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 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.
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We gratefully acknowledge Annella Boon for secretarial assistance, Jacqueline van Valen and Phary Hart for technical assistance, Professor Rob Benner for continuous support, and Marieke Comans-Bitter for preparation of the figures. We thank the pediatric oncologists of the Department of Pediatrics, Sophia Children's Hospital, Rotterdam, for the collection of samples at diagnosis and during follow-up. We acknowledge the Dutch Childhood Leukemia Study Group for kindly providing additional ALL cell samples. Board members of the DCLSG are PJ van Dijken, K Hählen, WA Kamps, ETh Korthof, FAE Nabben, A Postma, JA Rammeloo, GAM de Vaan, AJP Veerman and RS Weening. This study was supported by the Dutch Cancer Society/Koningin Wilhelmina Fonds (grants: SNWLK 97–1567 and SNWLK 2000–2268).
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van der Velden, V., Wijkhuijs, J., Jacobs, D. et al. 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, 1372–1380 (2002). https://doi.org/10.1038/sj.leu.2402515
- acute leukemia
- minimal residual disease
- T cell receptor gamma
- real-time quantitative
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