T cell receptor gamma gene rearrangements as targets for detection of minimal residual disease in acute lymphoblastic leukemia by real-time quantitative PCR analysis
Leukemia , Volume 16 - Issue 7 p. 1372- 1380
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 applicability 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 Jγ reverse primers and Jγ TaqMan probes, a reproducible sensitivity of ≤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 ≤10-4 in approximately 60% of cases was the non-specific amplification of TCRG gene rearrangements in normal T-lymphocytes. A maximal sensitivity of ≤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 ≤10-4. The usage of a touchdown PCR or the usage of an ASO reverse primer in combination with Vγ 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.
|Acute leukemia, Minimal residual disease, Real-time quantitative, Rearrangements, T cell receptor gamma, TCRG|
van der Velden, V.H.J, Wijkhuijs, J.M, Jacobs, D.C, van Wering, E.R, & van Dongen, J.J.M. (2002). T cell receptor gamma gene rearrangements as targets for detection of minimal residual disease in acute lymphoblastic leukemia by real-time quantitative PCR analysis. Leukemia, 16(7), 1372–1380. doi:10.1038/sj.leu.2402515