Droplet Digital PCR Improves IG-/TR-based MRD Risk Definition in Childhood B-cell Precursor Acute Lymphoblastic Leukemia

Abstract

Minimal residual disease (MRD) is the most powerful prognostic factor in pediatric acute lymphoblastic leukemia (ALL). Real-time quantitative polymerase chain reaction (RQ-PCR) represents the gold standard for molecular MRD assessment and risk-based stratification of front-line treatment. In the protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) and the Berlin-Frankfurth-Munschen (BFM) group AIEOP-BFM ALL2009 and ALL2017, B-lineage ALL patients with high RQ-PCR-MRD at day+33 and positive at day+78 are defined slow early responders (SERs). Based on results of the AIEOP-BFM ALL2000 study, these patients are treated as high-risk also when positive MRD signal at day +78 is below the lower limit of quantification of RQ-PCR (“positive not-quantifiable,” POS-NQ). To assess whether droplet digital polymerase chain reaction (ddPCR) could improve patients’ risk definition, we analyzed MRD in 209 pediatric B-lineage ALL cases classified by RQ-PCR as POS-NQ and/or negative (NEG) at days +33 and/or +78 in the AIEOP-BFM ALL2000 trial. ddPCR MRD analysis was performed on 45 samples collected at day +78 from SER patients, who had RQ-PCR MRD ≥ 5.0 × 10 –4 at day+33 and POS-NQ at day+78 and were treated as medium risk (MR). The analysis identified 13 of 45 positive quantifiable cases. Most relapses occurred in this patients’ subgroup, while ddPCR NEG or ddPCR-POS-NQ patients had a significantly better outcome ( P < 0.001). Overall, in 112 MR cases and 52 standard-risk patients, MRD negativity and POS-NQ were confirmed by the ddPCR analysis except for a minority of cases, for whom no differences in outcome were registered. These data indicate that ddPCR is more accurate than RQ-PCR in the measurement of MRD, particularly in late follow-up time points, and may thus allow improving patients’ stratification in ALL protocols.

Droplet Digital PCR for BCR–ABL1 Monitoring in Diagnostic Routine: Ready to Start?

“…Moreover, Fluidigm digital PCR assay has been used in the ENESTnext (NCT01227577) study, confirming that ddPCR is more sensitive than RT-qPCR in patients with confirmed MR4.5, since about 40% of analyzed samples had a detectable BCR-ABL1 [26]. Our data also corroborated results previously obtained by Della Starza et al in pediatric acute lymphoblastic leukemia, where they identify ddPCR as more accurate than RT-qPCR in the measurement of MRD, particularly in the advanced follow-up [27]. Moreover, we showed no differences between duplicate or quadruplicate analysis, even for the lowest levels of disease.…”

Digital Droplet PCR in Hematologic Malignancies: A New Useful Molecular Tool

“…[ 68 ]. Trying to address this issue, Dr. Della Starza and coworkers proposed ddPCR as an alternative method for MRD monitoring [ 11 , 69 ] in samples from patients enrolled in the GIMEMA LAL1308 and in the AIEOP-BFM ALL 2000 trials, finding a concordance rate of 70% between QT-PCR and ddPCR. The greater accuracy of ddPCR allowed to quantitate samples defined as PNQ by QT-PCR in a quarter of cases.…”

Immune Gene Rearrangements: Unique Signatures for Tracing Physiological Lymphocytes and Leukemic Cells

“…Although NGS and ddPCR have not yet been fully implemented into routine practice, there are hints that both methods possess appreciable advantages over RQ-PCR. Particularly samples which are positive below the quantitative range (pos < QR) of RQ-PCR can often be assessed more confidently: thanks to the more specific readout of NGS, false-positive RQ-PCR results which might occur due to the nonspecific amplification can be reliably distinguished from truly positive samples [ 101 ]; ddPCR is able to provide an accurate MRD quantification in 20–45% of pos < QR samples thanks to its absolute quantification without the need of a standard curve [ 102 , 103 , 104 , 105 ]. Another frequently mentioned advantage of the modern methods is the improved sensitivity.…”