急性髓系白血病患者白血病干细胞WT1基因表达水平及其临床意义

目的 探讨急性髓系白血病(AML)患者WT1基因高表达是否发生在白血病干细胞(LSC)阶段及其意义.方法 采用流式细胞术分选AML患者CD34~+ CD38~- CD123~+细胞,实时荧光定量RT-PCR方法 检测其总WT1、+17AA、+KTS异构体的表达,计算出WT1(+/+)、wT1(+/-)、WT1(-/+)、WT1(-/-)四种异构体的比例,并与来源于正常人和AML患者骨髓的CD34~+ CD38~- CD123~-干细胞比较;同时分析AML患者LSC WT1表达水平与缓解率、生存期的关系.结果 AML患者骨髓CD34~+CD38~-CD123~+细胞WT1相对表达水平最高(0.034±0.034),AML患者骨髓CD34~+CD38~-CD123~-细胞群表达次之,来源于正常人的骨髓CD34~+ CD38~- CD123~- 细胞表达最低,三组之间比较差异有统计学意义(P<0.05);三群细胞中均以+17AA为主,组间比较差异无统计学意义.+KTS异构体比例在正常干细胞中最高,为0.57±0.04,在AML患者CD34~+ CD38~- CD123~- 细胞群中所占比例最低,为0.50±0.12,两组比较差异有统计学意义(P<0.05);三种细胞群中四种异构体的表达比例各组之间比较差异无统计学意义(P>0.05),均以WT1(+/+)为主,WT1(-/-)最少.CD34~+ CD38~- CD123~+ LSC中WT1表达水平与患者性别、年龄、FAB分型及骨髓中原始细胞比例无明显相关性,但AML患者WT1高表达组原始细胞群CD34~+ 细胞比例明显高于WT1低表达组(P<0.01).WT1高表达组患者CR率为21.1%,低表达组CR率为59.1%,差异有统计学意义(P<0.05).对41例患者跟踪随访118(3-290)d,WT1 mRNA高表达组中位生存时间77[95%可信区间(CI)45-108]d,较低表达组中位生存时间158(95% CI 100-215)d短,差异有统计学意义(P= 0.041).结论 AML患者WT1高表达发生于LSC阶段,其异构体比例与正常干细胞比较差异无统计学意义.干细胞阶段WT1高表达常导致患者缓解率低、生存期短.

Expression of WT1 gene in CD34~+ CD38~- CD123~+ AML stem cells and its significance analysis

objective To investigate whether WT1 gene overexpressed in leukemic stem cells (LSCs)and its significance.Methods Expression of WT1(+17AA)and WT1(+KTS)gene isoforms in CD34~+ CD38~- CD123~+ cells(LSCs)of 47 AML patients were determined by fluorescence quantitative RTPCR.The ratio of the four splicing isoforms WT1(+/+),WT1(+/-),WT1(-/+)and WT1(-/-)in LSCs were calculated and compared with that in normal CD34~+ CD38~- CD123~- cells(HSCs).The relationship in AML patients between LSCs WT1 expression and remission rate.survival time and relapse rate was analyzed.Results The expression of WT1 gene was highest(0.034±0.034)in LSCs,and higher in CD34~+ CD38~- CD123~- AML cells as compared with that in HSCs(P<0.05).The proportion of + 17AA isoform was predominant over-17AA in all the three cell subsets with no difiefence.The proportion of + KTS isoform was the highest in HSCs(0.57±0.04).while the lowest in CD34~+ CD38~- CD123~- AML cells(0.50±0.12)(P<0.05).No significant difference in the four isoforms expression ratio was obserred among the three groups.WT1 expression in LSCs was not correlated with flex,age,FAB subtype and blast cell ratio,while the ratio of CD34~+ cells in blast cell was significantly higher in the WT1 high expression group than in the low expression group(P<0.01).The CR rate was significantly lower in WT1 hish expression group (21.1%)than in the WT1 low expression group(59.1%)(P<0.05).The follow-up data were available in 41 patients with a median follow-up duration of 118(3-290)days.The median overall survival(OS) for WT1 hjgh and low expression group were 77[95%confident interval(CI)45-108],158(95%CI 100-215)days respectively(P=0.041).Condusion WT1 gene overexpressed in AML LSCs and the ratios of four WT1 isoforms have no difiefence in LSC compared with HSC.Patients with higher LSC WT1 expression have lower CR rate and shorter survival time.