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目的评价实时定量 RT-PCR(Q-PCR)技术用于监测慢性粒细胞白血病(CML)患者异基因造血干细胞移植(HSCT)疗效的意义。方法采用基于 TaqMan 探针的 Q-PCR 技术检测112例CML 患者 HSCT 后不同时间共316份骨髓标本 bcr-abl mRNA 的表达。bcr-abl mRNA 水平以内参基因abl 进行归一化。采用荧光原位杂交法(FISH)评估 HSCT 后是否达细胞遗传学完全缓解(CCyR)。结果 Q-PCR 实验可重复敏感度为5个拷贝。abl 及 bcr-abl 基因C_T 值的批内差及批间差均<2.0%。HSCT 后1个月开始持续处于 CCyR 状态的101例患者,中位跟踪时间为12(6～60)个月的289份标本表现为 HSCT 后各时间段均有一定数量患者可检测出 bcr-abl mRNA,总体上随着 HSCT 时间延长,中位水平逐渐降低。HSCT 后1个月中位 bcr-abl 水平为0.035%(0～0.406%),较本室资料初治 CML 慢性期患者中位水平降低3个对数级,3个月时为0.006%(0～0.683%),6个月开始降至0%(0～0.225%),移植6个月后标本最高 bcr-abl 水平为0.068%。8例患者 HSCT 后未达 CCyR 或遗传学复发时 bcr-abl 水平为0.12%～13.45%,其中5例患者遗传学复发前的1份标本(复发前1～2个月) bcr-abl 水平为0.09%～3.42%。9例持续 CCyR 患者 bcr-abl 水平曾经>0.090%,但随后均下降至0。2例急变期患者 HSCT 后1.6和3个月内持续 CCyR,但分别于1个月和1.5个月后进展为血液学复发,bcr-abl 水平亦从0及0.140% 分别急剧增至46.900% 和75.900%。结论 Q-PCR 技术精确、可靠,对CML 患者 HSCT 后有必要定期系列监测 bcr-abl 水平,以及时了解病情变化。对急变期患者需要更密切的监测。
Objective To evaluate the value of real-time quantitative RT-PCR (Q-PCR) in monitoring the efficacy of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with chronic myeloid leukemia (CML). Methods The expression of bcr-abl mRNA in 316 bone marrow specimens from 112 CML patients after HSCT was detected by Q-PCR based on TaqMan probe. The level of bcr-abl mRNA was normalized with the reference gene abl. Fluorescence in situ hybridization (FISH) was used to assess cytogenetic complete response (CCyR) after HSCT. Results The reproducibility of Q-PCR assay was 5 copies. The intra-assay differences and inter-assay differences of abl and bcr-abl gene C_T values were all <2.0%. Of the 101 patients who remained in CCyR at 1 month after HSCT, 289 patients with a median follow-up time of 12 (6 to 60) months showed that a certain number of patients at each time after HSCT had detectable bcr-abl mRNA, the overall level with the HSCT time-lapse, the median level gradually decreased. At 1 month after HSCT, the median bcr-abl level was 0.035% (0-0.406%), which was 3 log lower than the median level of CML patients in the first trimester and 0.006% at 3 months ~ 0.683%), 6 months began to drop to 0% (0 ~ 0.225%), 6 months after transplantation the highest bcr-abl level of 0.068%. In 8 patients, the level of bcr-abl was 0.12% ~ 13.45% after CCCT or no genetic recurrence, and the level of bcr-abl in 5 patients before genesis (1 ~ 2 months before relapse) was 0.09% ~ 3.42%. The bcr-abl level was> 0.090% in 9 patients with persistent CCyR but then all decreased to 0.2% of patients with persistent CCyR who sustained CCyR at 1.6 and 3 months after HSCT but progressing to 1 and 1.5 months respectively Hematological relapse, bcr-abl levels also drastically increased from 0 and 0.140% to 46.900% and 75.900% respectively. Conclusion The Q-PCR technique is accurate and reliable. It is necessary to regularly monitor the level of bcr-abl in CML patients after HSCT, and to understand the changes of the disease. Patients in blastic need to be monitored more closely.