Expression And Clinical Significance Of CD74 And P65 In Acute Leukemia

Background and ObjectiveAcute leukemia is a clonal malignant disease of blood system, one of the most common malignant tumors in young people. To explore the pathogenesis of acute leukemia has been an important field of scientific research. The CD74/P65/TAp63 pathway defines a novel anti-apoptotic pathway, which plays an important role in apoptosis.CD74 is a trans-membrane protein that functions as a MHC class II chaperone. In the membrane, CD74 combined with MHC classⅡmolecules in a form of CD74 complex is translocated into the nucleus and lysosomes and leads the activation of the P65, a member of the NF-kB family. The transcription activity of P65/RelA dimmer and its costimulator is mediated by CD74 and the activation of P65 upregulates the expression of p53-related TAp63 protein, which activates the Bcl2 gene and induces the production of Bcl-2 protein resulting in cell survival. CD74 was expressed on a variety of malignant tumors. P65 was upregulated in malignant tumors, such as breast cancer, stomach cancer and colorectal cancer. But the expression of CD74 and P65 in primary leukemic cells and their possible role in pathogenesis of acute leukemia need further study. This experiment is aimed to detect the expression of CD74 mRNA and P64 mRNA in the bone marrow mono-nucleus cell from 49 cases of acute leukemia by RT-PCR. Their relationship will be analyzed. the expression of CD74 mRNA and P64 mRNA in 24 cases complete remission acute leukemia, and in 12 patients with non-malignant hematological diseases as control will be compared to these in acute leukemia in order to explore the possible role of CD74 and P65 in the development of acute leukemia.Materials and methods1 Research objects:Forty-nine cases of acute leukemia were enrolled for the research, which were collected in department of Hematology of the First Affiliated Hospital of ZhengZhou University from March to July,2010. The diagnosis of acute leukemia is based on the marrow cell morphology, and immunophenotypes and cytogenetic analysis, according to the diagnostic criteria. The patients with de novo or relapse acute leukemia have a mean age of 36 years old (ranged form 14 to 64 years old), with 18 males and 31 females. Among them,30 cases are AML (15 cases of M2,4 cases of M3,3 cases of M4,8 cases of M5) and 19 cases are ALL (12 cases of B-ALL,7 cases of T-ALL). Patients in complete remission of acute leukemia have a mean age of 39 years old (ranged form 17 to 68 years old), with 18 males and 6 females. Control group consists of 12 cases non-malignant hematological diseases that have a mean age of 25 years old (ranged form 18 to 81 years old), with 6 males and 6 females. All of the patients were hospitalized and in agreement to WHO (2000) diagnostic criteria.2 Experimental methods (RT-PCR):Bone marrow of patients were collected and the bone marrow mono-nucleus cells were harvested using the Ficoll-Hypack gradient and suspended in a concentration of 1×107/ml. Total RNA were extracted by TRIZOL method according to cDNA kit. The reaction system of retro-transcription is 20μl. Synthetic cDNA was used in PCR amplification for CD74 mRNA and P65 mRNA by specific primer. PCR reaction system included Taq Master MIX (2x), Forward primer (10μM), Reverse primer (10μM), Template, RNase-Free water in a total volume of 50μl. After 25 cycles of PCR amplification,3μl products of PCR were electrophoresed in 1.5% agarose gel with 5μl Sunbio DNA Marker I as molecular marker. The bands were viewed in ultraviolet and photographed. The densities of bands were analyzed.3 Statistics analysis:The data were analyzed using software SPSS16.0. Numeric data were expressed as mean±standard deviation (x±s). Qualitative data were analyzed by X2-test. The correlation analysis of CD74 mRNA and P65 mRNA was analyzed by spearman cascade correlation analysis. The standard of significant level wasα＝0.05,when P<0.05 has a statistical significance.Result1. Expression of CD74 mRNA in BMMNC of acute leukemiaThe positive rates were 81.6% in AL,80% in AML, and 84.2% in ALL, respectively. There was no statistically significance between them (P>0.05), which were significantly higher than that in control group (16.7%) (P<0.05). In 19 cases of ALL, the positive rate in B-ALL is significantly higher than that in T-ALL (100%, n=12 vs 57.1%, n=4) (P<0.05)2. Expression of P65 mRNA in BMMNC of acute leukemia.The positive rates were 59.2% in AL,53% in AML, and 68.4% in ALL, respectively. There was no statistically significant between them (P>0.05), which were significantly higher than that in control group (8.3%)(P<0.05). There was no statistically significance of P65 mRNA expression between B-ALL (n=12,83.3%) and T-ALL (n=7,42.9%) (P>0.05).3. Concomitant expression and correlation of CD74 mRNA and P65 mRNA in acute leukemia.Concomitant expression of CD74 mRNA and P65 mRNA in AL were 48.9% which was positive correlated (P<0.05). The CD74 mRNA and P65 mRNA were concomitantly expressed in 83.3% of ALL, which were positive correlated (P<0.05). The CD74 mRNA and P65 mRNA were concomitantly expressed in 46.7% of AML. which were positive correlated (P<0.05).4. Expression of CD74 mRNA and P65 mRNA and patients'clinical features.There is no significant difference between the expression of CD74 mRNA and P65 mRNA and patients gender, age, blasts in marrow, peripheral white blood count and extramedullary infiltration (P>0.05)5. Expression of CD74 mRNA and P65 mRNA in patients with CR.There were 24 cases of patients in CR, with 16 cases of AML and 8 cases of ALL. Ten out off 16 cases of AML were marginal positive of CD74 mRNA and P65 mRNA after 2 months of CR, and 6/16 cases were negative after 3 months of CR. Four out off 8 cases of ALL were marginal positive of CD74 mRNA and P65 mRNA after 6 months of CR, and 4/8 cases were negative after 6-10 months of CR.6. The relationship between the expression of CD74mRNA and P65mRNA and prognosis.Patients were arbitrarily divided into high expression group and low expression group of f CD74 mRNA and P65 mRNA based on the scanned value of mRNA bands (mean=0.8). Nine out off 17 cases relapsed with high expression of CD74; 3/23 cases relapsed with low expression of CD74; and none relapsed without expression of CD74. Five out of 8 cases relapsed with high expression of P65; 5/21 cases relapsed with low expression of P65; and 2/20 cases relapsed in P65 negative patients. The relapse rates in patients with high expression of CD74 or P65 were significantly higher than that in patients with low or none expression of CD74 or P65 (P<0.05). There was no significant difference of relapse rate between low and none expression patients (P>0.05).Conclusion1. The expressions of CD74 mRNA and P65 mRNA in acute leukemia were upregulated and positive correlated. The expression of CD74 mRNA and P65 mRNA were declined after complete remission.2. There is no statistical significance of CD74 mRNA and P65 mRNA expression in AML and ALL, it have statistical significance of CD74 mRNA between B-ALL and T-ALL.3. Prognosis of high expression group is poor.