title: T cell receptor gamma gene rearrangements as targets for detection of minimal residual disease in acute lymphoblastic leukemia by real-time quantitative PCR analysis.
abstract: Several studies have shown that quantitative detection of minimal residual disease (MRD) predicts clinical outcome in childhood acute lymphoblastic leukemia (ALL). In this report we investigated the applicablility of T cell receptor gamma (TCRG) gene rearrangements as targets for MRD detection by real-time quantitative PCR analysis. Seventeen children with precursor-B-ALL and 15 children with T-ALL were included in this study. Using an allele-specific (ASO) forward primer in combination with germline Jgamma reverse primers and Jgamma TaqMan probes, a reproducible sensitivity of < or =10(-4) (defined by strict criteria) was obtained in only four out of 19 (21%) TCRG gene rearrangements in precursor-B-ALL patients and in 10 out of 15 (67%) TCRG gene rearrangements in T-ALL patients. The main reason for not obtaining a reproducible sensitivity of < or =10(-4) in approximately 60% of cases was the non-specific amplification of TCRG gene rearrangements in normal T-lymphocytes. A maximal sensitivity of < or =10(-4) (defined by less strict criteria) was obtained in 42% of TCRG gene rearrangements in precursor-B-ALL patients. The number of inserted nucleotides was significantly higher in T-ALL (mean: 8.5) as compared to precursor-B-ALL (mean: 6.8) and appeared to be the most important predictor for reaching a reproducible sensitivity < or =10(-4). The usage of a touchdown PCR or the usage of an ASO reverse primer in combination with Vgamma member forward primers and TaqMan probes did not clearly improve the overall results. Nevertheless, RQ-PCR analysis of TCRG gene rearrangements in follow-up samples obtained from 12 ALL patients showed the applicability of this method for MRD detection. We conclude that RQ-PCR analysis of TCRG gene rearrangements can be used for the detection of MRD, but that sensitivities might be limited due to non-specific amplification. This method is applicable in the majority of T-ALL patients and in almost half of precursor-B-ALL patients, particularly when used as second-choice target for confirmation of the MRD results obtained via the first-choice target.
authors.affiliation: Department of Immunology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: V H J
authors.initials: VH
authors.lastname: van der Velden
authors.forename: J M
authors.initials: JM
authors.lastname: Wijkhuijs
authors.forename: D C H
authors.initials: DC
authors.lastname: Jacobs
authors.forename: E R
authors.initials: ER
authors.lastname: van Wering
authors.forename: J J M
authors.initials: JJ
authors.lastname: van Dongen
mesh_terms: D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; D019675:Genes, T-Cell Receptor gamma; D006801:Humans; D018365:Neoplasm, Residual; D016133:Polymerase Chain Reaction / Q000379:methods; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology*; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity