半定量逆转录-PCR检测骨髓eIF4E相对表达水平在急性髓细胞白血病疗效监测中的意义

Significance of eIF4E expression detected by semi-quantitative reverse transcription-PCR in monitoring therapeutic effect of acute myeloid leukemia

目的 通过比较初治、完全缓解（CR）、未缓解及复发的急性髓细胞白血病（AML）患者骨髓eIF4E相对表达水平,探讨其在AML疗效监测中的意义.方法 选择2012年8月至2014年5月,四川省人民医院血液科收治的152例初治AML患者为研究对象,纳入研究组.对研究组AML患者按照法国、美国和英国协作组（FAB）进行分型,M1型为18例,M2型为60例,M3型为28例,M4型为20例,M5型为24例,M6型为2例,分别纳入相应亚组.选择本科同期收治的20例非血液肿瘤疾病患者纳入对照组,其中,免疫性血小板减少症患者为14例,血液系统良性疾病导致的缺铁性贫血为6例.本研究遵循的程序符合四川省人民医院人体试验委员会制定的伦理学标准,得到该委员会批准,并征得受试对象的知情同意.收集研究组AML患者治疗前、后与复发前、后及对照组患者骨髓标本,采用半定量逆转录-PCR法检测骨髓eIF4E表达相对水平.以β-actin为内标物,采用PDQuest软件分析eIF4E/β-actin的比值,计算骨髓eIF4E相对表达水平.对研究组AML患者骨髓标本均采用半定量逆转录-PCR进行融合基因及突变基因检测.采用R语言软件对研究组与对照组、研究组各亚型之间化疗缓解前、后和复发前、后骨髓eIF4E相对表达水平及骨髓eIF4E与FLT3/ITD基因表达的相关性进行统计学分析.结果 研究组患者中,因各种原因未接受治疗者为21例.接受诱导化疗的131例患者中,获得CR的患者为95例（72.5％）.对获得CR的患者平均随访18个月后,34例（35.8％）复发.研究组与对照组患者骨髓标本的eIF4E相对表达水平分别为0.81±0.27与0.04±0.01,二者比较,差异有统计学意义（W=3 040,P＜0.05）.研究组各亚型AML患者中,M4和M5型患者的骨髓eIF4E相对表达水平较其他亚型高,但各亚型患者的骨髓eIF4E相对表达水平比较,差异无统计学意义（W=13,P＞0.05）.研究组经诱导治疗后获得CR的95例患者中,化疗缓解前骨髓eIF4E相对表达水平为0.87±0.25,化疗缓解后骨髓eIF4E相对表达水平降低至0.28±0.17.化疗缓解前、后骨髓eIF4E相对表达水平比较,差异仍有统计学意义（W=4 408,P＜0.05）.但缓解后骨髓eIF4E相对表达水平仍高于对照组,且二者比较,差异有统计学意义（W=1 898.5,P＜0.05）.获得CR各亚型AML患者的骨髓eIF4E相对表达水平比较,差异均无统计学意义（W=34,P＞0.05）.研究组34例复发AML患者中,复发前、后骨髓eIF4E相对表达水平分别为0.30±0.10和0.86±0.27,二者比较,差异有统计学意义（W=1 249,P＜0.05）.eIF4E与FLT3/ITD基因突变无相关性（x2=0.017,P＞0.05）.结论 eIF4E在AML的发病中起着重要作用.骨髓eIF4E相对表达水平在诱导治疗获得CR后明显下降,复发时升高.半定量逆转录-PCR检测骨髓eIF4E相对表达水平,可用于AML的疗效监测.

Objectives To explore the significance of the relative level of eIF4E in monitoring therapeutic effect of acute myeloid leukemia （AML）,by comparing the relative level of eIF4E in AML patients who received initial treatment,complete remission （CR）,non-remission and relapse.Methods From August 2012 to May 2014,a total of 152 AML patients who received initial treatment in Department of Hematology in Sichuan Provincial People＇s Hospital were enrolled in this study,as study group.According to France-America-Britain （FAB） assistance typing method,these patients included 18 cases of M1 subtype,60 cases of M2 subtype,28 cases of M3 subtype,20 cases of M4 subtype,24 cases of M5 subtype and 2 cases of M6 subtype.In the same period,20 cases with non-tumorous blood diseases patients were detected as control group,which included 14 cases with immune thrombocytopenia,6 cases with iron deficiency anemia.The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Sichuan Provincial People＆#39;s Hospital.Informed consent was obtained from all participants.Bone marrow samples were collected from study and control group patients,and the eIF4E expression was detected by semi-quantitative reverse transcription （RT）-PCR method,the mutation of FLT3/ITD was also detected by RT-PCR method.Results Among 152 newly diagnosed AML patients,only 131 cases accepted the chemotherapy.95 cases had acquired CR and the CR rate was 72.5%.In patients with CR,34 cases relapsed after an average follow-up of 18 months,and the relapsed rate was 35.8%.The eIF4E relative level in 152 cases of untreated AML was 0.81±0.27,which was significantly higher than that of control group 0.04±0.01 （W=3 040,P〈0.05）.Besides,there was no statistical significance among different subtypes of AML,although the eIF4E level in M4,M5 subtypes was higher than that of other subtypes （W=13,P〉0.05）.The eIF4E level decreased significantly in patients with CR 0.28±0.17 compared with untreated patients 0.87±0.25 （W=4 408,P〈0.05）,which was still higher than that of control group （W=1 898.5,P〈0.05）.The eIF4E levels increased in relapsed AML patients,from 0.30±0.10 to 0.86±0.27,which had a statistical significance （W=1 249,P〈0.05）.There was no correlation between eIF4E expression and FLT3/ITD gene mutation （x2 =0.017,P 〉 0.05）.Conclusions eIF4E plays an important role in AML,the relative level of eIF4E decreased obviously in CR patients and increased in relapsed patients.Detection of eIF4E expression with semi-quantitative RTPCR could be used for monitoring therapeutic effect in AML patients.