Quantification of measurable residual disease using duplex sequencing in adults with acute myeloid leukemia

Abstract

The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). Despite the correlation with clinical outcomes, MRD assessment has yet to be standardized or routinely incorporated into clinical trials and discrepancies have been observed between different techniques for MRD assessment. In 62 patients with AML, aged 18-60 years, in first complete remission after intensive induction therapy on the randomized phase III SWOG-S0106 clinical trial (clinicaltrials gov. Identifier: NCT00085709), MRD detection by centralized, high-quality multiparametric flow cytometry was compared with a 29-gene panel utilizing duplex sequencing (DS), an ultrasensitive next-generation sequencing method that generates double-stranded consensus sequences to reduce false positive errors. MRD as defined by DS was observed in 22 (35%) patients and was strongly associated with higher rates of relapse (68% vs. 13%; hazard ratio [HR] =8.8; 95% confidence interval [CI]: 3.2-24.5; P<0.001) and decreased survival (32% vs. 82%; HR=5.6; 95% CI: 2.3-13.8; P<0.001) at 5 years. DS MRD strongly outperformed multiparametric flow cytometry MRD, which was observed in ten (16%) patients and marginally associated with higher rates of relapse (50% vs. 30%; HR=2.4; 95% CI: 0.9-6.7; P=0.087) and decreased survival (40% vs. 68%; HR=2.5; 95% CI: 1.0-6.3; P=0.059) at 5 years. Furthermore, the prognostic significance of DS MRD status at the time of remission for subsequent relapse was similar on both randomized arms of the trial. These findings suggest that next-generation sequencing-based AML MRD testing is a powerful tool that could be developed for use in patient management and for early anti-leukemic treatment assessment in clinical trials.

Figure 1.

Clinical outcomes of S0106 acute myeloid leukemia patients analyzed for measurable residual disease. Rates of (A) non-relapse related mortality (NRM), (B) relapse, (C) relapse-free survival (RFS), and (D) overall survival (OS) are shown for the 62-patient cohort from the S0106 clinical trial analyzed for measurable residual disease (MRD) by duplex sequencing and multiparametric flow cytometry. CR: complete remission; No.: number.

Figure 2.

Mutational spectrum, measurable residual disease status, and clinical outcomes of patients in complete remission. The heatmap displays variants detected at diagnosis and the presence (divided into variant allele fraction [VAF] ≥ or <5%) or absence at the time of complete remission (CR) by duplex sequencing (DS), DS measurable residual disease (MRD) status, multiparametric flow cytometry (MFC) MRD status, and clinical outcome at 5 years (relapse, no relapse, or non-relapse mortality [NRM]). The presence of a mutation within a gene is denoted in the heatmap, with the color corresponding to the highest VAF within each gene per patient. Variants identified in remission that were not identified at diagnosis are also marked (*). pos: positive.

Figure 3.

Impact of measurable residual disease status on clinical outcomes. Rates of non-relapse mortality (NRM), relapse, relapse-free survival, and overall survival are shown based on measurable residual disease (MRD) status as determine by (A) multiparametric flow cytometry (MFC) and (B) duplex sequencing (DS). pos: positive; neg: negative; No.: number; CR: complete remission.

Figure 4.

Analysis of discordant measurable residual disease results by duplex sequencing and flow cytometry. (A) Number and percentage of patients called measurable residual disease (MRD)-positive (pos) versus MRD-negative (neg) by duplex sequencing (DS) versus multiparametric flow cytometry (MFC). (B) Clinical outcomes (non-relapse mortality [NRM], relapse, or no relapse) of MFC MRD versus DS MRD discordant cases. (C) Rates of relapse for patients grouped by MRD status as defined by MFC MRD and DS MRD. CR: complete remission; No.: number.

Figure 5.

Impact of treatment randomization and duplex sequencing measurable residual disease status on relapse. Rates of relapse for patients as defined by (A) treatment randomization to daunorubicin and cytarabine (DA) versus daunorubicin, cytarabine, and gemtuzumab ozogamicin (DA+GO) and (B) treatment randomization (DA or DA+GO) and duplex sequencing (DS) measurable residual disease (MRD) status. pos.: positive; neg.: negative; No.: number; CR: complete remission.