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  • Biotechnical Methods Section BTS
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Biotechnical Methods Section (BTS)

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, pages 1372–1380 (2002)Cite this article

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 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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Acknowledgements

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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  1. Department of Immunology, Erasmus MC, Rotterdam, The Netherlands

    VHJ van der Velden, JM Wijkhuijs, DCH Jacobs & JJM van Dongen

  2. Dutch Childhood Leukemia Study Group, The Hague, The Netherlands

    ER van Wering

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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

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