Fluorescence Quantitative PCR Detection Of ERG And MN1 Gene Expression In Acute Leukemias And Their Clinical Implication

BackgroudAcute leukemia(AL),including acute lymphoblastic leukemia(ALL) and acute myeloid leukemia(AML),is one of the top ten malignant tumors in China,also the highest incidence rates in teenagers malignant.Proliferation of leukemic stem cells without intermission and the encroachment of various organs,tissues,resulting in the normal hematopoietic function is inhibited,which led to clinical common infections, bleeding,anemia and other symptoms.AL is fatal and in a critical condition,progress rapidly,once it happened.In recent years because of factors such as environmental pollution,morbidity of AL has obviously upward trend.Although as the development of new chemotherapy drugs,the rational combination of applications,support treatment and Hematopoietic Stem cell Transplantation(HSCT),the treatment effect of acute leukemia have been significantly improved,but about 20%～30%AL patients remain Complete remission(CR) after routine chemotherapy,Known as the refractory.In addition,about 50%of CR patients will relapse.Refractory and Relapse of AL patients,meaning to be hard to induce remission again,predicts adverse outcome.Therefore,the refractory and relapse is the the most fundamental reason for the treatment failure for AL patients.The current criteria of diagnosis of refractory and relapse for AL are retrospective,can not be used to guide clinical treatment.If a set of early diagnosis method of refractory and relapse AL can be established,we can use it to predict refractory and relapse AL early and judge the patient's prognosis.More importantly, we can use it as a guide to change the conventional treatment modalities,using a more active individual treatment such as earlier HSCT,for those high-risk patients, and it may be useful for reducing the incidence of refractory and relapse of AL, improveing treatment effect and long-term survival of AL.Current study found that certain genetic abnormalities are related to the occurrence and development of leukemia,such as FLT3 internal tandem duplication mutation(FLT3/ITD) in AML,and the bcr/abl fusion gene expression in ALL.It is confirmed that 20%～30%AML patients with FLT3/ITD mutation and ALL patients with bcr/abl fusion gene expression are prone to refractory and relapsed,having poor prognosis,and these two genetic abnormalities have been a prognostic molecule markers.However,70%～80%AML and ALL patients can't be detected with these two genetic abnormalities.Therefor,it's urgent to found the other new marker for AL patients.Latest studies discover that the ERG and MN1 gene overexpress in AL patients, and they are probabley new prognostic markers.ETS-related gene(ERG),which is located at chromosome band 21q22,is frequently overexpressed in AML patients with complex karyotypes and cryptic amplification of chromosome 21.ERG can express in hematopoietic stem cells, primitive megakaryocyte cell line,Down's syndrome patients with primary leukemia cells.ETS transcription factors are known to be key players in lineage specific regulation during commitment and differentiation of lymphocytes.In particular,the ETS transcription factor ERG has been shown to modulate the maturation of lymphoid cells.In early T-cell development,ERG expression is induced at the time of T-lineage specification and shut off once T-cell commitment is complete.ERG shows specificity to early T-and B-cell lineages as well as myeloid cells.ERG plays a very important role in Proliferation,differentiation and apoptosis of leukemic cells.ERG overexpression in AL patients predicts an adverse clinical outcome.The meningioma(disrupted in balanced translocation) 1(MN1) gene,localized on human chromosome 22,was first cloned from a patient with meningioma with translocation t(4;22).MN1 is overexpressed in primitive hematopoietic stem cells and patients with ALL,and is the the down regulation of cell differentiation.MN1 is a unique oncogene in hematopoiesis that both promotes proliferation/self-renewal and blocks differentiation,and may become useful as a predictive marker in AML treatment.Althought ERG and MN1 expression have been related to the prognosis of AL patients,but little is known about the clinical significance of ERG and MN1 expression level in them.Considering the frequent expression of ERG and MN1 in leukemic and its functions as a regulator of differentiation and apoptosis and a modulator of the immune system,the role of the ERG and MN1 level in AL is not fully understood.In this study,we analyzed the expression level of the ERG and MN1 in 87 de novo AML and 80 ALL de novo ALL cases.We investigated correlations with cytomorphology,cytogenetics,flow cytometry and other clinical parameters.Objective1.To establish a method of detecting ERG and MN1 mRNA with quantitative real time PCR.2.To detect the level of ERG and MN1 mRNA of de nove AL patients and the discrepancy of that of FAB subtypes.3.To discuss the relationship of different karyotype,clinical character, expression of CD34,CD56,CD117 to the level of ERG and MN1 mRNA. 4.To anylyse the relation of the level of ERG and MN1 expression in AL patients at the same time.Methods:1.KASUMI-1 cells were cultivated and the RNA was abstracted.GAPDH was used as an endogenous control.After prmers were designed according to ERG,MN1 and GAPDH mRNA sequences,the target fragments were amplified,purified and then transferred to T vector.Plasmid with target fragment was constructed and used as positive quantitive standard templet.2.Cloned templet was diluted into 10⁶,10⁵,10⁴,10³,10²,10¹ copies/μl accordingly and amplified in the machine.After achieving the standard curves we verified their sensitivity and repeatability.3.Bone marrow samples were derived from 87 de novo AML patients and 80 de novo ALL patients,who were finally diagnosed by the means of cell morphology, hostochemical stain,flow cytometry and immunophenotype.16 non-malignant hemotologic disease patients borne marrow samples were set as control group.In comparision with standard curve,the amplification efficacy of three genes is close to each other.4.Total RNAs of bone marrow of AML,ALL patients and non-malignant hemotologic disease patients were abstracted and then reverse-transcripted into cDNAs by rea time quantitive PCR.The ERG and MN1 relative expression levels values were calculated using the mean ofâ–³C_T(â–³C_T=ERG or MN1-GAPDH) from the three replicates,and expressed as 2^-â–³CT.According to these data we analyzed the relationship of ERG and MN1 mRNA to FAB subtypes,karyotype,expression of CD34 CD56 CD117,clinical character,therapeutic effect and prognosis.5.Statistical anylysis:Mann-Whitney U test for distribution among 2 groups or the Kruskal-Wallis test and the Bonferroni test for distribution among more than 3 groups.Analysis of the distribution between 2 continuous variables was performed by using the Spearman rank correlation test.Analysis of frequencies was performed by using chi-square test.Survival probabilities were estimated by the Kaplan-Meier method,and differences in the survival distributions were evaluated by using the log-rank test.Single factor analysis and Multiplicity of survival were evaluated by using Cox Regression.For all anylyses,the Pvalues were 2-tailed,and a P value of less than 0.05 was considered statistically significant.Results:1.Total RNAsof KASUMI-1 extracted by Trizol were identified by formaldehyde agarose gel electrophoresis.Two bands of 28S and 18S were observed. End products of RT-PCR were also identified after agarose gel electrophoresis. Aband of 69bp(ERG),92bp(MN1),225bp(GAPDH) were observed under ultraviolet rays.The sequence of positive recombinated plasmid was totally consistent with according base sequence in Genebank.2.It showed perfect linear correlation between logarithm of different multiproportion dilution template for quantitation and cycle number(Ct)(r=0.9996, 0.997,0.997).The slopes of the PCR reaction are -3.245,-3.182 and -3.284.The applification effect of three genes were similar to each other,so we choose the 2^â–³CT to analyse the data.3.The mean of ERG transcripts in the 87 AML patients was 0.056(0～0.750),and the mean of MN1 transcripts in the 87 AML patients was 0.047(0～0.773),while those in the 16 control samples was 0.004(0～0.056)(ERG) and 0.004(0.001～0.014)(MN1).The expression of ERG and MN1 gene was significantly higher in AML cases than in healthy donors(Z=-4.379 and -5.098;P<0.001). The mean of ERG transcripts in the 80 ALL patients was 0.071(0～11.174),and the mean of MN1 transcripts in the 80 ALL patients was 0.002(0.000～0.043),while those in the 16 control samples was 0.004(0～0.056)(ERG) and 0.004(0.001～0.014) (MN1).The expression of ERG gene was significantly higher in AML cases than in healthy donors(Z=-5.043,P<0.0001)While the expression of MN1 gene was not significantly higer in AML cases than in healthy donors(Z=-1.435,P=0.151).4.At the same time,it revealed that ERG and MN1 gene expressions were equqlly distributed among the FAB subtypes(P=0.850;P=0.870,Kruskal-Wallis test). Amone all the groups,Spearman's rank correlation showed that leukocyte counts and LDH were significantly related to ERG expression (P=0.005,r=0.300;P=0.032,r=0.317),but not significant correlation was found between hemoglobin and platelet counts.Not significant correlation was found between WBC,HGB,PLT,LDH to MN1 expression in AML patients.In 66 cases whose karyotypes were identified,there were noticeable deviation amone karyotypes in the expression of ERG.The expression of cases with midlle and low risk group was lower that of cases with high risk group.And expression of MN1 gene was not significantly different in cases with karyotypes.In 51 cases with immunophenotype available,there was no significant difference of ERG and MN1 mRNA between patients positive and negative for CD34,CD56,CD117.Not significant correlation was found between WBC,HGB,PLT,LDH to ERG expression in 80 ALL patients(P=0.550,0.872,0.868,0.819).The expression of MN1 gene was not significantly higer in B-ALL cases than in T-ALL case (Z=-1.428,P=0.153)).There was no significant difference of ERG mRNA between patients positive and negative for bcr/abl and myloid antigen(Z=-0.438,P=0.662; Z=-0.721,P=0.471).5.Within 75 follow-up cases with AML,there was no significant difference of CR rates in high ERG expression and low ERG expression groups after chemical theropy(P=0.968),as well as MN1 expression(P=0.695).During the follow-up 365 days,the cumulative relapse rates of high ERG expression groups was significantly higher than that of low ERG expression groups(P=0.039),and survival curve gave the clues that high ERG expression cases have a significantly worse OS that low ERG expression cases(χ²=7.750,P=0.005).There was no significant difference of the cumulative relapse rates and OS between high MN1 expression groups and low MN1 expression(P=0.080).The results of Single fator analysis and Multiplicity of survival with AML patients reveal that the ERG expression may be a prognositic factor for CN-AML.The cumulative relapse rates of high ERG and/or MN1 expression groups was significantly higher than that of low ERG/MN1 expression groups(P=0.029),and survival curve gave the clues that high ERG and/or MN1 expression cases have a significantly worse OS that low ERG/MN1 expression cases(P=0.043) in those cases with normal karyotypes.6.Within 63 follow-up cases with ALL,there was no significant difference of CR rates,cumulative relapse rates in high ERG expression and low ERG expression groups after chemical theropy(P=0.968).The results of Single fator analysis and Multiplicity of survival with AML patients reveal that the ERG expression may be a prognositic factor for CN-AML.Survival curve gave the clues that high ERG expression cases have a significantly worse OS that low ERG expression cases in 63 ALL cases(P=0.033).Conclusions:1.A method of detection ERG and MN1 mRNA level by FQ-PCR was successfully established.2.The expression of ERG and MN1 gene was significantly higher in patents with AML than in control samples and ERG expression had not correlation with MN1 expression.The expression of ERG gene was significantly higher in patents with ALL than in control sample. 3.The expression of ERG gene had a positive correlation with WBC,LDH.It was significantly high in high risk groups but low in low and middle risk groups. Meanwhile,ERG mRNA level in AML patients had no correlation with expression of CD34,CD56,CD117 antigen.MN1 mRNA level in AML patients had no correlation with expression of CD34,CD56,CD117 antigen,FABs,karyotypes.Not significant correlation was found between WBC,HGB,PLT,LDH,karyotypes,immunity classification to ERG expression in 80 ALL patients.There was no significant difference of ERG mRNA between ALL patients positive and negative for bcr/abl and myloid antigen.4.Within 75 follow-up cases with AML,there was no significant difference of CR rates in high ERG expression and low ERG expression groups after chemical theropy,as well as MN1 expression.During the follow-up 365 days,the cumulative relapse rates of high ERG expression groups was significantly higher than that of low ERG expression groups,and survival curve gave the clues that high ERG expression cases have a significantly worse OS that low ERG expression cases.There was no significant difference of the CR rates,cumulative relapse rates and OS between high MN1 expression groups and low MN1 expression.Within 63 follow-up cases with ALL,there was no significant difference of CR rates,cumulative relapse rates in high ERG expression and low ERG expression groups after chemical theropy.Survival curve gave the clues that high ERG expression cases have a significantly worse OS that low ERG expression cases in ALL.5.Overexpression of ERG gene is a independent prognositic factor by using Cox regression model.6.Combined detection of ERG and MNI genes expression level is not better than detection of ERG gene expression level only for AML.