Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR

Abstract

Immunoglobulin gene rearrangements are used as PCR targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). We Investigated the occurrence of monoclonal immunoglobulin kappa-deleting element (IGK-Kde) rearrangements by Southern blotting and PCR/heteroduplex analysis at diagnosis, their stability at relapse, and their applicability in real-time quantitative PCR (RQ-PCR) analysis. In 77 selected children with precursor-B-ALL, Southern blotting detected 122 IGK-Kde rearrangements, 12 of which were derived from subclones in six patients (8%). PCR/heteroduplex analysis with BIOMED-1 Concerted Action primers identified 100 of the 110 major IGK-Kde rearrangements (91%). Comparison between diagnosis and relapse samples from 21 patients with PCR-detectable IGK-Kde rearrangements (using Southern blotting, PCR/heteroduplex analysis, and sequencing) demonstrated that 27 of the 32 rearrangements remained stable at relapse. When patients with oligoclonal IGK-Kde rearrangements were excluded, 25 of the 27 rearrangements remained stable at relapse and at least one stable rearrangement was present In 17 of the 18 patients. Subsequently, RQ-PCR analysis with allele-specific forward primers, a germline Kde Taq-Man-probe, and a germline Kde reverse primer was evaluated for 18 IGK-Kde rearrangements. In 16 of the 18 targets (89%) a sensitivity of less than or equal to10(-4) was reached. Analysis of MRD during follow-up of eight patients with IGK-Kde rearrangements showed comparable results between RQ-PCR data and classical dot-blot data. We conclude that the frequently occurring IGK-Kde rearrangements are generally detectable by PCR (similar to90%) and are highly stable MRD-PCR targets, particularly where monoclonal rearrangements at diagnosis similar to(95%) are concerned. Furthermore, most IGK-Kde rearrangements (similar to90%) can be used for sensitive detection of MRD (less than or equal to10(-4)) by RQ-PCR analysi