Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes
Mj. Pongers-willemse et al., Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes, LEUKEMIA, 12(12), 1998, pp. 2006-2014
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.