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

PCR-based detection of minimal residual disease (MRD) in children with prec ursor B cell acute lymphoblastic leukemia (pre-B ALL) by amplification of c lone-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 o f the immunoglobulin heavy chain (IgH) and T cell receptor (TcR) gene rearr angements characterized at the time of diagnosis or relapse, primers were d esigned and tested in a clone-specific, single-round PCR assay, then analyz ed by fluorescence staining of the PCR products. The most critical step inv olved an adherence to a new set of guidelines for the design of clone-speci fic primers. Application of this method to 54 IgH and 13 TcR (nine V delta 2D delta 3 and four D delta 2-D delta 3) gene rearrangements in 47 patients resulted in an intense band within the region of the predicted molecular w eight, confirming the reproducibility of the assay. Quantitative applicatio ns of the approach were examined by performing a 10-replicate limiting dilu tion clone-specific PCR on six diagnostic samples and an asymptotic respons e model of the Von Krogh form was found to fit the data well. From this mod el, estimation of leukemic cells of remission bone marrow samples was achie ved at a detection sensitivity of 2x10(-6). The method is demonstrated on 1 8 patients whose marrows were prospectively analyzed during therapy. We con clude this methodology is useful in the quick and accurate assessment of MR D in children with pre-B ALL, and could be applied to other DNA quantitatio n assays.