Expression And Significance Of ERG And BAALC In Acute Myeloid Leukemia Patients With Normal Cytogenetics

ObjectiveLeukemia is a group of heterozygous diseases of hematopoietic stem/progenitor cells that involves dynamic change in the genome, characterized by clonal expansion of hematopoietic cells with uncontrolled proliferation, decreased apoptosis and blocked differentiation. Approximately 45% of patients with acute myeloid leukemia (AML) have a normal karyotype without chromosomal aberrations on standard cytogenetic analysis, and this subgroup is heterogeneous with regard to molecular genetic alterations and therapeutic outcomes. Submicroscopic genetic lesions not only are relevant for the pathogenesis of the disease but also influence response to therapy and survival. The most frequent molecular abnormalities in cytogenetically normal AML (CN-AML) are mutations in the nucleophosmin 1 (NPM1) gene and internal tandem duplications (ITDs) of the fms-like tyrosine kinase 3 gene (FLT3). Other molecular aberrations found in smaller proportions of patients with CN-AML include mutations in the CEBPA gene, which convey a favorable prognosis, and partial tandem duplications (PTDs)of the MLL gene.More recently, quantitative differences in the expression levels of several genes (ERG,BAALC),measured by quantitative real-time polymerase chain reaction (qPCR), have been shown to carry prognostic information in patients with CN-AML. However, information on the interactions and relative importance of these risk factors is sparse. In this study, we used real-time fluorescence quantitative PCR to detect the transcript levels of ERG and BAALC in acute myeloid leukemia patients with normal cytogenetics and investigate correlations between them. This study will provide new theoretical basis in improving refined classification and stratified therapy of CN-AML patients. Methods1.Patients selectionCollect EDTA anticoagulated bone marrow samples 3-5ml from clinic and hospitalized newly diagnosed acute myeloid leukemia patients in Blood Disease Therapeutics Center of China Medical University affiliated Shengjing Hospital during January 2008 and January 2009, among which there were 30 males and 32 females, ages between 20 and 77, the median age was 54.5. All the cases were diagnosed as AML(FAB) through morphology, cytochemistry stain, immunology, cytogenetics and molecular biology(MICM). Standard cytogenetic analyzed at least 20 clones, all the karyotypes were normal. There was 1 case of AML-M0,3 cases AML-M1,19 cases of AML-M2,11 cases of AML-M4,24 cases of AML-M5,4 cases of AML-M6,62 cases totally as experimental group. Collect EDTA anticoagulated bone marrow samples 3-5ml from 20 cases of Benign blood disease patients as control group.The experimental group were received intensive induction and consolidation chemotherapy.2.Experimental methodMononuclearcell(MNC) was separated from anticoagulated bone marrow samples by Ficoll-Hypaque density gradient centrifugattion; extraction of total RNA; reverse transcription reaction system was disposed as 20μl to synthesize cDNA;primer was designed and synthesized by Takara company, real time quantitative RT-PCR SYBR Green I method was apllied to detect expression level of ERG and BAALC, the concrete procedure follows reagent instructions, GAPDH was detected as inner reference.3.Result determination10 fold attenuate the cDNA of standard samples in 5-6 concentration gradient, take 2μl as template to run real time PCR reaction respectively. Standard curve was drew by each Ct value of amplification curves. Sample's copy number was obtained according to standard curve. GAPDH was detected as inner reference, while ERG and BAALC were detected as objective genes. Relative quantitative comparisons among different samples were taken according to the copy number ratio of objective gene and inner reference gene.4.Statistical methods All the data were analyzed with SPSS for windows 13.0 software. Relative expressive quantity of objective gene was taken as reference values, we defined patient subgroups with high and low expression of each gene. High ERG expression was defined as greater than the 75th percentile, for BAALC, this dichotomization was performed at the median of expression values. The nonparametric Mann-Whitney U test was applied for continuous variables, baseline clinical features across groups were compared using theχ2 test. Survival curves were generated by the Kaplan-Meier method, and P value of less than 0.05 was considered as statistically significant.Results1.Reliability and accuration of method(1) purity and quality analysis of RNADetecting extracted total RNA by ultraviolet spectrophotometer,the ratios were between 1.8 and 2.0. further identification was done by agarose gel electrophoresis, bands were clearly, which shows no obviously degradation.(2) specificity of amplification and accuration of quantitationStandard samples undertook PCR reactions after gradient dilution,which was aimed to make standard curves. All the correlation coefficients were more than 0.998,slopes were between -3.3 and -3.5 of standard curves, indicating quantitative accuration and good amplified effects. Dissociation curves show the identical Tm value for all samples,and the sharp sole peak ensured the specific amplification of target sequence without primer dimer.2.Expression of ERG and BAALC gene in cytogenetically normal acute myeloid leukemiaERG and BAALC mRNA expression levels in 62 cases of CN-AML mononuclear cells were statistically higher than those in 20 cases of benign blood disease patients (P<0.05).3.Correlation of ERG and BAALC expression levels with clinical characteristics(1) High ERG expressers had and bone marrow blast counts(P<0.05). Patients with high levels of ERG frequently had AML with immature M0/M1 cytomorphology.(2) High expression of BAALC was strongly correlated with higher white blood count (WBC) and CD34 positivity(P<0.05), it also had AML with immature M0/M1 cytomorphology.4.Correlation of ERG and BAALC expression levels with of CN-AMLThe patient subgroups with high expression of ERG and BAALC had lower CR rates (40% vs 74%;P<0.05,46% vs 79%;P<0.05) and shorter 1-year OS rates(33% vs 69%;P<0.05,36% vs 72%;P<0.05).5.Interactions of ERG and BAALC expression levels and correlation with prognosisOnly for low BAALC levels, high ERG expressers had a shorter survival than low ERG expressers(P<0.05), but there were no significant differences between high and low ERG expressers when BAALC were high.Low ERG and low BAALC patients had a superior 1-year OS rate compared with patients with either high ERG and/or high BAALC expression(P<0.05).Conclusions1.The expression levels of ERG and BAALC in CN-AML were statistically higher than those in benign blood disease patients.2.High ERG and BAALC expression were associated with immature FAB cytomorphology and CD34 positivity.3. High ERG and BAALC expressers had lower CR rates and shorter 1-year OS rates, they were both predictors for unfavourable prognosis.4.Low ERG and low BAALC expression identified a highly favorable outcome, this study demonstrates that combining ERG and BAALC expression results in refined risk classification and stratified therapy for CN-AML patients.