Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia

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.