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Rapid and Reliable Quantification of Minimal Residual Disease in Acute Lymphoblastic Leukemia Using Rearranged Immunoglobulin and T-Cell Receptor Loci by LightCycler Technology¹

Institute of Human Genetics, University of Heidelberg, D-69120 Heidelberg, Germany

The detection of minimal residual disease (MRD) using immunoglobulin and T-cell receptor (TCR) rearrangements as PCR targets provides important prognostic information on the in vivo effectiveness of treatment in acute lymphoblastic leukemia (ALL). Here we report on the real-time quantification of MRD in 25 ALL patients using LightCycler technology. We designed and adapted allele-specific oligonucleotide (ASO)-PCR protocols that enabled the detection of >90% of the IGH, IGK, TCRD, and TCRG rearrangements observed in ALL patients. In all patients, at least two suitable markers could be identified (average, 3.4 markers/patient). We applied ASO-PCR with 35 immunoglobulin and TCR rearrangements (11 IGH, 6 IGK, 12 TCRG, and 6 TCRD) and compared the sensitivity and practicability of the LightCycler strategy with conventional ASO-PCR on a block thermocycler followed by quantification with gel electrophoresis. The LightCycler measured leukemia-specific PCR products at each cycle (real-time) by staining the PCR product with the DNA-binding dye SYBR Green I. LightCycler technology showed a higher sensitivity than the conventional method in eight cases, whereas the sensitivity of the other markers matched exactly. The detection level varied between 10^-4 and 10^-6 leukemic cells. Furthermore, we determined the MRD status of 27 bone marrow follow-up samples from 15 ALL patients by both methods and revealed comparable results. However, the LightCycler also allowed accurate quantification in samples containing relatively high levels (>10^-3) of residual leukemia cells. The conventional ASO-PCR technique comprises various laborious and time-consuming PCR experiments and post-PCR steps to determine the number of cycles with the optimal linearity and sensitivity of the PCR. Real-time quantification through LightCycler technology obviates these post-PCR steps, provides the highest sensitivity via software analysis, and therefore represents a rapid, reliable, sensitive, and cost-effective technique for the routine monitoring of MRD in ALL patients.

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