Quantitation of leukemia clone-specific antigen gene rearrangements by a single-step PCR and fluorescence-based detection method

Abstract

PCR-based detection of minimal residual disease (MRD) in children with precursor B cell acute lymphoblastic leukemia (pre-B ALL) by amplification of clone-specific antigen gene rearrangements has been labor-intensive. In this study we present a simpler, yet accurate, method to assess and quantitate MRD in pediatric pre-B ALL that utilizes these markers. From the sequence of the immunoglobulin heavy chain (IgH) and T cell receptor (TcR) gene rearrangements characterized at the time of diagnosis or relapse, primers were designed and tested in a clone-specific, single-round PCR assay, then analyzed by fluorescence staining of the PCR products. The most critical step involved an adherence to a new set of guidelines for the design of clone-specific primers. Application of this method to 54 IgH and 13 TcR (nine Vδ2Dδ3 and four Dδ2-Dδ3) gene rearrangements in 47 patients resulted in an intense band within the region of the predicted molecular weight, confirming the reproducibility of the assay. Quantitative applications of the approach were examined by performing a 10-replicate limiting dilution clone-specific PCR on six diagnostic samples and an asymptotic response model of the Von Krogh form was found to fit the data well. From this model, estimation of leukemic cells of remission bone marrow samples was achieved at a detection sensitivity of 2 × 10⁻⁶. The method is demonstrated on 18 patients whose marrows were prospectively analyzed during therapy. We conclude this methodology is useful in the quick and accurate assessment of MRD in children with pre-B ALL, and could be applied to other DNA quantitation assays.