Monitoring of measurable residual disease by next-generation sequencing in patients with acute myeloid leukaemia

Abstract

Measurable residual disease (MRD) is a strong prognostic factor in acute myeloid leukaemia (AML). Next-generation sequencing (NGS) offers promise but must distinguish true signal from background. We assessed MRD in 98 adult AML patients in first complete remission after intensive chemotherapy using a duplex unique molecular identifier (UMI)-based NGS capture panel. Error reduction analysis showed up to a 20-fold decrease in artefactual calls versus conventional sequencing. Linearity studies with serial dilutions confirmed accurate quantification down to 0.01% variant allele frequency. In this cohort, NGS-MRD positivity did not significantly affect overall survival (OS) or relapse-free survival (RFS) after one course of chemotherapy. However, NGS-MRD positivity >0.1%, excluding DNMT3A, TET2, ASXL1, IDH1 and IDH2 mutations, was significantly associated with inferior outcomes after two courses (OS: hazard ratio [HR] = 3.04, p = 0.0173; RFS: HR = 2.83, p = 0.0097). Combining multiparameter flow cytometry (MFC-MRD) with NGS-MRD identified a double-positive subgroup with particularly poor outcomes after the first course (OS: HR = 7.98, p < 0.001; RFS: HR = 7.87, p < 0.001). These findings underscore that duplex UMI-based NGS is a sensitive, quantitative approach for MRD assessment in AML, offering prognostic information complementary to MFC-MRD.

Keywords: AML; MRD; NGS.

FIGURE 1

Principle and performance of next‐generation sequencing‐measurable residual disease (NGS‐MRD) with duplex unique molecular identifier (UMI). (A) Overview of UMI and duplex UMI usage (A, B, C, D refer to simplex UMIs; ⍺, β, δ, ɣ, ζ, ϵ, θ, η refer to duplex UMIs). (B) Comparison of error rates across methods. R3 refers to the call in three replicates (C) Evaluation of artefactual calls.

FIGURE 2

Mutational landscape and variant persistence in complete remission. (A) Oncoplot of baseline variants at AMLdiagnosis. (B) Variant allele frequencies plotted on a logarithmic scale for all detected mutations at MRD time points (post‐course 1 and post‐course 2) in AML patients in first CR. Each dot represents an individual mutation. Horizontal lines with crosshairs indicate the median VAF and interquartile ranges. AML, acute myeloid leukaemia; CR, complete remission; MRD, measurable residual disease; TMB, tumour mutational burden; VAF, variant allele frequency.

FIGURE 3

Outcomes of next‐generation sequencing‐measurable residual disease (NGS‐MRD) positivity after excluding DNMT3A, TET2, ASXL1 and IDH1/2 (DTAI) variants. (A) Overall survival (OS) and relapse‐free survival (RFS) at post‐course 1 (PC1). (B) OS and RFS at post‐course 2 (PC2). (C) Combined impact of NGS‐MRD and multiparameter flow cytometry (MFC‐MRD) on OS and RFS at PC1. (D) Combined impact of NGS‐MRD and MFC‐MRD on OS and RFS at PC2.