title: Rapid and reliable quantification of minimal residual disease in acute lymphoblastic leukemia using rearranged immunoglobulin and T-cell receptor loci by LightCycler technology.
abstract: 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.
authors.affiliation: Institute of Human Genetics, University of Heidelberg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Nakao
authors.forename: J W
authors.initials: JW
authors.lastname: Janssen
authors.forename: T
authors.initials: T
authors.lastname: Flohr
authors.forename: C R
authors.initials: CR
authors.lastname: Bartram
mesh_terms: D000328:Adult; D052160:Benzothiazoles; D002051:Burkitt Lymphoma / Q000175:diagnosis* / Q000235:genetics; D002648:Child; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism; D015321:Gene Rearrangement; D015329:Gene Rearrangement, T-Lymphocyte / Q000235:genetics*; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics*; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000175:diagnosis* / Q000235:genetics; D018365:Neoplasm, Residual / Q000175:diagnosis*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D007136:Immunoglobulins; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I