急性淋巴系白血病微小残留病的动态监测

Dynamic Monitoring of Minimal Residual Disease in Acute Lymphoid Leukemia

本研究旨在建立免疫球蛋白重链基因重排的实时定量PCR(RQ-PCR)检测方法,并探讨其在急性淋巴系白血病(ALL)微小残留病(MRD)动态监测中的应用价值。采用多重PCR筛选初诊ALL患者的IgH基因单克隆重排,PCR产物测序并进行序列比对,据其连接区序列设计等位基因特异性寡核苷酸(ASO)前向引物,联合下游保守JH引物和保守JH探针,用Taqman探针法RQ-PCR监测ALL患者治疗过程中各时间点的MRD水平,分析其敏感性和特异性。结果表明,在51例ALL患者中鉴定出21例单克隆IgH重排,其中有条件定量的为15例,标准曲线的斜率在-3.1～-3.9,相关系数均>0.98,敏感性在10-4-10-5,仅1例敏感性为10-3,定量范围多在10-4以下,背景的非特异性扩增均在低水平,不影响定量。7例MRD水平在10-3以下的患者一直持续缓解,8例MRD水平>10-3,复发率较高。结论:RQ-PCR方法定量IgH基因单克隆重排用于监测ALL患者的MRD水平,具有敏感性高、特异性强、定量结果可靠的优点。IgH重排水平与ALL患者预后高度相关。

The aim of this study was to establish a method for quantitative detection of immunoglobulin heavy chain（IgH） gene rearrangements and to explore its clinical application in monitoring minimal residual disease（MRD） of acute lymphoid leukemia（ALL）.Clonal IgH gene rearrangements in 51 patients with ALL at diagnosis were identified by multiplex PCR assay.PCR products were sequenced and pairwised in IMGT data base.Allele-specific oligonucleotides primers were designed complementary to the junctional region.The conservative JH primers combined with TaqMan probes were used to monitor the level of MRD in ALL patients.The sensitivity and specificity were assessed as well.The results indicated that out of all the 51 ALL patients,IgH rearrangements were identified in 21 cases,and 15 patients out of them were quantified.The slope of the standard curves was-3.1 to-3.9 and the correlation coefficients of all standard curves were 0.98.The sensitivity was between 10^-4 and 10^-5,only one patient＇s sensitivity was 10^-3.Most of the quantitative range was less than 10^-4.The background＇s nonspecific amplification was detectable at a low level and had a little influence on results.7 patients whose MRD level below 10^-3 kept complete remission and another 8 patients whose MRD level above 10^-3 had a higher relapse rate.It is concluded that the analysis of IgH gene rearrangements with RQ-PCR is a highly sensitive,specific and reliable method for accurate evaluation of MRD in ALL.The data indicates a high correlation between the level of IgH rearrangments and the prognosis in ALL patients.