Detection Of Multiple Genes In Adult Patients With De Novo Acute Myeloid Leukemia By Fluorescence Quantitative PCR And Its Clinical Significance

BackgroundAcute myeloid leukemia (AML) is a kind of lethal disease with the characteristics of malignancy clone proliferation of myeloid blasts in hematopoietic system, also a group of diseases with high heterogeneity. AML is the most common type of acute leukemia (AL) in adults, and its incidence rate is on the increase in recent years. Although the development of Hematopoietic Stem Cell Transplantation (HSCT) as well as traditional chemotherapy, it’s really difficult to improve the curative effect and the outcome of AML effectively, the treatment of AML is in the bottleneck stage. Previous study showed that the abnormal expression of genes and transcription factors in some signal pathways is associated with the carcinogenesis, progression and prognosis of AML, the mechanisms may be relate to promoting proliferation, inhibiting apoptosis and blocking differentiation of leukemia cells. With the development of cytogenetic and molecular genetics, more and more gene mutations are acknowledged. The targeting therapy of genetic alterations related to the prognosis of AML combines with traditional chemotherapy could be the main measure to improve the curative effect of AML. The molecular mechanisms for the development of AML and targeting treatment of AML have been the research hotspot in both domestic and overseas.Be111a (B-cell CLL/lymphoma11A) gene is mapped at human chromosome band2p16.1, the full length of Be111a is102kb, which encodes4isoforms: Be111a-XL, Bc111a-L, Bc111a-S, Bc111a-XS. Be111a encodes a C2H2zinc finger transcription factor that was initially discovered as a retroviral insertion site (Evi9) in myeloid leukemia tumors in BXH-2mouse. Recent investigations have revealed that Be111a is expressed in most hematopoietic cells and is highly enriched in B cells, early T cell progenitors, common lymphoid progenitors, and hematopoietic stem cells (HSCs), which is essential for B lymphoid cell development. Bc111a was able to bind to a core motif in the HBG proximal promoter, recruit and ineract with partners to form a repression complex, leading to down-regulation of the HBG transcription. It’s reported that Be111a was associated with the development of leukemia, however, its mechanism remains uncertain.I-mfa has been identified as an inhibitor of MyoD and other related myogenic basic helix-loop-helix proteins. I-mfa is usually expressed in the cytoplasm, which is a transcription modulator that binds to and suppresses the transcriptional activity of transcription factor involved in several signal pathways. I-mfa contains a cysteine-rich C-terminal domain, and has been reported to function as transcriptional regulator of different pathways including Wnt signaling, c-jun N-terminal kinase signaling, and the regulatory properties of I-mfa depend on the C-terminal domain. Furthermore, recent studies have found that the I-mfa domain may have a close correlation with the development of myeloid neoplasms.Epidermal growth factor receptor pathway substrate8(EPS8) is a97kDa protein that is tyrosine phosphorylated following stimulation of receptor tyrosinc kinases, which is a substrate for the epidermal growth factor receptor kinase. EPS8is a multifunctional protein involves in regulating of several signal pathways, that is related to the deteriorate of normal cells and the proliferation, metastasis and invasion of tumor cells, but the mechanism remain unclear. It’s reported recently EPS8was highly expressed in hematologic malignancy cell lines, like KG-1a and ARH-77, and it might play an important role in the differentiation of them, EPS8promises to be a therapeutic target of hematological malignancies.Some scholars suggest that multidrug resistance(MDR) and minimal residual disease(MRD) are the roots of the refractory and relapse of AML, which is the most fundamental causes leading to the treatment failure of acute myeloid leukemia. MDR family in humans contains two members, MDR1and MDR2. Only can MDR1produce MDR phenomenon, although these two members are highly homologous. Recent studies revealed that MDR1is often expressed on leukemic cells at diagnosis in acute myelogenous leukemia, and is even more frequently present after treatment failure. Furthermore, the results of several larger-scale clinical trails at home and abroad show that high expression of MDR1is in high correlation with treatment failure of AML, and is associated with poor prognosis.Nuclear factor erythroid-2related factor-2(Nrf2), a helix-loop-helix basic leucine zipper transcription factor, is a key regulator in the cellular defense system against oxidative stress. Nrf2is binding with Keapl in a physiological manner, activation of Nrf2by ROS results in the induction of a series of anti-oxidative stress/detoxifying enzymes and proteins, such as HO-1, NQO-1, UGT and GST. Previous studies suggest that, Nrf2is aberrant activated in many cancer cells, which is playing an important role in the drug-resistance of the patients. What’s more, it was reported that there’s also an aberrant activation of Nrf2in the AML cells, which leading to the chemotherapy resistance of AML patients. Objective:Detect the multiple genes(Bcllla, I-mfa, EPS8, MDR1, Nrf2) mRNA level in110adult patients with de novo AML by quantitative real time reverse transcriptase polymerase chain reaction. Analyze the relation of clinical character with the level of multiple genes in adult patient with de novo AML at the same time. In order to provide guidance for the diagnostic classification and prognostic evaluation of AML, to provide potential new targets for the treatment of AML, and to provide theoretical basis for the clinical diagnosis and treatment of AML.Methods:1. Patients and controls Bone marrow samples were derived from110adult patients with de novo AML(66males and44females, mean age32years, range from12-77years), who were finally diagnosed by the means of cell morphology, histochemical stain, flow cytometry and immunophenotype in Nanfang Hospital, Southern Medical University among June2010to December2013. The patients with M3were excluded of this study considering its specific therapeutic regimen and prognosis. The diagnosis of110de novo AML patients was typing with FAB and WHO classification(M11, M249, M414, M528, M61, acute unclassified leukemia17).2. Method2ml bone marrow containing EDTA anticoagulant from110AML patient and20control cases were collected, mononuclear cells were isolated by Ficoll seperation. Total RNA was extracted using TRIzol reagent, and then the expression of multiple genes were detected by quantitative real time reverse transcriptase polymerase chain reaction. The multiple genes relative expression levels values were calculated using the mean of△Ct(△Ct=Ct Tartet-ct GAPDH) from the three replicates, and expressed as2"△Ct (2-△Ct=2Ct GAPDH-Ct Target).According to these data we analyzed the relationship of multiple genes mRNA to FAB subtypes, karyotype, immunophenotyping, clinical characters, therapeutic effect and prognosis.3. Statistical analysis The follow-up was started from the first visit to hospitalization and lasted for January20,2014(range from0.5months to43months, median time9months), and the follow-up rate was90%. Use the median value of multiple genes expression levels by110de novo AML patients as a cut-off point, all the patients were divided into two groups:low expression group, high expression group. Mann-Whitney U test for distribution among2groups or the Kruskal-Wallis test and the Bonferroni test for distribution among more than3groups. Analysis of the distribution between2continuous 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. The statistic use SPSS13.0software to analyze, for all analyses, the P values were2-tailed, and a P value of less than0.05was considered statistically significant.Results:1. Detection of multiple genes(Bcllla, I-mfa, EPS8, MDR1, Nrf2) by quantitative real time reverse transcriptase polymerase chain reaction, use the median value as a cut-off point,110de novo AML patients were divided into two groups:low expression group and high expression group, and every group had55patients.The median age of patients in high Be111a expression group is42years, which was much elder than in low Be111a expression group (42vs32years, P<0.05Mann-Whitney U test), and with no significant difference between different I-mfa(EPS8, MDR1, Nrf2) expression groups. What’s more there’s no significant difference of white blood count (WBC), hemoglobin B (Hb) level, platelets, peripheral blood and bone marrow blasts counts between different Be111a (I-mfa, EPS8, MDR1, Nrf2) gene expression groups (P>0.05, T test).Among the110adult patients with de novo AML, the median expression of Be111a gene for the male and female patients were0.032(0-0.443),0.032(0-0.443) separately; For I-mfa were0.018(0-9.726),0.013(0-31.476); For EPS8were0.022(0.001-11.677),0.022(0-1.527); For MDR1were0.022(0.001-26.715),0.014(0.001-8.223); For Nrf2were0.034(0-42.030),0.030(0.003-0.948). The results showed that there were no difference in the expression of multiple genes among different sex groups.The diagnosis of110de novo AML patients was typing with FAB and WHO classification, the statistical results obtained indicated that the expression of multiple genes had no statistical significance between different types of AML (P=0.775,0.169,0.601,0.678,0.385, Kruskal-Wallis H test).Immuophenotype was examined by flow cytometry in106adult patients with de novo AML, among them80patients expressed CD34and28patients CD34expression was negative. We compared the expression of multiple genes between the two groups, the results showed that there were also with no significant difference. In85cases whose karyotypes were identified, there was11patients in high-risk group,61patients in medial-risk group and13patient in low-risk group. The expression of Bcl11a, I-mfa, EPS8, Nrf2genes were no significantly different in cases with different risk groups of karyotypes (P=0.298,0.078,0.896,0.666, Kruskal-Wallis H test); there’s significant difference of MDR1gene expression between the different risk groups of karyotypes(P=0.019Kruskal-Wallis H test), a further intercomparison was made in the three groups, which showed that the expression of MDR1in case of medial-risk groups was signicant high compared with the low-risk group(P=0.007Mann-Whitney U test).2. There’s72.7%(80/110) AML patients achieved complete remission after induction chemotherapy. The CR rate of patients in low Be111a expression group was significant high campared with high Be111a gene expression(83.6%vs61.8%, P=0.010); For the low I-mfa gene expression group, the CR rate was also higher(81.8%vs63.6%, P=0.032); And slso for the low MDR1gene expression group(81.8%vs63.6%, P=0.032). Besides that, there were no significant difference in CR rate among different EPS8(Nrf2) expression groups(P>0.05).The follow-up was started from the first visit to hospitalization and lasted for January20,2014. The fellow-up rate was90%, and the survival rate was76.4%. The median survival period of patient in low Be111a expression group was377days, and in high Bcllla expression group was218days, there’s significant different of OS between the two groups (83.6%vs69.1%, P=0.009Kaplan-Meier analysis); the median survival period of patient in low MDR1expression group was410days and in high MDR1expression group was216days, the OS in low MDR1expression group was significantly higher than in high MDR1expression group(81.8%vs70.9%, P=0.045Kaplan-Meier analysis). There were no difference in survival rate among different I-mfa, EPS8, Nrf2genes expression groups(P>0.05).3. Cox regression analysis of several factors (including age, WBC, Hb level, PLT, bone marrow and peripheral blood blasts, mutiple genes expression levels, CD34expression, chromosome risk stratification) revealed that age, WBC and the expression of MDR1might be an independent prognostic factor for AML patients.Conclusions:1. The cases in high Bc111a group has a significantly worse CR rate, OS and survival period than in low Bc111a group, as well as different MDR1gene expression groups. Patients with high I-mfa expression get a better CR rate, both of the three gene may be an predictor of poor prognosis for de novo AML patients.2. To the de novo AML patients, there’re no difference in CR rate and OS rate among different EPS8(Nrf2) gene expression groups.3. Cox regression analysis of several factors show that age, WBC and the expression of MDR1might be an independent prognostic factor for AML patients.