BackgroundFlow‐cytometric minimal residual disease (FC‐MRD) monitoring is a well‐established risk‐stratification factor in B‐lymphoblastic leukemia/lymphoma (‐B‐ALL) and is being considered as a basis for deintensification or escalation in treatment protocols. However, currently practiced standard FC‐MRD has limited sensitivity (up to 0.01%) and higher false MRD‐negative rate. Hence, a highly sensitive, widely applicable, and easily reproducible FC‐MRD assay is needed, which can provide a reliable basis for therapeutic modifications.MethodsA 10‐color high‐event analysis FC‐MRD assay was studied for the evaluation of MRD status at postinduction, (PI; day‐35), postconsolidation, (PC; day‐78), and subsequent follow‐up time‐points (SFU) in bone marrow samples from pediatric B‐ALL.ResultsOne‐thousand MRD samples (PI‐62.2%; PC‐26.5%; and SFU‐11.3%) from 622 childhood B‐ALL patients were studied. High‐event analysis was performed with median 4,452,000 events (range, 839,000 to 8,866,000 events) and >4 million events in 71% samples. MRD was measurable in 43.2% of PI‐samples, in 29.4% PC‐samples, and in 32.7% SFU‐samples. To simulate comparison with standard FC‐MRD, we reanalyzed MRD results gating only first 500,000 and first 1000,000 events in 122 PI‐MRD positive samples with MRD levels <0.02%. Of these samples gated for 500,000 events and 1000,000 events, 32% and 21.3% were found to be falsely MRD‐negative, respectively.ConclusionsWe report an easily reproducible high‐sensitivity 10‐color FC‐MRD assay with the sensitivity of 2‐in‐106 (0.0002%). It allowed the detection of low‐level MRD in samples, which could have been reported negative using the standard FC‐MRD with limited event analysis. Thus, this high‐sensitivity MRD‐methodology can provide a reliable basis for therapeutic modifications in B‐ALL. © 2019 International Clinical Cytometry Society

We accrued 201 patients of adult AML treated with conventional therapy, in morphological remission, and evaluated MRD using sensitive error-corrected next generation sequencing (NGS-MRD) and multiparameter flow cytometry (FCM-MRD) at the end of induction (PI) and consolidation (PC). Nearly 71% of patients were PI NGS-MRD+ and 40.9% PC NGS-MRD+ (median VAF 0.76%). NGS-MRD+ patients had a significantly higher cumulative incidence of relapse (p = 0.003), inferior overall survival (p = 0.001) and relapse free survival (p < 0.001) as compared to NGS-MRD− patients. NGS-MRD was predictive of inferior outcome in intermediate cytogenetic risk and demonstrated potential in favorable cytogenetic risk AML. PI NGS-MRD− patients had a significantly improved survival as compared to patients who became NGS-MRD− subsequently indicating that kinetics of NGS-MRD clearance was of paramount importance. NGS-MRD identified over 80% of cases identified by flow cytometry at PI time point whereas FCM identified 49.3% identified by NGS. Only a fraction of cases were NGS-MRD− but FCM-MRD+. NGS-MRD provided additional information of the risk of relapse when compared to FCM-MRD. We demonstrate a widely applicable, scalable NGS-MRD approach that is clinically informative and synergistic to FCM-MRD in AML treated with conventional therapies. Maximum clinical utility may be leveraged by combining FCM and NGS-MRD modalities.

Detection of measurable residual disease (MRD) by mutation specific techniques has prognostic relevance in NPM1 mutated AML (NPM1mut AML). However, the clinical utility of next generation sequencing (NGS) to detect MRD in AML remains unproven. We analysed the clinical significance of monitoring MRD using ultradeep NGS (NGS-MRD) and flow cytometry (FCM-MRD) in 137 samples obtained from 83 patients of NPM1mut AML at the end of induction (PI) and consolidation (PC). We could monitor 12 different types of NPM1 mutations at a sensitivity of 0.001% using NGS-MRD. We demonstrated a significant correlation between NGS-MRD and real time quantitative PCR (RQ-PCR). Based upon a one log reduction between PI and PC time points we could classify patients as NGS-MRD positive (<1log reduction) or negative (>1log reduction). NGS-MRD, FCM-MRD as well as DNMT3A mutations were predictive of inferior overall survival (OS) and relapse free survival (RFS). On a multivariate analysis NGS-MRD emerged as an independent, most important prognostic factor predictive of inferior OS (hazard ratio, 3.64; 95% confidence interval [CI] 1.58 to 8.37) and RFS (hazard ratio, 4.8; 95% CI:2.24 to 10.28). We establish that DNA based NPM1 NGS MRD is a highly useful test for prediction of relapse and survival in NPM1mut AML.