儿童急性T淋巴细胞性白血病T细胞受体β链基因重排的特点及其在微小残留病定量检测中的意义

目的 建立以T细胞受体p链(TCRβ)基因重排为分子标志定量检测儿童急性T淋巴细胞性白血病(T-ALL)微小残留病(MRD)的实时定量聚合酶链反应(RQ-PCR)检测体系.方法 应用BIOMED-2欧洲协作组设计的PCR检测体系,检测26例初发T-ALL患儿的TCRB基因重排,并进行序列分析与比对.对其中6例患儿根据基因重排后连接区序列设计等位基因特异性寡核苷酸(ASO)引物,结合胚系TaqMan探针与引物,建立RQ-PCR检测方法,行缓解期标本MRD定量追踪检测.结果 92.3%的T-ALL患儿可检测到克隆性TCRβ基因重排84.6%具有Vβ-(Dβ)-Jβ完全重排,50%具有DB-Jβ不完全重排].通过序列分析,V片段使用频率最高的家族为BV5、BV6,J区使用频率最高的家族为Jβ2.7,J1.3、J2.4、J2.6未被使用.6例患儿ASO标准曲线的斜率为-3.54～-3.37,截距为19.35～20.51,相关系数＞0.98.定量范围均达到10-4.对随访期标本MRD检测发现,复发患儿的MRD水平在复发前显著升高.结论 以TCRβ基因重排为分子标志对T-ALL患儿行MRD定量检测,具有较高的敏感度和特异性,动态追踪检测随访期标本MRD水平具有重要的临床价值.

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(TCRβ)gene rearrangements in children with T-cell acute lymphoblagtic leukemia(T-ALL)and estabhsh a system of quantitative detection of MRD with real-time quantitative(RQ-PCR)targeted at TCRβ gene rearrangement.Methods Multiplex polymerase chain reaction(PCR)designed by BIOMED-2 wag used to detect TCRβ gene rearrangements in the bone marrow samples of 26 children with T-ALL Secquence of iunction 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 junetional region of TCRβ 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 TCRβ RQPCR assay to quangify MRD with germline Jβ 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(Vβ-Dβ-Jβ rearrangements in 84.6%,Dβ-Jβ rearrangements in 50%).Comparative sequence analysis of 42 TCRl3 recombinations revealed two downstream Vβ families(BV5,BV6)were preferentially used.The segment Jβ2.7 in childhood T-ALL was preferentially used.Jβ1.3,Jβ2.4.and Jβ2.6 were not found to be used.The slope of the standard curves wag 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 excelent(≥0.98).Au the RQ-PCR quantitative range reached 10-4.MRD analysis of follow up samples showed that MRD increased before relapse.Conclusion RQ-PCR analysis of TCRB gene rearrangements was highly sensitive and specific,it will be of hiish value for future T-ALL MRD studies.And quantitative and serial study of MRD may be of prognostic importance.