A New View on Minimal Residual Disease Quantification in Acute Lymphoblastic Leukemia using Droplet Digital PCR.

Real-time quantitative PCR (qPCR) using immunoglobulin/T-cell receptor gene rearrangements has been used as the gold standard for minimal residual disease (MRD) monitoring in acute lymphoblastic leukemia (ALL) for >20 years. Recently, new PCR-based technologies have emerged, such as droplet digital PCR (ddPCR), which could offer several methodologic advances for MRD monitoring. In the current work, qPCR and ddPCR were compared in an unbiased blinded prospective study (n = 88 measurements) and in a retrospective study with selected critical low positive samples (n = 65 measurements). The former included flow cytometry (Flow; n = 31 measurements) as a third MRD detection method. Published guidelines (qPCR) and the latest, revised evaluation criteria (ie, ddPCR, Flow) have been applied for data analysis. The prospective study shows that ddPCR outperforms qPCR with a significantly better quantitative limit of detection and sensitivity. The number of critical MRD estimates below quantitative limit was reduced by sixfold and by threefold in the retrospective and prospective cohorts, respectively. Furthermore, the concordance of quantitative values between ddPCR and Flow was higher than between ddPCR and qPCR, probably because ddPCR and Flow are absolute quantification methods independent of the diagnostic sample, unlike qPCR. In summary, our data highlight the advantages of ddPCR as a more precise and sensitive technology that may be used to refine response monitoring in ALL.

Prognostic value of low-level MRD in adult acute lymphoblastic leukemia detected by low- and high-throughput methods.

Persistence of minimal residual disease (MRD) after induction/consolidation therapy in acute lymphoblastic leukemia is the leading cause of relapse. The GMALL 07/2003 study used MRD detection by real-time quantitative polymerase chain reaction of clonal immune gene rearrangements with 1 × 10-4 as discriminating cutoff: levels ≥1 × 10-4 define molecular failure and MRD-negativity with an assay sensitivity of at least 1 × 10-4 defining complete molecular response. The clinical relevance of MRD results not fitting into these categories is unclear and termed "molecular not evaluable" (MolNE) toward MRD-based treatment decisions. Within the GMALL 07/03 study, 1019 consecutive bone marrow samples after first consolidation were evaluated for MRD. Patients with complete molecular response had significantly better outcome (5-year overall survival [OS] = 85% ± 2%, n = 603; 5-year disease-free survival [DFS] = 73% ± 2%, n = 599) compared with patients with molecular failure (5-year OS = 40% ± 3%, n = 238; 5-year DFS = 29% ± 3%, n = 208), with patients with MolNE in between (5-year OS = 66% ± 4%; 5-year DFS = 52% ± 4%, n = 178). Of MolNE samples reanalyzed using next-generation sequencing (NGS), patients with undetectable NGS-MRD (n = 44; 5-year OS = 88% ± 5%, 5-year DFS = 70% ± 7%) had significantly better outcome than those with positive NGS-MRD (n = 42; 5-year OS = 37% ± 8%; 5-year DFS = 33% ± 8%). MolNE MRD results not just are borderline values with questionable relevance but also form an intermediate-risk group, assignment of which can be further improved by NGS.