Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting

Abstract

Minimal residual disease (MRD) diagnostics is used for treatment stratification in childhood acute lymphoblastic leukemia. We aimed to identify and solve potential problems in multicenter MRD studies to achieve and maintain consistent results between the AIEOP/BFM ALL-2000 MRD laboratories. As the dot-blot hybridization method was replaced by the real-time quantitative polymerase chain reaction (RQ-PCR) method during the treatment protocol, special attention was given to the comparison of MRD data obtained by both methods and to the reproducibility of RQ-PCR data. Evaluation of all key steps in molecular MRD diagnostics identified several pitfalls that resulted in discordant MRD results. In particular, guidelines for RQ-PCR data interpretation appeared to be crucial for obtaining concordant MRD results. The experimental variation of the RQ-PCR was generally less than three-fold, but logically became larger at low MRD levels below the reproducible sensitivity of the assay (o10 À4 ). Finally, MRD data obtained by dot-blot hybridization were comparable to those obtained by RQ-PCR analysis (r 2 ¼ 0.74). In conclusion, MRD diagnostics using RQ-PCR analysis of immunoglobulin/T-cell receptor gene rearrangements is feasible in multicenter studies but requires standardization; particularly strict guidelines for interpretation of RQ-PCR data are required. We further recommend regular quality control for laboratories performing MRD diagnostics in international treatment protocols.

Screening for leukemia- and clone-specific markers at birth in children with T-cell precursor ALL suggests a predominantly postnatal origin

“…[12][13][14] Detection of the specific TCR rearrangements in DNA from neonatal blood spots was performed by real-time quantitative polymerase chain reaction (RQ-PCR) as used for minimal residual disease analysis. 15 All the other markers were amplified by a 2-round nested PCR (Table S1, available on the Blood website; see the Supplemental Tables link at the top of the online article). Identification of TCRD-LMO2 breakpoint regions was performed by long-range ligation-mediated PCR as previously described.…”

Quantification of free total plasma DNA and minimal residual disease detection in the plasma of children with acute lymphoblastic leukemia

“…MRD assessment in plasma and peripheral leukocytes MRD detection in PB leukocytes was conducted as published by others [23][24][25][26]. Briefly, MRD PCR targets were tested for specificity and sensitivity to enable on-time monitoring of remission samples.…”

Minimal residual disease (MRD) analysis in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: is it possible to avoid MRD testing?

“…Moreover, to guarantee the credibility of the results, it requires regular inter-laboratory quality controls. 9 For these reasons, this method is still inapplicable in many laboratories around the world.…”