| 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 malignant of teenagers and adults under 35 years old. Acute Myeloid Leukemia (AML) is the most common types of adult AL. In recent years because of factors such as environmental pollution, morbidity of AL has obviously upward trend. 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. If treated by standar treatment, AML patients could be complete remission, and even cured. 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. About 75% AML patients can get complete remission (CR), but about 20%-30% AML patients remain no remission (NR) after routine chemotherapy, known as refractory of leukemia. In addition, about 50% of CR patients will relapse after therapy. Refractory and Relapse of AML patients, meaning to be hard to induce remission again and even get the more aggressive therapy, predicts adverse outcome. Therefore, the refractory and relapse of AML patients are the most fundamental reason for the treatment failure for AML patients.The current criteria of diagnosis of refractory and relapse for AML patients as follows:(1) The conventional induction chemotherapy is invalid for the early treatment of primary AML patients; (2) Early relapse of leukemia in 6 months after complete remission; (3) The AML patients relapse after 6 months, but the original chemetheraphy is fail to make the patients get CR again; (4) AML patients relapse for 2 times or more than 2 times. These criterias are retrospective, can not be used to guide clinical treatment.If a set of early diagnosis method of refractory and relapse AML can be established, we can use it to predict refractory and relapse AML 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 AML, improveing treatment effect and long-term survival of AML.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. Others, such as ERG, MN1 and EVH, IDH1/2 gene expression are the prognostic indicator of AML, respectively affect the occurrence and development of AML through various signal pathways in the leukemia cell proliferation, apoptosis and inhibit differentiation. However,70%～80% AML patients can’t be detected with these two genetic abnormalities. Therefor, it’s urgent to found the other new marker for AML patients. Latest studies discover that the MDR1 and BAALC gene overexpress in AML patients, and they are probabley new prognostic markers.l,The role of MDR1 gene in AMLThe MDR1, which encodes for a transmembrane glycoprotein (P-170 or P-glycoprotein) capable of pumping several different drugs out of the neoplastic cell, is one of the main mediators for leukemia chemoresistance. An increased expression of the MDR1 gene or its protein has been detected in a large series of hematological malignancies and its negative prognostic role in AML has been frequently documented, several subsequent studies indicated the influence of MDR1 expression levels in the treatment response and survival of leukemia patients.The MDR1 gene is closely related to de novo drug resistance and relapses in AML patients. Idarubicin has been shown to be less affected by topoisomerase Ⅱ-related multidrug resistance than is daunorubicin in leukemia cells. Idarubicin also exhibited substantial growth inhibitory activity by overcoming Pgp-mediated multiple drug resistance in vitro. Pogliani et al. investigated 50 adults with de novo diagnosed AML using immunohistochemistry and found that in comparison with daunorubicin treated group, the Idarubicin -based treatment achieved significantly higher CR rate in P-gp positive patients. Therefore, it was unavoidable to speculate that the superiority of idarubicin to daunorubicin in induction remission in AML patients may also derived from a better efficacy in AML patients expressing high levels of MDR1.2.The role of BAALC in AMLBrain and acute leukemia, cytoplasmic (BAALC) gene located on chromosome 8q22.3, encodes a protein of yet unknown function. In hematopoietic cells, it is only expressed in progenitor cells whereas no expression was detected in mature bone marrow or circulating white blood cells. Baldus et al reported BAALC was associated with an immature leukemic phenotype in cytogenetically nomal AML (CN-AML) Our previous study demonstrated that BAALC may act as an adverse prognostic factor through prompting proliferation and inhibiting apoptosis in leukemia cells. It was also shown over expression of BAALC turned out to be predictive of inferior outcome and chemotherapy resistance in adult CN-AML patients, high BAALC expression of AML had a lower CR rate and OS.Althought MDR1 and BAALC expression have been related to the prognosis of AML patients, but little is known about the clinical significance of MDR1 and BAALC expression level in them. Considering the frequent expression of MDR1 and BAALC in leukemic and its functions as a regulator of differentiation and apoptosis and a modulator of the immune system, the role of the MDR1 and BAALC level in AML is not fully understood. In this study, we analyzed the expression level of the MDR1 and BAALC in 209 de novo AML cases.We investigated correlations with cytomorphology, cytogenetics, flow cytometry and other clinical parameters. Further more, we investigated whether idarubicin in cytarabine-based induction regimen was superior to daunorubicin in remission induction of de novo AML patients expressing high MDR1.Objective1. To establish a method of detecting MDR1 and BAALC mRNA with quantitative real time PCR.2. To detect the level of MDR1 and BAALC mRNA of de nove AML patients and the discrepancy of that of FAB subtypes.3. To discuss the relationship of different karyotype, clinical character, to the level of MDR1 and BAALC mRNA.4. To anylyse the relation of the level of MDR1 and BAALC expression in AML patients at the same time.5. To investigate whether idarubicin in cytarabine-based induction regimen was superior to daunorubicin in remission induction of de novo AML patients expressing high MDR1.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 MDRl, B 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 209 de novo AML patients, who were finally diagnosed by the means of cell morphology, hostochemical stain, flow cytometry and immunophenotype.30 non-malignant hemotologic disease patients bome 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 patients and non-malignant hemotologic disease patients were abstracted and then reverse-transcripted into cDNAs by rea time quantitive PCR. The MDR1 and BAALC relative expression levels values were calculated using the mean of ΔCT(ΔCT= MDR1 or BAALC-GAPDH) from the three replicates, and expressed as 2-ΔCT. According to these data we analyzed the relationship of MDR1 and BAALC mRNA to FAB subtypes, karyotype, clinical character, therapeutic effect and prognosis.5.189 cases of 209 patients with AML were treated by IA arm (idarubicin and cytarabine) chemotherapy or DA(daunorubicin and cytarabine) for remission induction therapy. To analyze the different treatment response rate of two treatment scheme and analyze the relationship with the expression of MDR1.6. Statistical anylysis:SPSS 19.0 statistical software was used to analyze the data. 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 RNAs of 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 62bp(MDR1),60bp(BAALC),225bp(GAPDH) were observed under ultraviolet rays. The sequence of positive recombinated plasmid was totally consistent with according base sequence in Genebank.2.1t showed perfect linear correlation between logarithm of different multiproportion dilution template for quantitation and cycle number (Ct)(r=0.997, 0.996,0.997).The slopes of the PCR reaction are-3.213,-3.208 and -3.251. The applification effect of three genes were similar to each other, so we choose the 2-ΔCT to analyse the data. Amplification efficiency were 97%,96%,97%, shows that the three amplification efficiency.3.The mean of MDR1 transcripts in the 209 AML patients was 0.016 (0-19.525),and the mean of BAALC transcripts in the 209 AML patients was 0.0470.043 (0-12.148), while those in the 30 control samples was 0.005 (0-0.098) (MDR1) and 0.004(0.001-0.013){BAALC). The expression of MDR1 and BAALC gene was significantly higher in AML cases than in healthy donors (P=0.000).4.At the same time, it revealed that MDR1 and BAALC gene expressions were equally distributed among the FAB subtypes (P=0.225;P=0.152,Kruskal-Wallis test). Amone all the groups, Spearman’s rank correlation showed that not significant correlation was found between ages and sexual distribution of the AML patients. Not significant correlation was found between WBC, percentage of blast celss of bone marrow,and karyotype(P>0.05).5.189 cases of 209 patients with AML were treated by IA arm (idarubicin and cytarabine) chemotherapy or DA(daunorubicin and cytarabine) for remission induction therapy.20 cases (include 12 cases MDS related AML and 8 cases older AML patients) were treated by other chemotherapy arms including CAG arm combined with or without decitabine. During the period of this analysis, patients received induction therapy with Ara-C at 100-200 mg/m2/d by continuous infusion for 7 days (day 1 to 7) and either daunorubicin at 35-45 mg/m2/d from d 1 to 3 (the daunorubicin arm) or idarubicin at 6-8 mg/m2/d from day 1 to 3 (the idarubicin arm). All patients were subjected to two courses of idarubicin or daunorubicin regimen treatment. The first course was the first induction course. The second course was given irrespective of remission status after the first course. Thus, it was either the second induction or the first postremission course. Supportive care, including administration of antibiotics and platelet transfusions, was provided if needed. Granulocyte colony-stimulating factor was administered for patients with life-threatening infections due to neutropenia.6. We collected bone marrow samples from a total of 209 AML patients in the current study, including 118 males and 91 females. The demographic and disease characteristics of these patients are shown in Table 2. Their ages ranged from 14 to 80 years (Mean 40.3±16.0). Cytogenetic analysis revealed that 28 (13.4%,28/209) patients were classified in the favorable-risk group,92 (44.0%,92/209) in the intermediate-risk group,19 (9.1%,19/209) in the adverse group, and 70 (33.5%, 19/209) in the undetected group. Median MDR1 expression of 209 AML bone marrow samples is 0.373(0-74.819), while median BAALC expression is 0.759(0-65.070). MDR1 expression levels were dichotomized at their median, and patients were classified as having low MDR1 (n= 104) if they had expression values within the lower 50% and as high MDR1 (n= 105) if they had MDR1 expression values within the upper 50% of all measured values. Simlarly,104 cases were classified as having low BAALC and 105 cases as high BAALC expression. The high and low MRD1 groups were well balanced with regard to pretreatment characteristics such as age, initial WBC counts, Bone marrow (BM) blasts, FAB classification, and cytogenetic prognostic grouping, so were the the high and low BAALC groups.High MDR1 expression was correlated with a high BAALC expression (r=0.487,P<0.001). However, there was no significant association between expression of MDR1 and BAALC and age, initial WBC counts, Bone marrow (BM) blasts, FAB classification, and cytogenetic prognostic grouping. To investigate the impact of MDR1 combined BAALC expression on clinical outcome in intermediate cytogenetic risk group, we further stratified AML patients by MDR1 and BAALC expression status and identified four different subgroups:high BAALC/high MDR1(n= 30), low BAALC/low MDR1(n= 30), low BAALC/hgh MDR1(n= 16) and high BAALC/low MDR1(n= 16). As shown in table 3 and Figure 1, there was no difference in the CR rate, relapse rate and OS among low MDR1/high BAALC, high MDR1/low BAALC, and high MDR1/high BAALC groups, The three groups were characterized with an similar clinical outcome and therefore can be considered as one entirety which we defined as "combined group". However, patients with low MDR1/low BAALC expression had a higher CR rate (93.3% vs 72.6%., P=0.021), lower relapse rate(7.1% vs.43.5%,P=0.000), longer OS (50.3% vs 17.8%, P=0.001) than the "combined group" (Table 4 and Fig 2A).To further evaluate prognosis of low MDR1/low BAALC expressers in intermediate-risk group, we compare the clinical outcome with those in favorable-risk group. As shown in table 5 and Fig 2b, no significant difference was observed in the CR, relapse rate and OS between the two groups.In an exploratory multivariable analysis the combined prognostic validity of MDR1 and BAALC expression was investigated. In this model, patients with combined group had a significantly higher risk of death (HR:5.703, P=0.002), once adjusting for WBC, the only other prognostic factor (Table 6).7. We collected bone marrow samples from a total of 189 AML patients treated by IA or DA arm in the current study. The 2 groups were well balanced with regard to pretreatment characteristics such as age, initial WBC counts, FAB classification, and cytogenetic prognostic grouping.92 patients received the idarubicin arm and 97 the daunorubicin arm. The two treatment arms were matched with respect to various baseline characteristics such as age, initial WBC counts, and FAB classification. Overall, among 189 patients,130 (68.8%,130/189) achieved CR after two courses of induction chemotherapy. Of 92 patients in the idarubicin group,76 (82.6%,76/92) achieved CR. Of 97 in the daunorubicin group,54 (55.8%,54/97) patients obtained CR. The CR rate of the idarubicin treatment arm was significantly higher than that of the daunorubicin treatment arm (P=0.000) We determined the expression of MDR1 in bone marrow cells from AML patients. At a cutoff value of 0.016, the median MDR1 expression of 189 AML bone marrow samples,94 patients were classified as high MDR1 expressors and 95 as low MDR1 expressors. Among high MDR1 expressors,56(59.6%,56/94) patients achieved CR and in low MDR1 expressors,74 (77.9%,74/95) patients achieved CR.We further investigated whether MDR1 expression impacted upon the therapeutic response of AML patients to idarubicin or daunorubicin. We stratified the response of AML patients who were high or low MDR1 expressors by treatment. We found that the CR rate in high MDR1 expressors treated with idarubicin was significantly higher than high MDR1 expressors treated with daunorubicin (P<0.05). By contrast, no significantly statistical difference was noted in the therapeutic response between patients expressing low levels of MDR1 treated with idarubicin and those treated with daunorubicin(P<0.05). These findings suggested that idarubicin is associated with a more favorable therapeutic response than daunorubicin in AML patients expressing high levels of MDR1. We were interested in whether cytogenetic abnormalities modulated the therapeutic response of AML patients to idarubicin and daunorubicin. We found that in AML patients with a favorable risk, idarubicin was associated with a higher CR rate than daunorubicin in AML patients. Similar finding was observed in AML patients with an intermediate risk (P<0.05). However, in AML patients with an unfavorable risk, no significant difference was noted in the CR rate in patients receiving idarubicin and daunorubicin(P>0.05). In addition, we found that patients exhibiting different genetic risk profiles showed no difference in MDR1 expressionWe further stratified AML patients in different genetic risk groups by MDR1 expression and examined their association with therapeutic response to idarubicin and daunorubicin. In AML patients exhibiting a favorable risk profile and expressing high levels of MDR1, idarubicin was associated with a significantly higher CR rate than daunorubicin (P<0.05). Similar findings were observed in AML patients exhibiting an intermediate risk profile and expressing high levels of MDRl(P<0.05). However, no significant difference in CR rate was observed between the two treatment arms in AML patients exhibiting either favorable or intermediate risk but expressing low levels of MDR1. Because of the extremely limited number of cases in the unfavorable risk group, no such analysis was carried out.Conclusions:1.A method of detection MDR1 and BAALC mRNA level by FQ-PCR was successfully established.2.The expression of MDR1 and BAALC gene was significantly higher in patents with AML than in control samples and MDR1 expression had not correlation with BAALC expression.3. MDR1 and BAALC gene expressions were equally distributed among the FAB subtypes (P=0.225;P=0.152,Kruskal-Wallis test). Amone all the groups, Spearman’s rank correlation showed that not significant correlation was found between ages and sexual distribution of the AML patients. Not significant correlation was found between WBC, percentage of blast celss of bone marrow,and karyotype.4. In conclusion, this study demonstrates the prognostic value of the combined assessment of MDR1 and BAALC expression in adult intermediate cytogenetic risk patients. The low MDR1 and low BAALC expression status identifies a highly favorable risk group for which post-remission therapy with SCT may not be indicated. Independent studies should be performed to confirm that the combined assessment of MDR1 and BAALC expression can improve treatment stratification in adult intermediate cytogenetically AML.5. Idarubicin is associated with significantly better remission induction of de novo AML patients with high MDR1 expression. |
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