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. Discrepancies have been observed between different techniques for MRD assessment and there remains a need to compare centralized, high-quality multiparametric flow cytometry (MFC) and ultrasensitive next-generation sequencing (NGS) in AML patients with diverse mutational profiles. In 62 patients with AML, aged 18-60, in first complete remission after intensive induction therapy on the randomized phase 3 SWOG-S0106 clinical trial, MRD detection by MFC was compared with a 29 gene panel utilizing duplex sequencing (DS), an NGS method that generates double-stranded consensus sequences to reduce false positive errors. Using DS, detection of a persistent mutation utilizing defined criteria was seen in 22 (35%) patients and was strongly associated with higher rates of relapse (68% vs 13% at year 5; HR, 8.8; 95% CI, 3.2-24.5; P<0.001) and decreased survival (32% vs 82% at year 5; HR, 5.6; 95% CI, 2.3-13.8; P<0.001). MRD as defined by DS strongly outperformed MFC, which was observed in 10 (16%) patients and marginally associated with higher rates of relapse (50% vs 30% at year 5; HR, 2.4; 95% CI, 0.9-6.7; P=0.087) and decreased survival (40% vs 68% at year 5; HR, 2.5; 95% CI, 1.0-6.3; P=0.059). Furthermore, the prognostic significance of DS MRD status at the time of remission was similar on both randomized arms of the trial, predicting S0106 clinical trial outcomes. These findings suggest that DS is a powerful tool that could be used in patient management and for early treatment assessment in clinical trials.

Test Then Erase? Current Status and Future Opportunities for Measurable Residual Disease Testing in Acute Myeloid Leukemia

“…A digital targeted RNA-sequencing-based approach was able to detect all ELN defined MRD targets, and two-thirds of patients in a single standardized assay with a sensitivity of 1/100,000 events, similar to that seen with qPCR [101]. Subsequently, duplex sequencing was tested on post-induction patient samples from the randomized phase 3 SWOG-S0106 clinical trial [101]. Duplex sequencing, which generates double-stranded consensus sequences to reduce false positive errors, outperformed MFC-MRD testing showing 35% of patients with MRD compared to 16% detected by MFC, and association with higher relapse rates [101].…”

“…More recent data suggests MFC-MRD adds little to NGS-MRD testing in specific molecular subsets. NGS-MRD is able to detect the majority of cases with MFC-MRD+ disease who relapse and many relapsing patients are MFC-MRD negative [99,101]. The most important prognostic time points are still being discerned.…”

“…There are various sequencing approaches being evaluated for molecular MRD detection [101,102]. A digital targeted RNA-sequencing-based approach was able to detect all ELN defined MRD targets, and two-thirds of patients in a single standardized assay with a sensitivity of 1/100,000 events, similar to that seen with qPCR [101]. Subsequently, duplex sequencing was tested on post-induction patient samples from the randomized phase 3 SWOG-S0106 clinical trial [101].…”

“…Subsequently, duplex sequencing was tested on post-induction patient samples from the randomized phase 3 SWOG-S0106 clinical trial [101]. Duplex sequencing, which generates double-stranded consensus sequences to reduce false positive errors, outperformed MFC-MRD testing showing 35% of patients with MRD compared to 16% detected by MFC, and association with higher relapse rates [101].…”

Droplet digital PCR for sensitive relapse detection in acute myeloid leukaemia patients transplanted by reduced intensity conditioning

“…9 Naturally, the benefits and limitations of each platform are changing with technological developments. Recent notable progress includes leukaemic stem cell (LSC) based assays in MFC, 42 the introduction of duplex sequencing in NGS, 43 and multiplex monitoring in ddPCR. 44 MRD by single-cell profiling is promising but currently, the price is out of reach for routine clinical use.…”