title: Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes.
abstract: Analysis of minimal residual disease (MRD) can predict outcome in acute lymphoblastic leukemia (ALL). A large prospective study in childhood ALL has shown that MRD analysis using immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets can identify good and poor prognosis groups of substantial size that might profit from treatment adaptation. This MRD-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 samples. However, current PCR methods do not allow easy and accurate quantification. We have tested 'real-time' quantitative PCR (RQ-PCR) using the TaqMan technology and compared its sensitivity with two conventional MRD-PCR methods, ie dot-blot and liquid hybridization of PCR amplified Ig/TCR gene rearrangements using clone-specific radioactive probes. In RQ-PCR the generated specific PCR product is measured at each cycle ('real-time') by cleavage of a fluorogenic intrinsic TaqMan probe. The junctional regions of rearranged Ig/TCR genes define the specificity and sensitivity of PCR-based MRD detection 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 design of patient-specific TaqMan probes at the position of the junctional regions. 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-blot 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 possibilities for semi-automation. We consider this novel technique as an important step forward towards routinely performed diagnostic MRD studies.
authors.affiliation: Dept of Immunology, University Hospital Rotterdam/Erasmus University Rotterdam, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Pongers-Willemse
authors.forename: O J
authors.initials: OJ
authors.lastname: Verhagen
authors.forename: G J
authors.initials: GJ
authors.lastname: Tibbe
authors.forename: A J
authors.initials: AJ
authors.lastname: Wijkhuijs
authors.forename: V
authors.initials: V
authors.lastname: de Haas
authors.forename: E
authors.initials: E
authors.lastname: Roovers
authors.forename: C E
authors.initials: CE
authors.lastname: van der Schoot
authors.forename: J J
authors.initials: JJ
authors.lastname: van Dongen
mesh_terms: D000328:Adult; D002648:Child; D015342:DNA Probes / Q000235:genetics; D015329:Gene Rearrangement, T-Lymphocyte; D005803:Genes, Immunoglobulin; D019672:Genes, T-Cell Receptor; D006801:Humans; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis* / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes