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

Abstract

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline J H TaqMan probes to be used in combination with one of six corresponding germline J H primers and one allele specific 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 least 10 −4 in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 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 concerned 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 cycles above the C T value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline J H TaqMan probes and six corresponding germline J H primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies. Leukemia (2000) 14, 1426-1435.

Genome-wide expression analysis of paired diagnosis–relapse samples in ALL indicates involvement of pathways related to DNA replication, cell cycle and DNA repair, independent of immune phenotype

“…[25][26][27][28][29] Clonal rearrangements were subsequently analyzed in the paired relapse sample of the patient by mixed PCR heteroduplex analysis. 30 In all patients who could be evaluated for Ig/TCR gene rearrangements, at least one rearrangement was preserved, indicating that the relapse did not result from de novo leukemia development.…”

Quantification of fusion transcript reveals a subgroup with distinct biological properties and predicts relapse in BCR/ABL-positive ALL: implications for residual disease monitoring

“…18,19. Quantification of clone-specific antigen-specific receptor gene rearrangements was performed using germline probes and reverse primers [29][30][31][32][33] as described previously. 13 The albumin gene was used to normalize DNA concentration and quality.…”

Minimal residual disease (MRD) analysis in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: is it possible to avoid MRD testing?

“…Family-specific reverse primers and probes for immunoglobulin heavy chain, immunoglobulin light chain k, T-cell receptor d and T-cell receptor g were described previously. [18][19][20][21] Ig/TCR RQ-PCR was performed using the iCycler IQt real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the ABI Prism 7700 real-time PCR System (Applied Biosystems, Foster City, CA, USA). Standard curves were prepared by diluting the diagnostic samples in polyclonal DNA from healthy donors.…”