Real-Time Quantitative PCR: Standardized Detection of Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia

PURPOSE: To develop a standardized real-time polymerase chain reaction (PCR) method of quantifying minimal residual disease (MRD) in patients with pre-B acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: In a series of 24 follow-up bone marrow (BM) samples in 11 patients (14 clonal markers), we performed real-time PCR assays using one consensus and one clone-specific primer for each marker. The markers analyzed included immunoglobulin heavy chain (IgH), T-cell receptor (TCR) and TEL-AML1 rearrangements. RESULTS: We achieved a detection limit of 3.3 x 10(-5) +/- 1.2 x 10(-5) and an accurate quantitation (r = -0.99) limit of 2.0 x 10(-4) +/- 8.8 x 10(-5) blasts. Both inter- and intra-assay reproducibility were exceptional. Additionally, we found comparable results to those of a "gold standard" limiting-dilution PCR assay (r = 0.62). CONCLUSIONS: The IgH, TCR and TEL-AML1 markers can be used as targets by real-time PCR under the same cycling profile, allowing quantitation of MRD in more 95% of patients with pre-B ALL. This standardized, real-time PCR technique should simplify monitoring MRD in clinical trials.