (MRD) negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for MRD assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-SNVs. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by NGS, evaluating the level of mutations detected at diagnosis. The predictive value of MRD status by NGS, multiparameter flow cytometry, or quantitative PCR was determined by survival analysis. The method achieved a sensitivity of 10-4 for SNV mutations and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnosis data set). NGS-determined MRD positive status was associated with lower disease-free survival (hazard ratio [HR] 3.4, p=0.005) and lower overall survival (HR 4.2, p<0.001). Multivariate analysis showed that MRD positive status by NGS was an independent factor associated with risk of death (HR 4.54, p =0.005) and the only independent factor conferring risk of relapse (HR 3.76, p =0.012). This NGS based method simplifies and standardizes MRD evaluation, with high applicability in acute myeloid leukemia. It also improves upon flow cytometry and quantitative PCR to predict acute myeloid leukemia outcome and could be incorporated in clinical settings and clinical trials.