Ojhm. Verhagen et al., Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia, LEUKEMIA, 14(8), 2000, pp. 1426-1435
Large-scale clinical studies on detection of minimal residual disease (MRD)
in acute lymphoblastic leukemia (ALL) have shown that quantification of MR
D levels is needed for reliable MRD-based risk group classification. Recent
ly, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied
for this purpose using patient-specific immunoglobulin (Ig) and T cell rec
eptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the po
sition of the junctional region and two germline primers. Now, we tested an
alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrange
ments, by designing three germline J(H) TaqMan probes to be used in combina
tion with one of six corresponding germline J(H) primers and one allele spe
cific oligonucleotide (ASO) primer complementary to the junctional region.
In nine cases in which both approaches were compared, at least similar (n =
4) or slightly higher (n = 5) maximal sensitivities were obtained using an
ASO primer. The ASO primer approach reached maximal sensitivities of at le
ast 10(-4) in 33 out of 35 IGH rearrangements. The reproducible range for a
ccurate quantification spanned four to five orders of magnitude in 31 out o
f 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions
had to be increased to remove or diminish background signals; this only co
ncerned the frequently occurring J(H)4, J(H)5 and J(H)6 gene rearrangements
. After optimization of the conditions (mainly by increasing the annealing
temperature), only occasional aspecific amplification signals were observed
at high threshold cycle (C-T) values above 42 cycles and at least six cycl
es above the C-T value of the detection limit. Hence, these rare aspecific
signals could be easily discriminated from specific signals. We conclude th
at the here presented set of three germline J(H) TaqMan probes and six corr
esponding germline J(H) primers can be used to develop patient-specific RQ-
PCR assays, which allow accurate and sensitive MRD analysis in almost all I
GH gene rearrangements. These results will facilitate standardized RQ-PCR a
nalysis for MRD detection in large clinical studies.