Study findings indicate that droplet digital polymerase chain reaction (ddPCR) may be more accurate than real-time quantitative PCR in measuring minimal residual disease (MRD) among pediatric patients with acute lymphoblastic leukemia (ALL), particularly in late follow-up time points.
Droplet digital polymerase chain reaction (ddPCR) may more superior to real-time quantitative PCR (RQ-PCR) for measuring minimal residual disease (MRD), say new study results, which tested the ability of the 2 assessments in risk stratifying patients with acute lymphoblastic leukemia (ALL).
Currently, RQ-PCR represents the gold standard for measuring MRD—the most important prognostic indicator in pediatric ALL—and subsequently risk stratifying these patients.
“Despite the high sensitivity of RQ-PCR method, a nonnegligible fraction of patients with very low MRD levels are classified as positive not-quantifiable (POS-NQ) according to the EuroMRD guidelines,” wrote the researchers. “In particular, MRD is defined POS-NQ when the delta cycle threshold of replicates is ≥ 1.5 or when the mean cycle threshold (CT) value of the replicates is greater than the highest CT value of the ‘quantitative range’. In such cases, treatment decision based on MRD risk stratification may be suboptimal.”
ddPCR has been suggested as a viable alternative since each sample is divided into droplets that are individually analyzed and with smaller changes in fluorescence intensity being more easily detected. Findings from this new study are supporting the use of this method, as they indicate that it may be more accurate, particularly in late follow-up time points.
The findings are based on more than 200 pediatric patients with B-lineage ALL classified as POS-NQ and/or negative (NEG) at days 33 and/or 78.
ddPCR was able to improve the stratification of slow early responders with POS-NQ MRD based on RQ-PCR and allow for distinguishing of true positive cases from NEG—groups that carry different clinical outcomes—based on 45 collected samples at day 78, which revealed that 13 (29%) were positive quantifiable cases.
The was also deemed more sensitive at day 78 compared with RQ-PCR after it identified several cases with positive MRD values that were considered RQ-PCR NEG cases. Overall, ddPCR confirmed MRD negativity and POS-NQ in 112 moderate risk patients and 52 standard risk patients, with the exception of a few cases in which no differences in outcome were registered.
“The greater accuracy of ddPCR allowed to discriminate very low/POS-NQ samples by RQ-PCR, turning them into POS-Q in 20% (21/105) of cases, or confirming them to be NEG in 56% (59/105),” wrote the researchers. “Of note, ddPCR was able to prove a more robust and precise quantification than RQ-PCR for samples with positivity < 10–4, the most challenging cut-off at both clinical and methodologic levels. Importantly, ddPCR MRD data were generated by three different laboratories, and all labs were able to precisely quantify RQ-PCR low positive samples ranging between 10–4 and 10–5, confirming the strength of the ddPCR assay.”
ddPCR does carry an overall cost that’s double that of RQ-PCR, although the researchers note that more samples per plate can be run by ddPCR (29 vs 25 on a 96-well plate, respectively).
Starza I, Nunes V, Lovisa F, et al. Droplet digital PCR improves IG-/TR-based MRD risk definition in childhood B-cell precursor acute lymphoblastic leukemia. Hemasphere. 2021;5(3):e543.