Screening And Identification Of Leukemia Associated Antigens By Serological Proteome Analysis And Pilot Study On Its Clinical Utility And Relevance To Disease

Serological proteome analysis (SEPRA) , which uses a combination of SEREX(serological analysis of recombinant cDNA expression libraries), proteomics and mass spectrometry to analyze and characterize proteins, provides major opportunities for the screening and identification of tumor associated antigens. The proteomic-based approach to uncover tumor-associated antigens is composed by following steps: (1) separate several thousands of individual celluar proteins from tumor tissue or tumor cell lines; (2) separated proteins are transferred onto membranes. Sera from cancer patients are used as primary antibody and sera from health volunteer are used as control for western blot analysis and are screened individually for antibodies that react against separated proteins; (3) Protein spots that specifically react with sera from cancer patients are extracted from the gel and identified by mass spectrometric analysis. This proteomic-based approach has several advantages. First, it can be used for individual screening of autoantibodies in a large number of patient sera. Second, the antigenic proteins identified by this approach have a potential clinical utility and may serve as novel cancer markers in screening, diagnosis or in prognosis. Finally, it may be powerful for the detection of autoantibodies against post-translational modifications of specific targets. This approach has been applied to different types of cancer and several autoantigens have been identified.In our study, SEPRA technology was applied to screen and identify leukemia-associated antigens (LAAs) in chronic myeloid leukemia (CML). We choose CML as study object is that no ideal antigen for immunotherapies in CML had been defined. The Wilms' tumor antigen 1 (WT-1) and proteinase 3 had been discussed as potential candidates for immunotherapy of CML. However, WT-1 and proteinase 3 are not only overexpressed in most patients with CML and AML, but also expressed in several normal tissues, normal granulocytes and progenitor cells of normal hematopoesis. Therefore, immunotherapies targeting WT-1 or proteinase 3 might elicit cytotoxic effects directed against normalhematopoesis. Thus, screening and identification of novel leukemia associated antigens may be of vital importance in antigen-based immunotherapy.In this study, five leukemia-associated antigens were screened and identified by SEPRA technology. These proteins include: (1) enzymes associated with tumors: alpha enolase and aldolase A; 2) chaperone protein: HSP70 protein 8; (3) structural proteins: beta-tubulin and tropomyosin isoforms. Most importantly, autoantibodies against alpha enolase had a high frequent (33%)in srea of patients with CML .The previous studies have found that alpha enolase is associated with tumor and the presence of autobodies against alpha enolase in sera of patients with CML may be related to differential expression levels and aberrant localization of the target proteins in tumors. Then the analysis of the expression levels of the antigenic proteins in cancer may provide explanation for antoantigenictity and might also reveal proteins with functional importance in cancer.Therefore, the expression level of alpha enolase in patients with CML was analyzed to study the clinical utility and relevance of alpha enoalse to CML. The results of RT-PCR revealed that compared with the expression of alpha enolase in the PBMNs of normal donors, the expression of alpha enolase in patients with CML in blast crisis was significant lower (P<0.05) and this expression was even lost in some patients(2/8), while in all patients in chronic and accelerated phase, the expression of alpha enolase had no significant difference with the normal. The results of immunohistochemical (IHC) analysis indicated that compared with the expression of alpha enolase in marrow cells of normal donors, the expression of alpha enolase in all (16/16) patients with CML in chronic and accelerated phage was light higher or even moderate higher, while in all (5/5) patients with CML in blast crisis, the expression of alpha enolse was extreme lower and showed a decrease of cytoplasmic and a specific loss of nuclear immunoreactivity. These results predict the expression of alpha enolase and the loss of nuclear localization is associated with the progress of disease. At the same time, we analyzed the presence of alpha enolase in the sera from normal donors and patients with CML by western blot. A band corresponding to the molecular mass of alpha enolase was observed with high intensity in sera from 40% (12 of 30) patients with CML. None of the normal sera exhibited a comparable intensity band, although a band at the limit of detect was present in some sera. These results suggest that alpha enolase is circulating at low levels in sera from some healthy individuals and athigher levels in sera from patients with CML.How does this protein exert its functions in the progression of CML? The down regulated of this protein may affect the proliferation of the leukemia and change the cell circle or even its anti-apoptosis ability? All of these need further investigation. Thus, RNA interference technology was applied to study the potential function of alpha enolase in the progression of CML. The recombinant retroviral vectors expressing siRNA to aim directly at alpha enoalse were designed and transfected to K562 cells. The results of RT-PCR revealed that transfected vector composing siRNA sequence knocked down the expression alpha enolase, but more studies are needed to investigate the effect of the silencing of alpha enolase on the functions of leukemia cells.