Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR

PCR of clonally rearranged immunoglobulin heavy chain (IgH) gene sequences is increasingly used for detection of minimal residual disease (MRD) in lym phoid malignancies. Inherent quantitating problems are the main drawbacks o f traditional PCR technologies. These limitations have been overcome by the recently developed real-time quantitative PCR (RQ PCR) technology. However , clinical application of the few published RQ PCR assays targeting immune gene rearrangements is hampered by the expensive and time-consuming need fo r individual hybridization probes for each patient. We have developed a new RQ PCR strategy targeting clonally rearranged IgH sequences that solves th is problem. The method uses only two different J(H) hybridization probes an d four downstream J(H) primers homologous to consensus germline J(H) gene s egments. In combination with an allele-specific upstream (ASO) primer the c onsensus J(H) probes and primers allow quantitation of about 90% of possibl e IgH rearrangements. In a series of 22 B-lineage ALL the new assay allowed the detection of one to 10 blasts in a background of 10(5) normal cells. T o prove the clinical utility we quantified MRD in 23 follow-up samples of s ix ALL patients with the new assay in comparison with a published RQ PCR te chnique that used individually designed primer/probe sets. We showed that t he sensitivity of the new RQ PCR assay was slightly higher for four of the six cases and about 100-fold higher for one case, enabling detection of an increasing MRD level as an indicator of subsequent relapse 44 weeks earlier compared to the ASO probe assay in this particular patient. The results su ggest, that the novel RQ PCR assay is a rapid, technically simple, reliable , and sensitive alternative to traditional quantification assays and simpli fies current approaches of monitoring MRD in clinical trials.