Droplet Digital PCR (ddPCR) for MRD Quantitation Using Ig/TCR Gene Rearrangements in Acute Lymphoblastic Leukemia: A Proposed Analytic Algorithm

In minimal residual disease (MRD), in which there are exceedingly low target copy numbers, droplet digital PCR (ddPCR) can improve the quantitation. However, we currently lack the standards for ddPCR data analysis and results for MRD interpretation in acute lymphoblastic leukemia. Here, for immunoglobulin/T-cell receptor-based MRD quantitation, we propose an objective, statistics-based analytic algorithm. In 161 postinduction samples from 79 children with acute lymphoblastic leukemia, we performed MRD quantitation by ddPCR and real-time quantitative PCR (qPCR) using the same markers and primer-probe sets. The ddPCR raw data were analyzed by using an automated algorithm. For assigning MRD-positive/negative status, ddPCR and qPCR results were highly concordant (P < 0.0001): 98% (50 of 51) of qPCR positive were positive by ddPCR, whereas 95% (61 of 64) of qPCR negative results were also negative by ddPCR. For MRD quantitation, both qPCR and ddPCR were tightly correlated (R2 = 0.94). Using more DNA (1 μg × 7 versus 630 ng × 3), ddPCR improved the sensitivity of MRD quantitation by one log10 (median MRD positive cutoff 1.6 × 10-5). With the improved sensitivity by ddPCR, 83% (29 of 35) of positive-not-quantifiable results by qPCR could be assigned positive/negative MRD status. We also determined that seven replicates of tested samples and negative controls were optimal for the assay. Compared with qPCR, for immunoglobulin/T-cell receptor-based MRD quantitation, ddPCR could improve MRD sensitivity by one log10. We proposed an automatable, statistics-based algorithm that minimized interoperator variance for ddPCR MRD.