Pathogenesis and Immunpathology of LCAL

The general aim of this proposal is to elucidate pathogenetic mechanisms in anaplastic large cell lymphomas (ALCL) and the potential differences of the known subtypes. ALCL were first described as a separate lymphoma entity in 1985. They are now a well-recognized entity accounting for more than 2% of all adult NHL and about 13% of paediatric NHL. 3 entities of ALCL have been identified: primary systemic anaplastic lymphoma kinase (ALK)-positive ALCL, primary systemic ALK-negative ALCL and primary cutaneous ALCL. To analyze RNA expression patterns in lymphoma derived cell lines, we generated cDNA microarrays using clones form the IMAGE consortium. Using this method some apparently cell line-specific marker genes could be identified. In addition, the effects of CD30 stimulation were studied in Hodgkin`s lymphoma derived cell lines. In the proposed project cell lines derived from different forms of ALCL, other T -NHL and (FACS flow sorted) normal CD4+ and CD8+ cells shall be compared to identify candidate genes which differentiate cell types. In addition, subtractive hybridization will be used for the identification of additional differentially expressed genes. After validation using immunohistochemistry, quantitative RT-PCR or Northern blotting, this information will provide the basis for the investigation of the following questions. Which genes are involved in the pathogenesis of different types of ALCL ? From which normal cells do ALCL cells arise? Can their immunological function be deduced from gene expression analysis? W hat mechanisms are involved in the deregulation of growth and apoptosis in ALCL ? Are there differences in the response to CD30 stimulation ? W hat is the molecular basis of dissemination of ALCL? Is there an elicitation of an immune response? W hat are the molecular targets of ALK overexpression ? The information gained in the project might help to understand the pathology of ALCL. It might also further the understanding of the immunological function of normal cellular counterparts of ALCL cells. In addition, it will aid in the diagnosis of "borderline cases", which is of great importance since treatment strategies and prognosis are very different. The understanding of basic pathophysiological and immunological functions might also facilitate the development of new treatment strategies for non-Hodgkin`s lymphoma.

Anaplastic large cell lymphoma (ALCL) comprises 30% of all childhood lymphomas and 3% of adult lymphomas. It is characterized by a very rapid growth. In approximately 50% of this hematologic neoplasia, the translocation (2;5)(p23;35) resulting in the fusion gene Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK) can be detected. This translocation, however, is missing in a subgroup of this lymphoma which shows an even more aggressive course. To investigate the consequences of the activation of this kinase and, thereby, the differences between the two subgroups, we analyzed the mRNA expression pattern in various tissue culture systems using "suppression subtractive hybridization (SSH)" and "cDNA-arrays". In a fibroblast system, the active form of the kinase elicits significant changes in the expression of 38 genes. ALK-positive lymphoma cell lines differ from their ALK- negative counterparts in the expression of 102 genes. The majority of these genes were isolated from gene libraries constructed in our lab and play a role in the cell cycle or in apoptosis. A subset of these genes points to an activation of AP-1 transcription factors. This NPM-ALK dependent increased AP-1 DNA binding activity was verified using "electrophoretic mobility shift" assays. "Supershift" assays identified JUNB as the transcription factor to be strongest involved. Therefore, we could demonstrate that NPM-ALK induces AP-1 activation via JUNB. The exact pathway conferring this activation was analysed in various inhibitor studies. We could show that the amount of JUNB protein is regulated on transcriptional level via the RAF-MEK-ERK pathway. In addition, a translational regulation via the AKT pathway was also established. This activation is the basis for the rapid growth of tumor cells and can be reversed using the immunosuppressant rapamycin. In summary, our data point to a thus far unknown regulatory mechanism in the activation of AP-1. Its modulation might provide a novel strategy in the treatment of malignant lymphoma.