Quantitative Assessment of Minimal Residual Disease in Childhood Lymphoid Malignancies Using an Allele-Specific Oligonucleotide Real-Time Quantitative Polymerase Chain Reaction

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Abstract
We developed an assay using a real-time quantitative polymerase chain reaction (RQ-PCR) for the quantitative assessment of minimal residual disease (MRD) in childhood lymphoid malignancies by using a consensus V-region probe combining a allele-specific oligonucleotide (ASO) reverse primer. Our strategy employs a set consisting of a consensus V-region probe, an ASO reverse primer, and a patient-specific forward primer for clonal antigen-receptor (IgH, immunoglobulin heavy chain; TCR, T-cell receptor) gene rearrangements (IgH-ASO and TCR-ASO RQ-PCR assays). The limit of detection in both assays was 5 copies of the target/105 cell equivalents. We tested the assays in 17 childhood malignancies (14 cases of acute lym-phoblastic leukemia and 3 of non-Hodgkin’s lymphoma). High correlation coefficients of the standard curves (>0.980) and PCR efficiency (>0.95) were achieved with all primer/probe sets. In 2 (12%) of the 17 patients, ASO primers could not be designed because there was no junctional N-sequence. The quantitative data suggest that the copy number of clonal antigen receptors markedly decreased after induction therapy in 15 of 17 patients and that 1 patient relapsed and died of the disease. Consensus probes make it possible to examine a large number of patients with only a limited number of probes. The strategy used for IgH-ASO and TCR-ASO RQ-PCR assays is accurate and reliable in the clinical prospective study of MRD in childhood lymphoid malignancies.