Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes

Analysis of minimal residual disease (MRD) can predict outcome in acute lym phoblastic leukemia (ALL). A large prospective study in childhood ALL has s hown that MRD analysis using immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets can identify good and poor prognosis gro ups of substantial size that might profit from treatment adaptation. This M RD-based risk group assignment was based on the kinetics of tumor reduction . Consequently, the level of MRD has to be defined precisely in follow-up s amples. However, current PCR methods do not allow easy and accurate quantif ication. We have tested 'real-time' quantitative PCR (RQ-PCR) using the Taq Man technology and compared its sensitivity with two conventional MRD-PCR m ethods, ie dot-blot and liquid hybridization of PCR amplified Ig/TCR gene r earrangements using clone-specific radioactive probes. In RQ-PCR the genera ted specific PCR product is measured at each cycle ('real-time') by cleavag e of a fluorogenic intrinsic TaqMan probe. The junctional regions of rearra nged Ig/TCR genes define the specificity and sensitivity of PCR-based MRD d etection in ALL and are generally used to design a patient-specific probe. In the TaqMan technology we have chosen for the same approach with the desi gn of patient-specific TaqMan probes at the position of the junctional regi ons. We developed primers/probe combinations for RQ-PCR analysis of a total of three IGH, two TCRD, two TCRG and three IGK gene rearrangements in four randomly chosen precursor-B-ALL. In one patient, 12 bone marrow follow-up samples were analyzed for the presence of MRD using an IGK PCR target. The sensitivity of the RQ-PCR technique appeared to be comparable to the dot-bl ot method, but less sensitive than liquid hybridization. Although it still is a relatively expensive method, RQ-PCR allows sensitive, reproducible and quantitative MRD detection with a high throughput of samples providing pos sibilities for semi-automation. We consider this novel technique as an impo rtant step forward towards routinely performed diagnostic MRD studies.