Expression And Clinical Significance Of AF1q Gene,ILP-2 And Smac In Leukemia

Objectives: The AF1q gene, located on chromosome 1q21, is a main fusion partner of MLL gene in AML. AF1q is highly expressed on CD34+ stem cells, and this expression declines by differentiation and is nearly undetectable in peripheral blood. It has been shown that AF1q gene expression level intimatly correlates with therapeutic efficacy and clinical outcome.ILP-2 is a new potent member of inhibitor of apoptosis proteins(IAP)family. It is a weak inhibitor of caspase 9, and exerts their antiapoptotic effects through the neutralization of Smac. It has been shown that ILP-2 is highly expressed in MDS, but studies on its expression in leukemia are very limited. Smac (second mitochodria-derived activator of caspase, Smac) is a proapoptosis gene releasing from mitochondrion, which induces cell apoptosis by relieving the inhibition of IAP and release activated caspase.This study intended to investigate the expression and clinical significance of AF1q,ILP-2 and Smac in leukemia.Methods 1 Patients:In the study there were 100 adult acute leukemia patients (including 64 de novo acute leukemia patients, 6 relapsed patients and 30 complete remission patients), 16 chronic myeloid leukemia (CML) patients (9 in chronic phase, 5 in accelerated phase and 2 in blastic phase), and 20 samples of normal controls (NC). There were 48 acute myelogenous leukemia (AML) patients and 16 acute lymphocytic leukemia (ALL) patients in the 64 de novo acute leukemia patients (11M2, 15M3, 10M4, 9M5, 3M6, 12L2) (according to FAB classification).2 The expression of AF1q gene,ILP-2 and Smac mRNA were measured by semi-quantity reverse transcription polymers chain reaction (RT-PCR).Results1 Expression of AF1q mRNA1.1 Expression of AF1q mRNA in acute leukemia patients44 of 64 initial treatment acute leukemia (AL) patients were detected the expression of AF1q mRNA. The positive rate of AF1q mRNA in initial treatment acute leukemia (AL) patients was 68.8%. The positive rate of AF1q mRNA in normal controls (NC) patients was 15.0%. The average expression level of AF1q mRNA in initial treatment AL patients was higher than that of normal controls (0.2188 versus 0.0208) (P<0.05). 35 of 48 acute myelogenous leukemia (AML) patients were detected the expression of AF1q mRNA, the positive rate was 72.9%,and the average expression level of AF1q mRNA in acute myelogenous leukemia (AML) was 0.2266. 9 of 16 acute lymphocytic leukemia (ALL) patients were detected the expression of AF1q mRNA, the positive rate was 56.3%,the average expression level of AF1q mRNA in ALL was 0.1953. The expression level of AF1q mRNA in AML was higher than that of ALL without statistical significance (P>0.05). Both of them were higher than that of normal controls (P<0.05). 5 of 6 relapsed patients were detected the expression of AF1q mRNA, the AF1q positive rate was 83.3%, and the average expression level in relapsed patients (0.3517) was significantly higher than that of normal controls (P<0.05). It was higher than initial treatment AL patients with no statistical significance (P>0.05). 11 of 30 patients in remission were detected the expression of AF1q mRNA, the positive rate (36.7%) and average expression level (0.0763) of patients in remission were lower than AL group with statistical significance (P<0.05). And the AF1q level of patients in remission was higher than that of NC group with no statistical significance (P>0.05).In FAB classification, the difference of expression level of AF1q mRNA in M2, M3, M4, M5, M6 and L2 had no statistical significance(P>0.05).1.2 Expression of AF1q mRNA in chronic myeloid leukemia patientsThe positive rate (66.7%) and average expression level (0.2031) of AF1q mRNA in CML-CP patients were higher than that in normal controls (NC) with statistical significance (P<0.05). The expression of AF1q in CML-AP/BP (positivity 71.4%, mean level 0.2598) was higher than that of NC (P<0.05). The expression level of AF1q mRNA in CML-AP/BP was higher than that in CML-CP,but the difference had no statistical significance (P>0.05).1.3 Expression of AF1q mRNA and CR rate in initial treatment acute leukemia patientsThe therapeutic efficacy in 45 of 64 newly diagnosed acute leukemia patients was evaluated. 13 of 24 AF1q+ AL patients achieved complete hematological response (CR) after induction therapy with a CR rate of 54.17%. 19 of 21 AF1q - AL patients achieved CR with a CR rate of 90.48%. The CR rate in AF1q- AL was significantly higher as compared with AF1q+AL (χ~2=7.188, P<0.01).2 Expression of ILP-2 mRNA2.1 Expression of ILP-2 mRNA in acute leukemia patients39 of 64 initial treatment acute leukemia patients were detected the expression of ILP-2 mRNA, the positive rate of ILP-2 mRNA was 60.9%. The positive rate of ILP-2 mRNA in normal controls (NC) was 20.0%. The average expression level of ILP-2 mRNA in initial treatment AL patients was significantly higher than that of normal controls (NC) (0.2842 versus 0.0112) (P<0.05). 27 of 48 acute myelogenous leukemia (AML) patients were detected the expression of ILP-2 mRNA, the positive rate and average expression level of ILP-2 mRNA in AML were (56.3% and 0.2577) respectively. 12 of 10 acute lymphocytic leukemia (ALL) patients were detected the expression of ILP-2 mRNA, the positive rate and average expression level of ILP-2 mRNA in ALL were (75.0% and 0.3638) respectively, it was higher than that of AML without statistical significance (P>0.05). Both of them were higher than that of normal controls (P<0.05). 5 of 6 relapsed patients were detected the expression of ILP-2 mRNA, the ILP-2 positive rate (83.3%) and average expression level (0.4612) in relapsed patients were significantly higher than that of normal controls (NC) (P<0.05). It was higher than initial treatment AL patients,but the difference had no statistical significance (P>0.05). 10 of 30 patients in remission were detected the expression of ILP-2 mRNA, the positive rate (30.0%) and average expression level (0.0727) of patients in remission were lower than AL group with statistical significance (P<0.05). And the ILP-2 level of patients in remission was higher than that of normal controls (NC) group, but with no statistical significance (P>0.05).In FAB classification, the difference of expression level of AF1q mRNA in M2, M3, M4, M5, M6 and L2 had no statistical significance(P>0.05).2.2 Expression of ILP-2 mRNA in chronic myeloid leukemia patientsThe positive rate (55.6%) and average expression level (0.2298) of ILP-2 in CML-CP were higher than that of NC group with statistical significance (P<0.05). The positive rate (55.6%) and average expression level (0.4292) of ILP-2 in CML-AP/BP were higher than that in CML-CP. The difference between CML-CP and CML-AP/BP had no statistical significance (P>0.05). But the expression level of ILP-2 in CML-AP/BP was significantly higher than that of NC group (P<0.05).2.3 Expression of ILP-2 mRNA and CR rate in initial treatment acute leukemia patients8 of 19 ILP-2+ AL patients achieved complete hematological response after induction therapy with a CR rate of 42.11%. 24 of 26 ILP-2- AL patients achieved CR with a CR rate of 92.31%. The CR rate of ILP-2- AL group was significantly higher than that of ILP-2+ group, (χ~2=13.468,P<0.01).3 Expression of Smac mRNA3.1 Expression of Smac mRNA in acute leukemia patients46 of 64 initial treatment acute leukemia (AL) patients were detected the exprssion of Smac mRNA. The positive rate of Smac mRNA in AL patients was 71.9%. The positive rate of Smac mRNA in normal controls (NC) patients was 15.0%. The average expression level of Smac mRNA in initial treatment AL patients was significantly higher than that of normal controls (0.4852 versus 0.0025) (P<0.05). The positive rate (72.9%) and average expression level (0.4969) of Smac mRNA in acute myelogenous leukemia (AML) were higher than that of acute lymphocytic leukemia (ALL) (68.8% and 0.4621) respectively without statistical significance (P>0.05). Both of them were significantly higher than that of normal controls (P<0.05). The Smac positive rate (83.3%) and level (0.5929) in relapsed patients were higher than that of normal controls with statistical significance (P<0.05). It was higher than AL patients with no statistical significance (P>0.05). The positive rate (30.0%) and level (0.2177) of patients in remission were lower than AL group with statistical significance (P<0.05). The level of Smac in remission patients was still higher than that of NC group with statistical significance (P<0.05).In FAB classification, the difference of expression level of Smac mRNA in M2, M3, M4, M5, M6 and L2 had no statistical significance (P>0.05).3.2 Expression of Smac mRNA in chronic myeloid leukemia patientsThe positive rate (44.4%) and average expression level (0.4031) of Smac in CML-CP were higher than that of NC group with statistical significance (P<0.05). The positive rate (57.1%) and average expression level (0.5981) of Smac in CML-AP/BP were higher than that in CML-CP .The difference between CML-CP and CML-AP/BP had no statistical significance (P>0.05). But the expression level of Smac in CML-AP/BP was significantly higher than that of NC group (P<0.05).3.3 Expression of Smac mRNA and CR rate in initial treatment acute leukemia patients92.00% of Smac- AL patients (23/25) achieved CR, only 45.00% of Smac+ AL patients (9/20) achieved CR. The CR rate of Smac- AL group is significantly higher than that of Smac+group, (χ2=11.557,P<0.01).4 The relationship between expression of ILP-2 and Smac in acute leukemiaIn initial treatment acute leukemia (AL) patients, the level of ILP-2 was positively correlated with Smac (r=0.2605, P<0.01). Only 5 of 13 ILP-2+Smac+ AL patients achieved complete hematological response with the CR rate 38.46%, all of the 15 ILP-2-Smac- patients achieved complete hematological response group, the CR rate was 100%. The CR rate of ILP-2-Smac- was significantly higher than that of ILP-2+Smac+ (χ~2=13.062,P<0.01). In 6 relapsed cases, only 1 achieved CR. The remaining 5 cases with positive ILP-2 and Smac failed to response.Conclusions1 The expression level of AF1q mRNA in initial treatment acute leukemia patients was significantly higher than that of normal controls and patients in remission. The level of relapsed patients was higher than that of normal controls and patients in remission. The level of relapsed patients was higher than initial treatment group with no significance. The CR rate of AF1q- group was higher than that of AF1q+ cases. AF1q may be involved in the pathogenesis and progression in AL. Its high expression predicts poor prognosis and more possibility to relapse.2 The expression level of ILP-2 mRNA in initial treatment acute leukemia and relapsed patients was significantly higher than that of normal controls and patients in remission. The CR rate of ILP-2- group was higher than that of ILP-2+ cases. ILP-2 may be involved in the pathogenesis and progression in AL. Its high expression level depresses the sensitivity to therapy and leads to poor CR rate.3 The level of Smac mRNA in initial treatment acute leukemia patients was also significantly higher than that of normal controls. The CR rate of Smac - group was higher than that of Smac+ cases. The high expression of Smac mRNA was associated with high ILP-2, suggesting that Smac performs its proapoptotic acting through inversion of the antiapoptotic effect of ILP-2.4 There was a positive correlation beween ILP-2 and Smac. The high expression of Smac mRNA was associated with high ILP-2, suggesting that ILP-2 exert their effects through the neutralization of Smac, Smac performs its proapoptotic act through inversion of the antiapoptotic effect of ILP-2. There was intimate connection between them.5 The level of AF1q,ILP-2 and Smac mRNA in chronic myeloid leukemia chronic phase,accelerated phase and acute transformation phase was higher than that of normal controls (P<0.05).