| 【objective】1. To evaluate the clinical and cytogenetic characteristics of 447 acute myeloid leukemia(AML) patients admitted in our center by different EVI1 expressions.2. To retrospectively assess the impact of different EVI1 expressions to the prognosis of 151 patients of AML in first remission underwent myeloablative allogeneic hematopoietic stem cell transplantation.3. To screen micro RNAs associated with EVI1 expression and explore potential mechanism of 11p15 rearrangement resulting in high EVI1 expression.【Methods】1. We established an absolute quantity method by preparing standard plasmid of EVI1 and its splices. A total of 447 AML patients in our center were analyzed for EVI1 expression, demographics data, blood cells count, morphology, cytogenetic, and mutations in AML. Their remission rate and leukemia free survival were also analyzed. Additionally, cytogenetics feature of high EVI1 expression was analyzed separately.2. We retrospectively conducted research of 151 AML patients in first remission who underwent myeloablative allogeneic hematopoietic stem cell transplantation in our center to evaluate the impact of different EVI1 expressions. Their characteristics of clinical feature, morphology, cytogenetic abnormalities and types of transplantation were compared. Univariate and multivariate models were constructed to investigate the impact of different EVI1 expression on leukemia free survival and overall survival..3. Micro RNA profile was screened to find special mi Rs closely associated with EVI1 expression. Mi R-181 a and mi R-128 a were screened and verified to be negatively related with EVI1 expression by RQ-PCR. Their target genes were predicted. Gene expression profiles and RQ-PCR were performed to explore EVI1 and its splices’ expression in ten 11p15 rearrangement samples. Preliminary study on mechanism between 11p15 rearrangement and high EVI1 expression was conducted by recombinant transfection in vitro.【Results】1. We successfully established quantitative assay to measure EVI1 and its splices by constructing standard plasmids. The working curve of RQ-PCR was examined. Normal monocytes extracted from volunteers bone marrow were tested to measure expression of EVI1 and its splices and indicate that normal expression of EVI1-1d was 99.812±94.823/10000 PBGD, MDS1EVI1 186.171 ± 188.678/10000 PBGD and c EVI1 1198.428±1266.043/10000PBGD(mean±standard deviation). The cutoff value of c EVI1 was ten-fold of above-mentioned value. We identified 80 AMLs with high EVI1 expression among 447 patients(17.9%). No significant differences in age, sex, hemoglobin level, white blood cell count and platelet count were found between patients EVI1 expression high and low. M5 and M6 subtypes were observed predominantly in EVI1 high group(p=0.0038 and 0.01 separately). A trend of M3 dominant in EVI1 low group was shown(p=0.08). High EVI1 expression was found mostly in cytogenetic abnormalities of 11q23/MLL rearrangement, followed by 3q26,-7/7q-, t(9;11)(p<0.0001, <0.0001, =0.0006, =0.0135 separately). Of note 5 patients with recurrent rearrangement of 11p15 were found high expression of EVI1(p<0.0001). Ph chromosome showed a trend of predominance of high EVI1 expression(p=0.093). Normal karyotype, inv(16), t(8;21) were mostly seen in high EVI1 group(p=0.001, 0.009, 0.002 separately) and a trend of t(15;17)/PML-RARα was noticed(p=0.088). Low risk and middle risk group of karyotype occurred more frequently in low EVI1 group(p<0.0001, =0.044 separately) while high risk group in high one(p<0.0001). NPM1 mutation was associated with EVI1 low group(p=0.0003). According to NCCN 2014 category criteria of AML risk, the high risk group associated more frequently with EVI1 overexpression(p<0.0001) and low risk one more frequently with EVI1 low expression(p<0.0001). Remission rate in patients younger than 60 years was significantly lower in high EVI1 expression group when compared with the low one(CR rate: 37.5% v.s. 69.4%, p<0.0001). Leukemia-free survival of patients with EVI1 overexpression was significantly inferior compared with EVI1 low AML patients(p=0.031). We separately analyzed the subset of 11q23/MLL rearrangement. M5 subtype was slightly predominant in high EVI1 group(p=0.231). Karyotypes of t(11;19)(q23;p13) and t(6;11)(q27;q23) showed a trend to more associate with high EVI1 AML(p=0.077). The CR rate was inferior in high EVI1 AML compared with low one for all 11q23/MLL rearrangement patients(p=0.061). Abnormality of-7/7q- without additional cytogenetic alteration was more frequently seen in EVI1 overexpression for all-7/7q- karyotypes(4/7). The CR rate showed no difference. Some infrequent breaking way were detected in 3q26 rearrangement and cytogenetic abnormalities of 3q27 or 3q29 could be latent 3q26 rearrangement when detecting with fluorescence in situ hybridization(FISH) technique. A total of twelve AMLs with 11p15 rearrangement was detected to over express EVI1 after a broadened search.2. A total of 151 AML patients in first remission underwent myeloablative allogeneic hematopoietic stem cell transplantation(allo-HSCT). EVI1 overexpression was found in 32 AMLs(21.2%). No significant differences in age, sex, white blood counts and blasts in bone marrow were observed at diagnosis between EVI1 high and low expression groups. Higher proportion of M5 subtype was found in high EVI1 expression group(p=0.0015).. 11q23/MLL rearrangement occurred more frequently in high EVI1 group(p<0.0001) on the contrary normal cytogenetic occurred more frequently in low group(p<0.0001). Middle and low risk category of cytogenetic were more likely to associated with EVI1 low expression(p<0.0001). Distribution of mutations showed no difference between the two groups. To account for transplantation features, no differences were observed in distribution of priming regimen, sibling donor, bone marrow(BM) or peripheral blood(PB) or cord blood(UC) resources in the two groups. There were more patients receiving mixed infusion of BM plus PB in EVI1 overexpression group(p=0.0039). More partial-matched allo-HSCT were more widely used in higher EVI1 expression group compared with fully matched one(p=0.0333). All patients were successfully grafted and full chimerism was achieved as well. Cumulative incidence of a GVHD(II-IV°) after 100 days was 21%±5%. Seven patients were diagnosed as severe a GVHD(III-IV°). Cumulative incidence of c GVHD was 30%±10%. 15 patients were diagnosed as extensive c GVHD in 2 years. Overall survival rate(OS) was 68.5%(59.9-76.5%). Event free survival was 67.4%(58.5-74.6%). A median follow-up was 26 months(2-60 months). OS in EVI1 high group at 24 months was 52.8%(32.5-69.5%) and significant lower than EVI1 low group [72.4%(63-80%), p=0.0122]. LFS showed a similar result [56.7%(34.9-73.7%), v.s. 76.1%(66.6-83.2%), p=0.0083]. Death without relapse was considered as competing factor. Cumulative incidence of relapse was higher in EVI1 overexpression group compared with lower expression [9.5%(20.9- 57.6%) v.s. 22.5%(15.2-30.7%), p=0.013]. In univariate analyses, low EVI1 expression and middle risk cytogenetic group predicted superior LFS(p<0.05) while young age, low EVI1 expression, wild type of FLT3-ITD and/or NPM1 predicted superior OS(p<0.05). In multivariate analysis showed low EVI1 expression was the only prognostic factor predicting for superior LFS(HR=0.44, 95%CI 0.23-0.84, p=0.014). Wild type of FLT3-ITD and low or middle risk karyotype predicted superior OS [(HR=0.31, 95%CI 0.15-0.63, p=0.001),(HR=0.11, 95%CI 0.01-0.86, p=0.03),(HR=0.43, 95%CI 0.24-0.78, p=0.005) separately]. NCCN 2014 AML risk category criteria combined cytogenetic with mutations to predict disease hazard. Using NCCN AML risk criteria we confirmed EVI1 status and NCCN risk category significantly affected LFS in univariate analysis. In multivariable analyses, NCCN AML risk category outweigh all other risk factors for OS(HR=0.37, 95%CI 0.21-0.64, p=0.0004). When stratified by cytogenetic group, EVI1 expression showed no significant influence for both LFS and OS.3. We screened several important micro RNAs associated with EVI1 expression using micro RNA microchip profile in 107 AML patients Mir-181a(Fold change 0.368, p=0.00018) mi R-128b(Fold change 0.465, p=0.002), mi R-181c(Fold change 0.470, p=0.001) and mi R-128a(Fold change 0.479, p=0.002) were significantly negatively associated with EVI1 expression. Mi R-151-3p, mir-500, mi R-143, mi R-199a-5p, mi R-363, mi R-148 a, mi R-151-5p, mi R-125a-5p, mi R-99 b were positively associated with EVI1 expression. Of above, mi R-181 a and mi R-128 a were verified to have significant difference between EVI1 high and low expression groups in 71 AML patients with RQ-PCR technique(p=0.05 and 0.0015)..We predicted potential target gene of mi R-128 was HOX family members(such as HOXA9) while target gene of mi R-181 was Bcl-2 by Targetscan software. Gene expression profile(GEP) of ten 11p15 rearrangement samples was analyzed to demonstrate EVI1 d, not MDS1/EVI1 was one of important splices of EVI1 driven by NUP98 rearrangement. GEP also indicated expression of HOXA3、HOXA4、HOXA5、HOXA7、HOXA9、HOXA10、HOXAB in NUP98 rearrangement were higher than other HOX family members. It was proved that c EVI1 and EVI1 d, not MDS1/EVI1 over expressed in NUP98 rearrangement patients after RQ-PCR verifying. We transfected plasmid containing NUP98/HOXA9 or NUP98/HOXD8 fusion gene into K562 or 293 T cell line and indicated relative expression of c EVI1 elevated in 293 T cells with NUP98/HOXA9 not others. CCK8 method indicated cell vitality decreased after the transfection.【Conclusions】1. High EVI1 expression occurred in 17.9% AML patients. More AMLs were associated with M5 subtype. Most common cytogenetic abnormalities associated with high EVI1 expression were 11q23/MLL rearrangement, 3q26 rearrangement, monosome 7 or 7q-, t(9;11) and 11p15 rearrangement. Remission rate was significantly lower in EVI1 overexpression group2. The prognosis of AML patients with high EVI1 expression was poorer in LFS, OS or CIR than low EVI1 expression in the first remission undergoing myeloablative allo-HSCT. In multivariate analysis, EVI1 expression status was the solely prognostic factor for LFS.3. Mi R-128 a and mi R-181 a were negatively associated with expression of EVI1. Their target gene was HOXA9 and Bcl2 separately by software prediction. EVI1 splices type of 11p15 rearrangement was different from others. The level of EVI1 expression elevated in 293 T cells after transfection with recombinant plasmid of NUP98/HOXA9. The cell vitality decreased after transfection. |
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