Co-expression And Significance Of CD20、CD52in B-lineage Lymphoblastic Tumors

Background and ObjectiveB-lineage lymphoblastic tumors is a kind of mainly for the origin of B lymphocytes malignant diseases, including B-ALL, B-NHL and most CLL, etc. B-lineage acute lymphocytic leukemia(B-ALL) is a kind of malignant cloned disease which expresses the early differentiation antigen of white blood cells, and its incidence were higher in malignant tumor of both children and adults. Chronic lymphocytic leukemia (CLL) is a monoclonal small lymphocyte malignant disease originating from B cells. In the European and American countries CLL is the most common type of leukemia, while it also increases year by year in China. B-lineage acute lymphoblastic leukemia (ALL) may express CD52and CD20. Alemtuzumab (ALM) and rituximab (RTX) are therapeutic antibodies directed against CD52and CD20respectively. Compared with traditional chemotherapy agents, Alemtuzumab and Rituximab have the obvious advantages of low toxicity and strongly targeted. Both of them have been used in the clinical treatment for B-lineage lymphocytes tumors and have obtained the certain effect, but did not reach expected ideal treatment response, especially Alemtuzumab or rituximab was used alone in B-lineage lymphocytes tumor when curative effect was limited. The mechanisms for the impaired responses remained unclear. However, Distinct CD52negative (CD52-) subpopulations were detected in most cases as the result of defective glycophosphatidyl-inositol (GPI) anchoring. While ALM efficiently eradicated CD52positive (CD52+) cells in NOD/scid mice engrafted with primary human B-lineage lymphoblastic tumors, CD52-subclones escaped therapy. In the same model, RTX showed limited activity due to occurrence of CD20down modulation. Davis et al. fristly reported between CD20(+) and CD20(-) of recurrence the homology of IgvH (Complementarity determining regions, CDR) in the Rituximab treated tumor cells is100%; Chu, et al. and Alvaro-Naranjo et al. also confirmed that CD20(+) and CD20(-) malignant B lymphocytes were from the same cloning origin before and after Rituximab treatment. However, CD52(-) cells concurrently lacked the GPI anchored complement regulators CD55and CD59and showed increased susceptibility to RTX mediated complement dependent cytotoxicity in vitro. At the same time, ALM was shown to inhibit down modulation of CD20in response to RTX by depleting the trogocytic capacity of phagocytic cells. Likely because of these complementary mechanisms, combined administration of ALM and RTX induced complete responses in vivo. Based on these data, we propose a mechanistic rationale for combined application of RTX and ALM in ALL. Based on the above materials, if co-expressed CD52and CD20were found in most cases when we studied expression of CD52and CD20on cells of B-lineage lymphoblastic tumors, we could put forward the above two kinds of combination of monoclonal antibodies treatment theory in B-linage acute lymphocytic leukemia and chronic lymphocytic leukemia. This experiment is to use Flow cytometry technique (Flow cytometry, FCM) to study cell differentiation related antigen of the two malignant disease leukemia, thus further to define the CD20and CD52in two kinds of leukemia cell surface of joint expression level, which could provide the theoretical foundation for application of monoclonal antibody targeted therapy in clinical in the future.Materials and method1. Research objectives: The erperimental group:(1) De novo B-linage acute lymphocytic leukemia (B-ALL) group:Samples from45cases of de novo B-linage acute lymphocytic leukemia were collected. Patients were14-58years (median39years old) with23males and22females.(2) Chronic lymphocytic leukemia (CLL) group(n=22): Patients were55-76years (median60years old) with12males and10females.The control group(n=10):bone marrow samples from non-malignant hematologic diseses or HSCT donors.All patients enrolled for the research were who hospitalized in the Department of Hematology of the First Affliated Hopsital of Zhengzhou Univesrity from February to December,2011. The diagnosis of acute leukemia was made based on complete blood count, bone marrow morphology, cytochemistry, cytogenetic and immunophenotype, according to the criteria for diagnosis of B-linage acute lymphocytic leukemia and Chronic lymphocytic leukemia.2. Method:This experiment detects expression level of CD20and CD52of bone marrow cells by flow cytometry instrument technique (FCM). In the research bone specimens were obtained from the research objects through techniques of bone marrow. Using CD20-FITC/CD52-PE/CD45-Pc5as three colors immune parting antibody mix, the membrane antibody marker method and flow cytometric analysis software were employed to collect and analyze the data, with15cases of normal bone marrow samples from non-malignant hematologic diseses or hematopoietic stem cells transplantation (HSCT) donors as control group.3. Statistical analysis:The data were analyzed using free Software of SPSS17.0version. Quantitative data were expressed as mean±standard deviation (x±SD); Pair-sample means were analyzed by Independent-Samples t test, while multi-sample means were analyzed through variance analysis. Qualitative data were analyzed by chi-square test(including Fisher’s accurate probability test). And correlation analyses were carried out using Spearman method, with a=0.05as the significant level.Result1. Expression of CD20、CD52in De novo B-linage acute lymphocytic leukemia (B-ALL) groupThe positive rate of CD20expression in Do nove B-ALL group was40%,18patients that relapsed out of45expressed CD20. The positive rate of Do nove group was higher than that of control group, but the difference was not statistically significant(p>0.05). Compared with control group, the difference of the positive rate of CD52between Do nove B-ALL group and control group was also not statistically significant(p>0.05).2. Co-expression and Correlation of CD20and CD52in Do nove B-ALL groupThe rate of CD20+CD52+in Do nove B-ALL group was37.8%, the difference was statistically significant compared with control group(p<0.05). There was a positive correlation between CD20and CD52expression in Do nove B-ALL group(p <0.001,r=0.673).3. Expression of CD20、CD52in Chronic lymphocytic leukemia (CLL) groupThe positive rate of CD20expression in CLL group was77.3%,17patients that relapsed out of22expressed CD20. That of CD52expression in Do nove CLL group was86.4%and19patients that relapsed out of22expressed CD52. Compared with control group, the difference of the positive rate of CD2、CD52in Do nove CLL group were both statistically significant (p<0.05).4. Co-expression and Correlation of CD20and CD52in CLL groupThe rate of CD20+CD52+in CLL group was68.2%, the difference was statistically significant compared with control group(p<0.05). There was a positive correlation between CD20and CD52expression in CLL group(p<0.05, r=0.711).5. There was significant difference between Do nove B-ALL group and CLL group in for CD20+CD52+(p=0.019)6. There was not significant relationship between CD20expression and clinical features(p>0.05).Conclusions1.The expression of CD20and CD52in CLL group is significantly higher than the control group. CD20and CD52can be used as the therapeutic targets of CLL. 2. The level of co-expression of CD20and CD52in B-lineage lymphocytes tumor is higher.3.The level of co-expression of CD20and CD52in different B-lineage lymphocytes tumors is different.4. The combined application of the two kinds of targeted antibodies (RTX and ALM) in the treatment of B-lineage lymphocytes tumors including B-ALL and CLL could be considered.5.The positiveness of CD20is the only basis for rituximab application.