Multi-target measurable residual disease assessed by error-corrected sequencing in patients with acute myeloid leukemia: An ALFA study

Abstract

The evaluation of measurable residual disease (MRD) in acute myeloid leukemia (AML) using comprehensive mutation analysis by next-generation sequencing (NGS) has been investigated in several studies. However controversial results exist regarding the detection of persisting mutations in DNMT3A, TET2, and ASXL1 (DTA). Benchmarking of NGS-MRD taking into account other molecular MRD strategies has to be done. Here, we performed error-corrected-NGS-MRD in 189 patients homogeneously treated in the ALFA-0702 study (NCT00932412). Persistence of non-DTA mutations (HR = 2.23 for RFS and 2.26 for OS), and DTA mutations (HR = 2.16 for OS) were associated with poorer prognosis in multivariate analysis. Persistence of at least two mutations in complete remission (CR) was associated with a higher cumulative incidence of relapse (CIR) (HR = 3.71, p < 0.0001), lower RFS (HR = 3.36, p < 0.0001) and OS (HR = 3.81, p = 0.00023) whereas persistence of only one mutation was not. In 100 analyzable patients, WT1-MRD, but not NGS-MRD, was an independent factor for RFS and OS. In the subset of 67 NPM1 mutated patients, both NPM1 mutation detection (p = 0.0059) and NGS-MRD (p = 0.035) status were associated with CIR. We conclude that detectable NGS-MRD including DTA mutations correlates with unfavorable prognosis in AML. Its integration with alternative MRD strategies in AML management warrants further investigations.

Fig. 1. Flow chart of the study.

CR1: Complete remission or complete remission with incomplete recovery; DTA: mutations in DNMT3A, TET2, or ASXL1; NGS-MRD: measurable residual disease using Next Generation Sequencing assay with a 0.1% threshold for positivity; neg: negative; pos: positive; NPM1-MRD: measurable residual disease for NPM1 mutation measured by Next Generation sequencing assays with a threshold of detection over 10⁻⁴ (see methods).

Fig. 2. Mutational profile at diagnosis and CR1 according to functional categories and gene mutations.

Diagnosis variant allele frequency (VAF) is plotted in x-axis (range 0–60% or 0–100% for genes with loss of heterozygosity) and VAF in CR1 in the y-axis (log-scale). The dotted red-line represents the retained threshold of detection of 0.1%. Mutations with VAF in CR1 under 0.01% were plotted at 0.01%. The “Epigenetic” plot summarizes data on DNMT3A, TET2, ASXL1 IDH1, IDH2, BCOR and ASXL2, ATRX, BCORL1, CTCF, CUX1, EP300, EZH2, KDM6A, KMT2C. The “Splice” plot summarizes data on SF3B1, SRSF2, U2AF1, and ZRSR2. The “Transcription” plot summarizes data on GATA2, RUNX1, CEBPA, WT1, PHF6, ETV6, and IKZF1. The “signaling” plot summarizes data on FLT3, KIT, MYC, PTPN11, NRAS, KRAS, NF1 and JAK2, JAK3, CHEK2, CSF3R, CBL and BRAF. The “All mutations” plot contains combined data of all mutated genes. A few mutations with uncertain VAFs at diagnosis or CR1 were not plotted (see methods).

Fig. 3. Prognosis according to NGS-MRD.

Prognosis according to NGS status (A–C) and the number of persistent mutations (D–F). p-values are for log-rank tests for Relapse Free Survival and Overall Survival and for Gray test for Cumulative incidence of Relapse. Data were not censored at allogeneic hematopoietic stem cell transplantation. NEG no mutation detected in CR1, DTA detection of only DNMT3A, TET2 or ASXL1 mutation in CR1; other: detection of other mutation than DNMT3A, TET2 or ASXL1 in CR1.

Fig. 4. Prognosis of NGS-MRD evaluation when compared to WT1 expression in CR and NPM1 MRD.

Panels A–C represent the cumulative incidence of relapse, relapse-free survival, and overall survival according to NGS-MRD in CR1 status in the 84 patients with low WT1 expression in CR1. Panels D–F represent the cumulative incidence of relapse, relapse-free survival, and overall survival according to NGS-MRD in CR1 and NPM1 MRD assessed by NGS in the 67 NPM1 mutated patients. p-values are for the log-rank test for RFS and OS and for the Gray test for CIR. Data were not censored at allogeneic hematopoietic stem cell transplantation.