The Clinical Prognosis Correlation Of The Complex Phenotype,Cytogenetics And Expression Of APCcdh1and NEK2mRNA Level In Cells From Patient With Acute Myeloid Leukemia

Background and objective:Acute myeloid leukemia is the most commen type of acute leukemia. Chemotherapy has been a traditional treatment of AML,The current standard chemotherapy regimen can gain60%-80%of complete remission (CR), even as part of patients are difficult to achieve CR and become refractory leukemia, eventually death in invalid treatment, most of them result from multi-drug resistance. The biological and cytogenetic characteristics were considered to be affirmative factors to drug resistance of AML.The minority of acute myeloid leukemia (AML) includes more than one hematopoietic differentiation characteristics, such as Mixed-phenotype acute leukemia, MPAL. Mixed-phenotype acute leukemia is characterized with the capability of differentiating into myeloid and lymphoid cells, It is hard to make a appropriate diagnosis and treatment, and not like acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), which can be classified clearly. whereas,the clear dignosis of MPAL needs morphological,immunology and genetics classification. There are complicated and diverse criteria for the diagnosis of this disease. Most studies in the last decade have used the EGIL scoring system to diagnose biphenotypic acute leukemia. Even when the correct EGIL criteria are applied, strict application of the point system can lead to an inaccurate classification. For these reasons, the2008WHO has replaced the EGIL scoring system with a simpler diagnostic algorithm that relies on fewer markers to define mixed phenotype acute leukemia, it grouped this disease together under a new heading, MPAL. The WHO2008criteria combines with flow cytometry, Immunohistochemistry, cytochemistry, and immunoeletrophoresis to define MPAL, a specific reference is now made to exclude cases that of chronic myelogenous leukemia (CML) in blast crisis, that with a history of MDS or recurrent genetic abnormalities, and therapy-related AML Although the diagnosis of MPAL is simpler with the2008WHO system, it is restricted for the units which has not develop the technology above,and it is not yet clear whether the new criteria actually refine this diagnosis in comparison with the EGIL criteria. lymphoid surface antigen-positive acute myeloid leukemia(Ly+AML) is a special subtype of AML, which is also increasingly taken seriously. There are confilicting reports about progonosis of Ly+AML, and the clinical characteristics and progonosis has not been compared between MPAL and Ly+AML.Genome integrity has becoming a focus of tumor study. Ubiquitin-proteasome Path-way (UP Path-way) regulates almost all the vital activates in animal and plant bodies, APC/C is one of the main polyubiquitin ligases E3,it have two substrate-linked proteins, APCcdc20and APCcdhl. APCcdhl Stays in active state from mitosis anaphase to G1phase. APCcdhl, the anaphase-promoting complex in mitosis, participates in regulation of accurate DNA repair from G1phase to S phase, acting as cell-cycle regulatory protein. Expression of APCcdhl exists in the cells of solid tumors such as non-small cell lung cancer, gastrointestinal stromal tumor, breast cancer and colorectal tumor; moreover, there is high expression of APCcdhl in hematological tumors, such as in chronic myeloid leukemia and acute myeloid leukemia. Studies have also reported that APCcdhl is closely associated with maintenance of genetic stability in the trophoblast and fibroblast cells. According to our findings of previous research,the expression of APCcdhl in the patient with CML-BC is different, low expression is associated with additional chromosome and ABL gene mutations,which suggest the expression of cdhl is probably associated with maintenance of genetic stability in the CML. In addition, Abnormal expression of NIMA-related kinase2(NEK2) exists in the cells of solid tumors such as breast cancer, multiple myeloma and leukemia cell, moreover, NEK2is closely associated with poor prognosis, genome instability and chemotherapy resistance in the tumors.According to the heterogeneity of immune phenotype and chromosome karotype of acute myeloid leukemia, in order to further understand the characteristics of clinical and outcome of these disease, we collected the information of the patients with AL in our hospital in resent10years. Patients wedre dignosed mixed-phenotype leukemia by EGIL1998and WHO2008criteria, and AML with or without lymphoid immunophenotype by WHO2008. The coincidence rate of diagnosis and therapeutic response of patients dignosed by the two diagnosis standards were compared. And we explored APCcdhl and NEK2gene expression level in AML by RQ-PCR in order to further confirm the influences of APCcdhl and NEK2to the therapeutic, prognosis of AML, and the diference gene expression and significance in different genectic background of AML.METHODS1. the correlation of the complex immunophenotype,chromosome karoytype and response to chemotherapy of AML.1) Study subjects:42patients were dignosed MPAL,59patients were Ly+AML and84patients were Ly-AML by WHO2008criteria,45patients were dignosed MPAL by EGIL1998criteria. All patients have been followed up to February in2013. The coincidence rate of diagnosis and therapeutic of two diagnosis standards were compared.2. Explore the influences of the APCcdhl and NEK2gene expression level in AML by RQ-PCR on the complex karotype, phenotype and the prognosis of the patient with AML.1) Study subjects:All the AML bone marrow specimens were taken from61cases of diagnosed patients from the previous analytical group,which include43patients with normal karyotype and18patients with complex karyotype. Control group are age, sex matched21patients excluding hematologic malignancies.2) Primer design and synthesis:In accordance with the GeneBank sequence we design APCcdhl and NEK2gene and the housekeeping genes β-actin primers.3) Extraction of bone marrow mononuclear cells from newly diagnosed AML and non-hematologic malignancies patients, then extraction of total RNA, reverse transcription into cDNA, doing real-time fluorescence quantitative PCR finally. To determine the specificity of PCR product through the melting curve. Take an average of two Ct values2-ACT value, as the specimen relative mRNA expression levels, ACT=(target gene CT value-reference gene CT value). Compare the APCcdhl and NEK2gene expression differences of patients bone marrow mononuclear cells from AML with normal karyotype group and with complex karyotype group, normal karyotype group and control group, complex karyotype group and control group. And relative gene mRNA expression levels are divided into high expression group and low expression groups. Compare the number of cases from each treatment group in the CR cases and NR cases after two course of chemotherapy, refractory cases and non-refractory cases, alive within3years, the association of the two gene expression level.3. statistical analysesAll statistical analyses were performed with the SPSS17(SPSS Inc, Chicago, IL, USA). Mann-Whitney U test was used to analyze the difference in the expression of each gene between each group. Rate compared using χ2test Correlations between variables were assessed by the Spearman rank correlation. Kaplan-Meier estimation was used to plot survival curves, and log-rank tests were used to test the difference between groups. Two-sided P values<0.05were considered statistically significant.Results:l.From juanuary in2002to August in2012, we collected45patients who were dignosed MPAL by EGIL1998criteria,.42patients were dignosed MPAL by WHO2008criteria,35cases met both criteria. In the subtypes of M/B,M/T and B/T, the coincidence rate is81.6%(31/38),66.7%(4/6), and100%(1/1) respectively. The complete remission rate of the patients meeting both EGIL1998and WHO2008 criteria,EGIL1998but excluded by WHO2008and by WHO2008but excluded by EGIL1998after two course of chemotherapy was72.0%,62.5%,57.14%, respectively, with no significant difference between any two groups based on χ2test (P>0.05), as also is the overall survival between groups.2.42patients were dignosed MPAL by WHO2008criteria, while comparing clinical feature of the patients with MPAL and two control groups, which are age, sex matched59patients with Ly+AML and84patients with Ly-AML,respectively, we found that the proportion of bone marrow blast and the incidence of liver, spleen and lymph node invasion is significantly higher in the MPAL group than in the two control group, while, the incidence of CD34positive expression in the MPAL and Ly+AML group is significantly higher than the Ly-AML group. Cytogenetic result shows that the incidence of ph+or BCR/ABL+and complex karoytype in MAPL is higher than the two control group (P<0.05).The CR rate after two course of chemotherapy was67.5%,68.5%and64.5%in MPAL,Ly+AML and Ly-AML group respectively, with no significant difference between any two groups based on χ2test (P>0.05). The overall survival is significantely shorter in the MPAL group than Ly+AML group and the Ly-AML group. There were no statistically significant differences in the clinical characters and prognosis between Ly+AML and Ly-AML group. In above each group, While comparing the overall survival between the normal karoytype and complex karoytype group, we find the overall survival is all significantely shorter in the complex karoytype than normal karoytype group. As to the patients with normal karoytype in MPAL, Ly+AML and Ly-AML group, the overall survival is significantely shorter in the MPAL group than Ly+AML and Ly-AML group, While the chromosome karoytype is complex, there is no significant difference among the three groups.3. Analysis of purity and quality of RNA and cDNA:total RNA and cDNA were detected by UV Spectrophotometer, and the ratio are between1.8and2.0. The same cDNA sample was diluted by2-fold, was setted by6gradients. The concentration gradients were measured, as the scope is8.1～258.8ng/μl. The housekeeper gene and target genes were amplified respectively. The result is that6gradient ACT differences are very small, thus amplification efficiency of the housekeeper gene and target genes are identical at the concentration range. Melting curve was done after amplification. It showed sharp single peak and no other specific peak with each gene. So we considered PCR products were homogeneous,no other non-specific amplification and primer dimmer. We can see that a single strip, no primer dimer and non-specific amplification by agarose gel electrophoresis,Which meets the requirement of experiment.4. Gene expression:There is no significant difference of APCcdhl and NEK2expression level in Ly+AML and Ly-AML group. The APCcdhl gene expression in complex karoytype group was significantly lower than normal karoytype and control group (Z=-2.918, P=0.004; Z=-3.493, P=0.000). To the contrary, NEK2expression level in the complex karoytype group was significantly higher than normal karoytype and control group (Z=-3.89, P=0.000; Z=-3.409, P=0.001).5. Relationship between gene expression level and clinical indicators: Correlation analysis was done between expression level of APCcdhl and NEK2gene and clinical indicators such as age, white blood cells count and the proportion of leukemic cells in peripheral blood and bone marrow leukemic cells of newly diagnosed patients. The results showed that age, white blood cells, bone marrow leukemic cells were not significantly related to APCcdh1expression level, the absolute value of correlation coefficients were less than0.3, P>0.05. APCddhl and NEK2expression level between man and woman had no significantly difference Z=-0.155,P=0.877; Z=-1.211, P=0.226). while NEK2expression level maybe positive correlated with the proportion of leukemic cells in bone marrow leukemic cells. And both APCcdhl and NEK2expression level is closely related to cytogenestic. As the karoytype is normal, the APCcdhl expression is high and the NEK2is low, while the karoytype is complex, the APCcdhl expression is low and the NEK2is highThere is no correlation between APCddhl and NEK2expression level(r=-0.213, P=0.099),which may result from the insufficient cases.6. Relationship between gene expression level and efficacy:Patients are divided into two groups according to the mediam of each expression level. There are56cases can be used to analyze the respose to chemotherapy after removing the lost cases. APCcdhl are28cases of high expression group,28cases of low expression group.24cases (85.7%) in high-expression group and14cases (50.0%) in low-expression group achieved CR, respectively, with significant diference(P<0.05).And there are10refractory cases (35.7%),19ases (70.4%) in high-expression group and low-expression group, respectively,with significant diference(P<0.05).While NEK2are29cases of high expression group,27cases of low expression group.15cases (51.7%) in high-expression group and21cases (77.8%) in low-expression group achieved CR, respectively, with significant diference(P<0.05). there are19refractory cases (65.5%),10cases (37.0%) in high-expression group and low-expression group, respectively, also with significant diference(P<0.05).7. The influence of gene expression level on the outcome:Univariate analysis of the survival time showed that white blood cells count, the chromosome karoytype and the expression level of NEK2gene of patients were found to have significant influence. But multivariate analysis showed that only white blood cells count and NEK2expression has significance influence on the survival time(P<0.05).Conclusion:1.Compared with EGIL1998criteria, the cases diagnosed by WHO2008criteria may conform to the characters of mixed cell phenotype better.2. the overall survival is significantely shorter in the MPAL group than Ly+AML and Ly-AML group, which may related to the high proportion of leukemic cells in bone marrow leukemic cells and complex karoytype. There were no statistically significant differences in the clinical characters and prognosis between Ly+AML and Ly-AML group.3.Complex chromosome karoytype is one of the most important factors of the prognosis of the AML patients with different phenotype.4. NEK2expression level is positive correlated with the proportion of leukemic cells in bone marrow leukemic cells.The low expression level of APCcdhl or high expression of NEK2may suggest a high incidence of complex karoytype and poor outcome..5.there are no differences of the APCcdhl or NEK2gene expression level between the patient with Ly+AML and Ly-AML.