[Characteristics of T cell receptor beta gene rearrangements and its role in minimal residual disease detection in childhood T-cell acute lymphoblastic leukemia].
OBJECTIVE
To explore the characteristics of T cell receptor beta (TCRbeta) gene rearrangements in children with T-cell acute lymphoblastic leukemia (T-ALL) and establish a system of quantitative detection of MRD with real-time quantitative (RQ-PCR) targeted at TCRbeta gene rearrangement.


METHODS
Multiplex polymerase chain reaction (PCR) designed by BIOMED-2 was used to detect TCRbeta gene rearrangements in the bone marrow samples of 26 children with T-ALL. Sequence of junction region were then compared and analyzed in IMGT database. Allele specific oligonucleotide (ASO) upstream primers were designed complementary to the V-D-J or D-J junctional region of TCRbeta gene rearrangements. Samples at diagnosis were serially diluted in DNA obtained from mononuclear cells (MNC) from a pool of 20 healthy donors to generate the patient specific standard curves. Subsequently, a TCRbeta RQ-PCR assay to quantify MRD with germline Jbeta primer/probe combinations was applied in six patients. To check the quantity and quality of DNA, the investigators used RQ-PCR analysis for the N-ras gene.


RESULTS
Clonal rearrangements were identified in 92.3% childhood T-ALL (Vbeta-Dbeta-Jbeta rearrangements in 84.6%, Dbeta-Jbeta rearrangements in 50%). Comparative sequence analysis of 42 TCRbeta recombinations revealed two downstream Vbeta families (BV5, BV6) were preferentially used. The segment Jbeta2.7 in childhood T-ALL was preferentially used. Jbeta1.3, Jbeta2.4, and Jbeta2.6 were not found to be used. The slope of the standard curves was from -3.54 to -3.37 and the intercepts were from 19.35 to 20.51. The correlation coefficients of all 6 standard curves were excellent (> or = 0.98). All the RQ-PCR quantitative range reached 10(-4). MRD analysis of follow up samples showed that MRD increased before relapse.


CONCLUSIONS
RQ-PCR analysis of TCRbeta gene rearrangements was highly sensitive and specific, it will be of high value for future T-ALL MRD studies. And quantitative and serial study of MRD may be of prognostic importance.
Liu
Jie-yu
JY
Hematology Center, Beijing Children's Hospital Affiliated to Capital Medical University, Beijing 100045, China.
Li
Zhi-gang
ZG
Gao
Chao
C
Cui
Lei
L
Wu
Min-yuan
MY
D000293:Adolescent; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D005260:Female; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor / Q000235:genetics*; D006801:Humans; D007223:Infant; D008297:Male; D008969:Molecular Sequence Data; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA