Background of the application:
Gastric cancer is the second leading cause of cancer related mortality worldwide end clinical prognosis is very poor. Epstein-Barr virus (EBV) is clonally present in all tumour cells of approximately 10% of gastric adenocarcinomas worldwide, independent of geographic regions and race. Although Helicobacter pylori infections are generally accepted to play a major role in the early onset of gastric cancers, the pathogenic role of EBV in the development of EBV carrying gastric cancers is undefined. Our preliminary results showed that the EBV expression pattern in EBV carrying gastric cancers is different from that observed in other EBV associated lymphomas and nasopharyngeal carcinomas (NPCs). EBNA1, LMP2A, EBERs end BARF0 specific transcripts have been found, whereas the viral oncogene LMP1 was not expressed. We showed that BARF1 RNA was specifically expressed in gastric cancers end NPCS, and not in lymphomas using a newly developed NASBA essay. Since LMP1 is not expressed in gastric cancers end recently in vitro immortalizing capacities of BARF1 in primate primary epithelial celis ware demonstrated, BARF1 appears to be an important gene in the pathogenesis of EBV carrying gastric cancers. Moreover, it might be an important target for immunotherapy. At present there are no data available about the host immune response against BARF1. In this project we want to investigate the expression of BARF1 at the single cell level in gastric carcinomas, and identify the immunogenic role of BARF1 in order to address its potential role as target for immunotherapy.
Aims of investigation:
1. To study the expression of BARF1 transcripts end its encoded protein in EBV carrying gastric cancers at the single cell level.
2. To investigate host cellular and humoral immune responses to the BARF1 protein in EBV positive gastric carcinoma patients and controls.
Plan of investigation:
1. Gastric cancer specimens will be analyzed for EBV using EBER RNA in situ hybridization (RISH). EBV positive gastric cancers will be subjected to transcript analyses of BARF1 using RT-PCR and novel NASBA essays. RISH will be used to identify BARF1 transcripts at the single cell level of EBV positive gastric cancer cases.
2. Monoclonal antibodies will be generated directed against bacterial fusion proteins and specific peptides of BARF1. Moabs will be analyzed for their specificity and evaluated on EBV carrying gastric cancers to study its expression at the protein level.
3. Humoral end cellular immune responses will be analyzed by a number of approaches employing purified recombinant BARF1 produced in E. coli, the Baculovirus expression system or vaccinia virus. Host immune responses will be analyzed in EBV positive gastric cancer patients and controls of which tumour tissues, sera end lymphocytes have already/are being collected.
Possible results / relevance for cancer research:
The results of this project will lead to a novel specific viral target, BARF1, for future immunotherapy studies in EBV carrying gastric carcinomas. This project fulfils all the requirements needed to set up vaccination studies with BARF1 in EBV carrying gastric carcinomas. |
