Digital PCR for Minimal Residual Disease Quantitation Using Immunoglobulin/T-Cell Receptor Gene Rearrangements in Acute Lymphoblastic Leukemia: A Proposed Analytic Algorithm

Abstract

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