Study On Expression And Clinical Characteristics Of Acute Leukemia With MLL Gene Rearrangement Detected By Multiplex Nested RT-PCR And The Set-up Of The Novel Detection Method Of Break-fusion Events Related With RARα AML1 And MLL Genes

Objective1 To detect the expression of mixed-lineage leukemia(MLL)gene rearrangement using multiplex nested RT-PCR method,and study the biological and clinical feature in acute leukemia (AL)with MLL rearrangement.2 To study the role of MLL gene rearrangement in monitoring minimal residual disease in AL patients with MLL gene rearrangement.3 To set up a novel RT-PCR detection method of break-fusion events related with RARα, AML1 and MLL genes.Methods1 To detect MLL gene rearrangements1.1 Multiplex nested RT-PCR was used to detect MLL gene rearrangements in 171 cases of acute myeloid leukemia(AML)and 76 cases of acute lymphoblastic leukemia(ALL),with E2A as internal positive control.1.2 Immunophenotype detected by flow cytometry(FCM),1.3 cytogenetics analyzing.2 To set up B/C method2.1 Several pairs of primers were designed for the major breakpoint(B)region and control (C)region on cDNA consequences of RARα,AML1 and MLL respectively.The fragment length and melting temperature was close to each other for primers on B region and C region.2.2 Seven group of PCR productions from a normal control respectively amplificated for 24, 26,28,30,32,34 or 36 cycles were analyzed by UVP system.Then curves were drawn according to OD of production in every group.2.3 The cells ofNB4 cell line and K562 cell line were mixed for 11 groups according to different ratios of NB4 to K562(0:100,10:90,20:80,30:70,40:60,50:50,60:40,70:30,80:20, 90:10,100:0).Then genomic RNA was extracted in each group and RT-PCR was performed to detect RARαgene.The OD of B/C was analyzed using SPSS software.The bone marrow cells of normal control and a patient who had 96.4%bone marrow blast cells were mixed for 6 groups according to different ratios of APL cells(15%,30%,45%,60%, 75%,90%).Then genomic RNA was extracted in each group and RT-PCR was performed to detect RARαgene.The OD of B/C was analyzed using SPSS software.1.1 ntial monitoring of MLL gene rearrangements show that persistent PCR positive test during clinical CR are predictive of anticipate the occurrence of hematological relapse.2 MLL rearrangement in ALL2.1 Eight cases of ALL with MLL gene rearrangements were found in 76 ALL patients (10.5%),including pro-B ALL in 5,pre-B/common-B ALL in 3 cases.All of the patients with MLL gene rearrangements expressed lymphoblastic markers,including CD19,CD22 and CD79. Two of them had myeloid marker.MLL gene rearrangements were related with young age and high WBC in pro-B ALL.2.2 There were 4 cases of MLL/AF4 fusion gene,1 case of MLL/ENL fusion gene, MLL-PTD,MLL/AF6 and MLL/AF10.2.3 Six of the 8 cases were treated with induction chemotherapy,and all got complete remission(CR),but relapse in short period.2.4 SThe B/C of patients with PML/RARα,AML1/ETO or MLL fusion(FG)gene was compared with normal control.Results3 MLL rearrangement in AML3.1 Thirteen cases of AML with MLL gene rearrangements were found in 171 AML patients (7.6%),including M2 in 2,M4 in 4 and M5 in 7 of the 13 cases,so MLL gene rearrangements were mainly in M4 and M5.All of the patients with MLL gene rearrangements had myeloid markers and three cases were co-expressed lymphoblastic marker.MLL gene rearrangements were related with high WBC in AML.3.2 There were 2 cases of MLL/AF6 fusion gene,4 cases of MLL/AF9 fusion gene,1 case of MLL/AF17 fusion gene,2 cases of MLL/AF10 fusion gene,1 cases of MLL/ELL fusion gene and 3 cases of MLL-PTD.3.3 Twelve of the 13 cases were treated with induction chemotherapy,and eight got complete remission(CR),but relapsed in short period and the median overall survival time was shorter than AML with t(8;22)or t(15;17).3.4 Sequeequential monitoring of MLL gene rearrangements show that persistent PCR positive test during clinical CR are predictive of anticipate the occurrence of hematological relapse.4 The setup of B/C method4.1 The best cycle count for amplification of RARαand AML1 were 30 cycles,and of MLL was 31 cycles.4.2 The B/C was significantly lower in mixed cells which NB4 cells proportion was under 30%than in 100%K562 cells(p=0.032),so the lowest leukecyte proportion is 30%of blast cells for this method in cell line.We got the same result in mixed cells with APL bone marrow blast cells and control bone marrow cells in deferent ratio.4.3 The B/C was significantly lower in PML-RARαpositive patients with more than 30% BM blast cells than in normal controls(p＜0.05).The B/C values was significantly lower in AML1-ETO and MLL FG patients with more than 30%BM blast cells than in normal controls(p＜0.05).Conclusion1 Multiplex RT-PCR method is a relatively accurate and sensitive method for detection of MLL gene rearrangements.2 MLL rearrangement is commonly genetic alternation detectable in M4 and M5,and MLL/AF4 is associated with pro-B ALL.3 Finding out and monitoring MLL gene rearrangement is important in guiding personal therapy regimen and predicting prognosis in AL.4 B/C method is an available and convenient method for detecting fusion gene with RARα,AML1 or MLL,and could use for Screenning test in AL patients.The method also can be used for detecting other fusion genes which have stable breakpoint region.