| 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 inhibition of normal hematopoietic function, which gave rise to clinical common infections, bleeding, anemia and other symptoms. AML, once it happened, is fatal and progress rapidly in a critical condition. Morbidity of AL has obviously upward trend rencently because of factors such as environmental pollution.Although the development of Hematopoietic Stem cell Transplantation (HSCTas well as the improvement of AML treatment effect, about 20%～30% AML patients remain the refractory one because of the poor performance of routine chemotherapy.. In addition, about 50% of CR patients will relapse. Therefore, the refractory and relapse is the the most fundamental reason for the treatment failure of AML patients.The current diagnosis criteria of refractory and relapse for AML are retrospective, which can not be used to guide clinical treatment. If a set of early diagnosis methods of refractory and relapse AML can be established, we can use it to predict AML refractory and relapse earlier and judge the patient's prognosis. More importantly, it can guide us to perform a more active indivual treatment such as earlier HSCT, instead of conventional ones for those high-risk patients.Besides, it may be helpful for reducing the incidence of refractory and relapse of AML, improving treatment effect and leading to long-term survival of AML.Current studies shown that certain genetic abnormalities, including FLT3 internal tandem duplication mutation (FLT3/ITD) in AML and the bcr/abl fusion gene expression in ALL, are related to the occurrence and development of leukemia. It is confirmed that 20%～30% AML patients with FLT3/ITD mutation are prone to be refractory and relapsed, having poor prognosis. So these two genetic abnormalities have been a prognostic molecule marker. However,70%～80% AML and ALL patients cannot be detected with these two genetic abnormalities. Therefore, it is of great urgence to found other new markers for AL patients. Latest studies discover that the MDR1 and BAALC gene overexpress in AML patients, and they are probably new prognostic markers.Chemotherapy is one of the main treatments for leukemia, but the existence of MDR(Multidrug resistance) often leads to chemotherapy failure. It is reported that MDR family in humans contains two members, MDR1 and MDR2. Only can MDR1 produce MDR phenomenon, althouth these two members are highly homologous. It has been revealed that MDR1 is located at chromosome band 7q21.1, whose mRNA and protein are detected in various human cancers.BAALC(brain and acute leukemia, cytoplasmic), which located on human chromosome 8 at q22.3, encodes at least 8 alternatively spliced transcripts in humans, whose according translation product share no homology with each other. Functionally speaking, BAALC is able to represent a marker of early hematopoietic progenitor cells for the in vitro studies showing it is up-regulated in CD34+ bone marrow (BM) cells and down-regulated as subsequent cell differentiation. With regard to such function, it may become useful as a predictive marker in AML treatment.Although MDR1 and BAALC expression have been related to the prognosis of AML patients, little is known about the clinical significance of MDR1 and BAALC expression level in AML patients. Considering MDR1 and BAALC function multifacetedly, such as highly expressed in leukemia, regulator of differentiation and apoptosis as well as modulator of the immune system, we are going to further decipher the role of the MDR1 and BAALC level in AML, which is remain unclear as far as we know. In this study, we analyzed the expression level of the MDR1 and BAALC in 160 AML cases.Objective:1. Detect the MDR1 and BAALC mRNA level in AML patients by quantitative real time PCR.2. Anylyse the relation of the level of MDR1 and BAALC expression and discuss the relationship of clinical character with the level of MDR1 and BAALC mRNA in AML patients at the same time.Methods:1. KASUMI-1 cells were cultivated and the RNA was extracted. GAPDH was used as an endogenous control. After primers were designed according to MDR1 and BAALC 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 templat.2. Cloned template 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 160 AML patients, who were finally diagnosed by the means of cell morphology, hostochemical stain, flow cytometry and immunophenotype. 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 extracted and then reverse-transcripted into cDNAs for realtime quantitive PCR. The MDR1 and BAALC relative expression levels values were calculated using the mean ofΔCT(ΔCT= ERG or MN1-GAPDH) from the three replicates,and expressed as 2-ΔCT.According to these data we analyzed the elationship of MDR1 and BAALC mRNA to FAB subtypes, karyotype, clinical character, therapeutic effect and prognosis5. 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. The band of MDR1,BAALC,GAPDH were observed under ultraviolet light. The sequence of positive recombinated plasmid was totally consistent with according base sequence in Genebank.2.The mean of MDR1 transcripts in the 160 AML patients was 0.016(0～81.865),and the mean of BAALC transcripts in the 160 AML patients was 0.012 (0～59.507) There is a correlation between the expression level of MDR1 and BAALC gene expression (r=0.351,P<0.001 Spearman rank correlation test)3. In all the groups, Spearman's rank correlation showed that WBC,HGB,PLT and LDH were not significantly related to MDR1 and BAALC gene expression in AML patients.4.Within 160 follow-up cases with AML, there was a significant difference of CR rates in high MDR1 expression group and low MDR1 expression group after chemical theropy (61.3% vs.80.0%,P=0.009), but not as BAALC expression(64.2% vs.77.2%,P=0.072). During the follow-up years or more,the cumulative relapse rates of high MDR1 expression group was significantly higher than that of low MDR1 expression group (46.9% vs.23.4%,P=0.009), and survival curve gave the clues that high MDR1 expression cases have a significantly worse OS that low MDR1 expression cases (32.5% vs.56.3%, P=0.007). There was no significant difference of the cumulative relapse rates between high BAALC expression group and low BAALC expression group(40.4% vs.27.9%,P=0.160),and OS (37.0% vs.51.9%, P=0.057)4..Combined detection of MDR1 and BAALC gene expression level show:high expression of MDR1 and BAALC group has the lowest CR rate, the higtest relapse rate and the worst over-all survive. Low expression of MDR1 and BAALC group, has the best prognosis.Conclusions:1. There is a correlation between the expression level of MDR1 and BAALC in AML patients.. 2.Two groups which are divided by expression level of MDRl genes,have a significant differences on CR rate, relapse and OS.BAALC gene does not reveal it.3. Combined detection of MDRl and BAALC genes expression level is better than detection of MDRl or BAALC gene expression level alone for AML.4. Singal detection of MDR1 genes expression level is better than BAALC gene for the AML patients. |
