成人急性T淋巴细胞白血病T淋巴细胞受体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

Characteristics of TCR β gene rearrangements in adult patients with T-lineage acute lymphoblastic leukemia and its significance in quantitation of minimal residual disease

目的 建立以TCR β基因重排为分子标志,基于ASO引物的RQ-PCR方法,检测成人T-ALL患者的TCR β基因重排,以利于动态监测患者的MRD.方法 20例成人T-ALL患者的初诊DNA标本经多重PCR检测方法设计38条引物,分成3管,分别扩增TCR β基因完全重排和不完全重排,克隆产物经电泳和双色基因扫描鉴定.对其中4例患者进行基因测序和序列比对分析,根据克隆特异性序列信息,利用软件设计4条ASO引物,作为RQ-PCR检测的上游引物,合成通用的Jβ下游引物和TaqMan探针,对患者的随访标本定量监测MRD.结果 20例患者中有17例可检测到克隆性TCR β基因重排,克隆检出率为85.0%（17/20）.其中16例可检测到至少1个Vβ-Jβ完全重排（94.1%,16/17）,7例患者有Dβ-Jβ的不完全重排（41.2%,7/17）,完全Vβ-Jβ和不完全Dβ-Jβ重排之间的比例约为2∶1.双色基因扫描分析显示Jβ2家族的使用频率为73%,Jβ1家族的使用频率为27%,Jβ2家族的使用频率明显高于Jβ1家族.4例患者使用ASO引物RQ-PCR扩增TCR β重排靶基因的标准曲线的斜率为-3.60～-3.27,相关系数＞0.99,敏感性达4×10-5μg/μl,荧光背景信号不显示或较低.监测4例T-ALL患者在疾病治疗各随访时间点的TCR β重排靶基因水平,包括诱导完全缓解、巩固治疗等时间点,RQ-PCR显示其MRD水平随病情的缓解呈逐渐下降的趋势.结论 以克隆性TCR β基因重排为靶分子的特异性寡核苷酸引物RQ-PCR方法可对T淋巴细胞系统的恶性克隆进行准确定量,适用于成人T-ALL患者MRD的随访监测.

Objective To develop allele specific oligonucleotide（ASO） -PCR assay based on TCR βgene rearrangements and provide a screening method for minimal residual disease （MRD） in adult patients with T-lineage acute lymphoblastic leukemia （T-ALL）.Methods DNA samples from newly diagnosed 20 adult T-ALL patients were obtained.The TCR β gene rearrangements were detected by multiplex PCR,which included 38 paired of primers in 3 reaction tubes.Gel electrophoresis and two-color Gene Scanning was also applied for clonality analysis of TCR β followed by sequencing and subsequent blasting for monoclonal PCR products in four patients.ASO primers were designed based on the sequence of junction regions.MRD were detected in the bone marrow by RQ-PCR with ASO upstream primers, consensus Jβprobes and downstream primers.Results The detection rate of the clonal TCR β rearrangements was 85.0% （17/20）.At least one complete Vβ-Jβ rearrangement could be detected at the time of diagnosis in 16 out of 17 patients（94.1%, 16/17）.Incomplete Dβ-Jβ rearrangement could be detected in 7 patients （41.2% ,7/17）.The positivitity rate of Vβ-Jβ to Dβ-Jβ was 2∶1 （94.1% versus 41.2% ）.Two-color Gene Scanning analysis showed the Jβ2 family was used more frequently than the Jβ1 family （73% versus 27% ）.The slopes of the standard curves ranged from - 3.60 to - 3.27.The correlation coefficients of all four standard curves were more than 0.99.The detection sensitivity of ASO-PCR was 4 × 10 -5 μg/μl.The fluorescence background were detected at a low level.Quantitative MRD values of TCR β rearrangement in sequential BM specimens of 4 adult T-ALL patients were monitored during the treatment, including complete remission after induction and after consolidation therapy. RQ-PCR showed the MRD values of TCR β rearrangement were gradually decreased in response to the treatment.Conclusions The quantification of TCR β rearrangement by ASO-PCR approach is sensitive, specific and reliable for the accurate evaluation of malignant clones.It is suitable for the monitoring of minimal residual disease of adult T-ALL patients.