Multicolor Flow Cytometry and Multigene Next-Generation Sequencing Are Complementary and Highly Predictive for Relapse in Acute Myeloid Leukemia after Allogeneic Transplantation

Abstract

Minimal residual disease (MRD) in acute myeloid leukemia (AML) is typically measured using multiparameter flow cytometry (MFC). Detection of leukemia mutations using multigene next-generation sequencing (NGS) can potentially be used to measure residual disease. We used a targeted 28-gene NGS panel to detect mutations and different-from-normal 10-color MFC to measure MRD in AML patients before allogeneic hematopoietic stem cell transplantation (HCT). Residual disease was defined when any abnormal blast population was detected using MFC and when any leukemia allele was detected with a variant allele frequency (VAF) ≥ 5% using NGS. We tracked the clearance of leukemia alleles between AML diagnosis and immediately before HCT and found that mutations in DNMT3A, TET2, and JAK2 were less likely to be cleared than NPM1, IDH 1/2, and FLT3-ITD. Despite varying sensitivities, the concordance rate of residual disease detection before HCT using the 2 assays was 44 of 62 (71%) evaluable cases. Discordance could be explained by residual mutations in DNMT3A and TET2 that were not detected by MFC and presence of residual leukemia mutations with VAF below the established thresholds for mutation calling. Presence of flow MRD and residual mutations immediately before HCT using the 2 assays was associated with relapse risk (MFC: hazard ratio, 4.62; 95% confidence interval [CI], 1.32 to 16.09; P = .016 and NGS: hazard ratio, 4.35; 95% CI, 1.63 to 11.6; P = .003) and survival (MFC: hazard ratio, 2.44; 95% CI, 1 to 5.97; P = .05 and NGS: hazard ratio, 2.1; 95% CI, .97 to 4.55; P = .059) after HCT. Residual disease detected concurrently by MFC and NGS conferred the highest relapse risk compared with patients who were either negative by both assays or had discordant status (overall, P = .008). Although MFC is universally applicable, a multigene NGS approach to measuring residual disease in AML provides additional information on differential clearance of disease alleles and can assess clonal architecture before transplantation.

Keywords: Acute myeloid leukemia; Flow cytometry; Minimal residual disease; Next-generation sequencing.

Copyright © 2017 The American Society for Blood and Marrow Transplantation. All rights reserved.

Figures

Figure 1A:

Relative change in mutant gene VAF Mutant gene VAF was compared between diagnosis and immediately before HCT in patients who had mutations identified at AML diagnosis (n=40). A significant reduction in VAF was noted for NPM1 (n=9), IDH1 and 2 (n=11), RUNX1 (n=7), TET2 (n=10) and TP53 (n=6) while no change in VAF was seen in mutant JAK2 (n=4), N/KRAS (n=6) and DNMT3A (n=11) alleles.

Figure 1B:

Comparison of residual mutation and flow MRD burden pre HCT Residual disease in the blast compartment quantified by MFC was matched to the VAF of residual mutations quantified by NGS on the same bone marrow sample for patients who were in hematologic CR/CRi at the time of transplant but who had residual mutations called and had MFC residual disease assessment performed (n=17). The VAF (%) of residual mutations (residual mutations defined by VAF ≥5%) and the percentage of aberrant blasts are plotted on the same Y-axis grouped by leukemia gene. In the vast majority of cases MFC was quantified to be

Figure 2:

Transplant outcome stratified by residual disease status. (A) Cumulative incidence of relapse and (B) overall survival by MFC MRD, (C) Cumulative incidence of relapse and (D) overall survival by NGS, (E) Cumulative incidence of relapse and (F) overall survival by tandem assessment using MFC and NGS. In (E) and (F), patients with discordant assessment are grouped together as their outcome tracked with patients in the F−/M− group. F denotes MFC, M denotes mutation analysis by NGS and status is categorized as +/−