CD34^＋ CD38^＋急性髓细胞白血病相对端粒长度与端粒酶活性及其临床意义

Expression and clinical significance of relative telomere length and telomerase activity in CD34^＋ CD38^＋ acute myeloid leukemia

目的探讨相对端粒长度及端粒酶活性及在CD34＋ CD38＋ AML诊断、治疗及预后中的临床意义及其指导作用。方法随机选择2015年4月至7月，于中南大学湘雅医院血液科住院的20例初发CD34＋ CD38＋ AML患者为研究对象，纳入研究组（n＝20）。随机选择同期于病例收集医院门诊或血液科因怀疑为血液系统恶性疾病进行骨髓穿刺的10例受试者，纳入对照组（n＝10）。采用相对定量PCR检测研究组与对照组受试者相对端粒酶长度，采用端粒重复序列扩增法（TRAP）-银染法与绝对定量PCR分别定性与定量检测2组受试者的端粒酶活性；记录研究组患者经化疗后完全缓解（CR）率；采用流式细胞仪检测获得CR患者的MRD。对2组受试者的相对端粒长度及端粒酶活性，研究组患者相对端粒长度及端粒酶活性与其临床资料、疗效、MRD及预后之间的关系进行回顾性分析，并且进行统计学比较。本研究遵循的程序符合中南大学湘雅医院人体试验委员会所制定的伦理学标准，得到该委员会批准，征得受试对象或受试对象监护人的知情同意，并与之签署临床研究知情同意书。2组受试者平均年龄、性别构成比等一般临床资料比较，差异均无统计学意义（P〉0.05）。结果① TRAP-银染法定性检测端粒酶活性结果显示，研究组CD34＋ CD38＋ AML患者的端粒酶活性显著高于对照组。端粒相对定量PCR扩增与端粒酶绝对定量PCR扩增结果显示，研究组患者的相对端粒长度为1.72±0.42，显著短于对照组的4.68±1.89，并且差异有统计学意义（t＝－6.906，P〈0.001）。研究组患者的端粒酶活性为（405 242.01±357 412.39）copies/μL，显著高于对照组的（17 356.14±7 021.03） copies/μL，并且差异有统计学意义（t＝3.357，P＝0.002）。研究组患者相对端粒长度与端粒酶活性呈负相关关系（r＝－0.508，P〈0.05）。②研究组不同性别CD34＋ CD38＋ AML患者的相对端粒长度、端粒酶活性相比，差异均无统计学意义（P〉0.05）。而CD7＋患者、血红蛋白（Hb）水平≤50 g/L患者的相对端粒长度均显著短于CD7－者、Hb水平为（〉50-120）g/L者，并且差异均有统计学意义（t＝－2.217、－3.011，P＝0.040、0.008）；其端粒酶活性均显著高于CD7－者、Hb水平为（〉50-120）g/L者，并且差异亦均有统计学意义（t＝3.253、2.255，P＝0.010、0.037）。骨髓原始幼稚细胞比例≤50%患者、白细胞计数为≤50×109/L患者的相对端粒均显著长于骨髓原始幼稚细胞比例〉50%者、白细胞计数〉50×109/L者，并且差异均有统计学意义（t＝4.218、2.637，P＝0.001、0.018）；其端粒酶活性均显著低于骨髓原始幼稚细胞比例〉50%者、白细胞计数〉50×109/L者，并且差异亦均有统计学意义（t＝－3.052、－3.281，P＝0.009、0.008）。③研究组中，获得CR的CD34＋ CD38＋ AML患者相对端粒长度为1.95±0.19，显著长于未获得CR者的1.28±0.38，并且差异有统计学意义（t＝5.336，P〈0.001）；其端粒酶活性为（197 670.43±92 077.14）copies/μL，显著低于未获得CR者的（790 732.07±347 624.01）copies/μL，并且差异亦有统计学意义（t＝－8.379，P〈0.001）。④对研究组中13例获得CR的CD34＋ CD38＋ AML患者进行MRD检测的结果显示，7例相对端粒长度≤1.95的患者中，MDR呈阳性者为5例，呈阴性者为2例；6例相对端粒长度〉1.95的患者中，MRD呈阳性者为1例，呈阴性者为5例；7例端粒酶活性≤197 670.43 copies/μL的患者中，MRD呈阳性者为1例，呈阴性者为6例；6例端粒酶活性〉197 670.43 copies/μL的患者中，MDR呈阳性者为5例，呈阴性者为1例。2015年8月至2016年3月随访检测其中5例获得CR的患者结果显示，其中3例长期处于CR的患者相对端粒长度与端粒酶活性均与对照组均值相近。而2例获得CR后复发患者相对端粒长度再次缩短、端粒酶活性再次增高。结论相对端粒长度及端粒酶活性能够作为CD34＋ CD38＋ AML的诊断、预后及复发判定指标之一，并为治疗方案的确定提供一定的理论依据。

Objective To discuss the clinical and guiding signification of relative telomere length and telomerase activity in diagnosis, prognosis and treatment of CD34＋ CD38＋ acute myeloid leukemia （AML）. Methods From April to July 2015, a total of 20 cases of newly diagnosed CD34＋ CD38＋ AML patients who were hospitalized in Department of Hematology, Xiangya Hospital, Central South University were selected randomly as study subjects and enrolled in study group （n=20）. And 10 cases of participants who were conducted bone marrow puncture due to suspected blood system malignant diseases were selected as control group, randomly （n= 10）. Relative quantitative PCR was conducted to detect relative telomere length of participants＇ bone marrow specimens in two groups, and telomere repeat sequence amplification method （TRAP）-argentation and absolute quantitative PCR were conducted to detect telomerase activity; rate of complete remission （CR） after chemotherapy was collected in study group. MRD was obtained by flow cytometry. Relative telomere length, telomerase activity of two groups, relationship between relative telomere lengths, telomerase activity and clinical data, curative effect, MRD and prognosis of study group were analyzed retrospectively and compared statistically. The study protocol was approved by the Ethical Review Board of Investigation in Human at Xiangya Hospital, Central South University. Informed consent was obtained from all participants. There were no significant differences in average age and constituent ratio of gender between two groups （P〈0. 05）. Results ① As TRAP-argentation showed, the telomerase activity of CD34＋ CD38＋ AML patient in study group was higher than that of control group. Relative quantitative PCR of telomere and absolute quantitative PCR of telomerase showed, relative telomere length of study group was 1.72±0.42, and significantly shorter than that of control group which was 4.68-1.89 （t=-6. 906, P=0. 002 0）. Telomerase activity of study group was （405 242.01＋357 412.39） copies/μL, and significantly higher than that of control group which was （17 956.53 ± 6 952.03） eop ies/ptL （t= 3.357, P=0. 002）. There was a negative correlation between relative telomere length and telomerase activity in study group （r=-0. 508, P〈0.05）. ② In study group, there were no significant differences in relative telomere length and telomerase activity between patients of different gender （P〉0. 05）. Compared with CDT- patients and patients with hemoglobin （Hb） level≤50 g/L, relative telomere length of CD7＋ patients and patients with Hb level within （〉50-120） g/L were significantly shorter（t =- 2. 217, -3. 011; P = 0. 040, 0. 008）, and their telomerase activity were significantly higher （t= 3. 253, 2. 255; P= 0. 010, 0. 037）. Compared with patients with immature cells ratio〉50%, white blood cell count〉50×10^9/L, relative telomere length and telomerase activity of patients with immature cells ratio≤50%, white blood cell count≤50×10^9/L were significantly longer （t = 4. 218, 2. 637; P = 0. 001, 0. 018）, their telomerase activity were significantly lower （t= -3. 052, -3. 281; P = 0. 009, 0. 008）. ③ In study group, relative telomere length of CD34＋ CD38＋ AML patients who achieved CR was 1.95±0.19, and significantly longer than that of patients who didn＇t achieve CR, which was 1. 28±0. 38 （t= 5. 336, P〈0. 001）. Their telomerase activity was （197 670.43±92 077.14） copies/μL, and significantly lower than that of patients who didn＇t achieve CR, which was （790 732. 07-347 624.01） copies/μL （t=-8. 379, P〈0. 001）. @ According to the results of MRD detection of 13 cases of CD34＋ CD38＋ AML patients who achieved CR in study group, among the 7 cases with the relative telomere length≤1.95, MRD of 5 cases were positive, and 2 cases were negative. Among the 6 cases with relative telomere length〉1.95, MRD of 1 case was positive, and 5 cases were negative. Among 7 cases with telomerase activity≤197 670.43 copies/ptL, MRD of 1 case was positive, and 6 cases were negative. Among the 6 cases with telomerase activity〉197 670.43 copies/μL, MRD of 5 cases were positive, 1 case was negative. From August 2015 and March 2016, follow-up of 5 patients who achieved CR showed, relative telomere length and telomerase activity of the 3 cases with long-term in CR were similar to these of control group. Relative telomere length shortened,and telomerase activity elevated again in 2 cases with recurrence. Conclusions The relative telomere length and telomerase activity can be used as one of the diagnostic, prognostic and recurrent criteria of CD34＋ CD38＋ AML, and provide some theoretical basis for the determination of treatment options.