| Background and ObjectivesAcute myeloid leukemia(AML) is high heterogeneous on the clinical and cytogenetic level.The molecular biology and cytogenety are the most important prognostic factors.About 50%～60%of patients with genetic chromosoma abnormalities like t(15;17),t(8;21)and inv(16)/t(16;16) according to FAB classcification,which are considered as markers and reliable for minimal residual disease(MRD) monitoring.But other 40～50%of AML patients in different clinical trials,who showed normal karyotype at conventional cytogenetics,especially falling in the intermediate group,have different clinical characters.NPM1,also called B23,numatrin or NO38,is a protein that shuttles between the nucleus,nucleoplasm and cytoplasm.NPM gene mutations and aberrant cytoplasmic NPM localization have been recently described in acute myelogenous leukemia.NPM1 plays an important role in regulation p53 activation and cell cycle arrest,throμgh binding to ARF.This present study aim to establish a method of detecting NPMmRNA with real time quantitative PCR,detect the level of NPM1-mutA of incipient patients of AML and the discrepancy of that of FAB subtypes,to investigate correlations with cytomorphology,white blood counting and the expression of CD 34 to the level of NPM1-mutA,as well as to discuss the application of NPM in the prognosis of AML and preliminaryevaluate it in the detection of MRD.Methods1.The expression level of NPM1-mutAmRNA by real time quantitative polymerase chain reaction(RQ-PCR) assay:The Taqman probe was chosen for real-time PCR detection of NPM1-mutA,The house-keeping gene ABL served as a control.Standard curves were constructed by performing modified real-time PCR on the standard template after 10-fold serial dilutions of cells cDNA.After achieving the standard curves for cDNA quality.The relative expression level of NPM1-mutAin each sample was calculated by comparing the copy number of NPM1-mutA with ABL and was subjected to analysis.The sensitivity,stability and repetitiveness of this method was determined.2.Bone marrow samples were derived from 142 de novo AML patients,who were finally diagnosed by the French-American-British(FAB) Cooperative Group morphologic criteria,Karyotype analysis using R-banding method.and immunophenotype detected by flow cytometry.Patients of MDS and secondary AML were excluded.Ten normal bone marrow samples from volunteers were set as control group.3.Total RNAs of bone marrow of AML patients and healthy donors was extracted with Trizol reagentaccording to the manufacturer's instructions,.and then cDNA was synthesized from each RNA4.To discuss the application of NPM1-mutA in the prognosis of AML and preliminary accordingly to amplified in the machine,as well as to investigate correlations with cytomorphology,white blood counting and the expression of CD 34 to the level of NPM1-mutA.5.Homogeneity test for variance and test of normality were carried out as a routine.Differences in median variables were analyzed with the T test for univariate among 2 groups and single factor analysis of variance for distribution among more than 3 groups.Nonparametric tests were performed for heterogeneity of variance.Chi-square test was performed for numeration data.The statistical analyses were performed with SPSS 12.0.It was considered statistically significant when P value was less than 0.05.Results1.It showed perfect linear correlation between logarithm of different multiproportion dilution template for quantitation and cycle number(Ct)(0.9963).The sensitivity of real-time quantitative PCR for detecting NPM1-mutA was about 10-5. Repeatability and reproducibility of the method were satisfying as intra- and inter-assay coefficient variation(CV) were1.8%,2.1%.2.NPM1 mutations were present in 21.8%f the overall 142 population,amounting 50.0%(1/2) of M0,25.0%(1/4) of M1,21.7%(10/46) of M2,0%(0/44) of M3, 41.7%(10/24) of M4,47.1%(8/17) of M5 and 20.0%(1/5) of M6.3.NPM1 gene mutations were more prevalent in patients with a normal karyotype 45.3%(9/64),when compared with patients with karyotypic abnormalities 2.6% (P＜0.05).4.NPM1 mutations were significantly associated with high white cell count (P＜0.05),and FABM4/M5,and associated with low expression of CD34(P＜0.05).Conclusions1.Establishing the Real time Fluorescence quantitative PCR assays for detecting the NPM1-mutA mRNA Successfully.2.NPM1 mutations also represent a common genetic abnormality in adult AMLs, especially in the presence of a normal karyotype.3.The occurrence of NPM1 mutations commonly genetic alternation detectable in M4 and M5,and associated high white cell as well as low expression of CD34.4.The expression level of NPM1-mutAmRNA was suit for detecting minimal residual leukemia of AML.5.Real time Fluorescence quantitative PCR assays are an excellent tool for detecting the presence,persistence and reappearance of leukemic cells in adult AML with normal karyotype.
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