Rapid and reliable quantification of minimal residual disease in acute lymphoblastic leukemia using rearranged immunoglobulin and T-cell receptor loci by LightCycler technology

The detection of minimal residual disease (MRD) using immunoglobulin and T- cell receptor (TCR) rearrangements as PCR targets provides important progno stic information on the in vivo effectiveness of treatment in acute lymphob lastic leukemia (ALL), Here we report on the real-time quantification of MR D in 25 ALL patients using LightCycler technology. We designed and adapted allele-specific oligonucleotide (ASO)-PCR protocols that enabled the detect ion of >90% of the IGH, IGK, TCRD, and TCRG rearrangements observed in ALL patients. In all patients, at least two suitable markers could be identifie d (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 t he sensitivity and practicability of the LightCycler strategy with conventi onal ASO-PCR on a block thermocycler followed by quantification with gel el ectrophoresis, The LightCycler measured leukemia-specific PCR products at e ach cycle (real-time) by staining the PCR product with the DNA-binding dye SYBR Green I. LightCycler technology showed a higher sensitivity than the c onventional method in eight cases, whereas the sensitivity of the other mar kers 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 compar able results. However, the LightCycler also allowed accurate quantification in samples containing relatively high levels (>10(-3)) of residual leukemi a 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 rep resents a rapid, reliable, sensitive, and cost-effective technique for the routine monitoring of MRD in ALL patients.