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Molecular characterization and identification of biological risk factors in mantle cell lymphoma

Deliverables

The co-workers of workpackage 4 (Universities of Nymegen, Kiel and Copenhagen) have developed methods to study and analyse the biological function of mantle cell lymphoma cells and its value in response to therapy. Thus, we established a significant role of CD40 ligand for long lived primary tumor cultures. We confirmed in larger scale the importance, not only of genetic changes, but also of unbalances in the apoptotic machinery (including many members of the bcl-2 family, caspases and IAPs) for both the pathogenesis and the resistance to therapy of MCL cells. We tested up-front new therapeutical agents (CD40L, TRAIL, NFkappaB-inhibitors) on primary cell cultures and developed methods to test their applicability and potential efficiency in the individual patients suffering from the otherwise incurable MCL lymphoma subtype.
The investigators focused on two aspects of mantle cell lymphoma: the translocation t(11;14) with deregulation of the cyclinD1 gene as a primary genetic abnormality and the occurrence of somatic hypermutations (SHM) in the immunoglobulin (Ig) genes. - The t(11;14) was studied by polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) assays in CD34+ bone marrow progenitor cells, the mature tumor cells and a case of transformed mantle cell lymphoma. FISH methods included FISH on paraffin tissue sections and Fiction with double staining for CD34. Cyclin D1 expression was measured by quantitative RT-PCR and immunohistochemistry and related to the molecular structure of the translocation. - Ig gene somatic hypermutations (SHM) were analysed by DNA sequencing and were related to class switch recombination (CSR) attempts and the level of Activation Induced Deaminase (AID), a key protein in the induction of SHM and Ig class switch recombination (CSR). We conclude that purified CD34+ bone marrow stem cells do not contain the t(11;14) after vigorous depletion of CD20+ cells. This supports the clinical use of anti CD20 antibodies in purging of autologous stem cell. The distribution of BCL1 breakpoints in MCL was slightly different from reported in literature. No correlation between cyclin D1 expression and the position of the breakpoints was observed. In one case of transformed MCL we identified an intact t(11;14) but an additional downstream MYC breakpoint at the functional IgH allele. In one quarter of the MCL cases we found evidence for SHM, however, without any correlation with AID expression or signs of CSR.
The main objective to characterize secondary genetic alterations on three different independent levels: DNA, RNA and protein, has been successfully carried out. Combination of these different approaches and the exchange of material and results between the network partners have contributed extensively to identifying the critical master genes in the malignant transformation and progression of MCL. One group has used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to analyse total cellular protein extracts to identify qualitatively differentially expressed proteins before and after treatment with 5-aza-dC. Using a high-density cDNA microarray, 128 primary tumours including 56 MCL cases, small lymphocytic lymphoma (31) and marginal zone lymphoma (37) have been analysed. Several clusters of genes involved in distinct biological functions such as cell-cycle control, cell differentiation, and intracellular signalling, significantly discriminated the three histological subtypes. Different groups succeeded in substantially narrowing down the critically deleted region in 1p and 13q. Although up to now the tumour suppressor genes in 1p and 13q have not been identified, a number of candidate genes have been studied for down regulation and mutations. Our comprehensive approach has given important new insights into genes and proteins that trigger the malignant transformation and progression in MCL.
The different partners of the project (Universities of Barcelona, Munich, and Würzburg) have developed a comprehensive analysis of cell cycle regulators in the pathogenesis of MCL and its potential values in the management of the patients. Thus, we have demonstrated that deletions of INK4/ARF locus and p53 mutations are the most common alterations in highly proliferative MCL. p15INK4b is highly hypermethylated but p16INK4a methylation is rare. BMI-1, an upstream regulator of INK4a/ARF locus, is amplified and over expressed in MCL with no alterations in this locus. Amplification and over expression of CDK4 seems to be an alternative mechanism to INK4a/ARF locus inactivation but occurs in cases with p53 mutations. Tetraploidization is a frequent phenomenon in MCL but rare in other lymphomas. Anomalies in the number and conformation of centromeres have been demonstrated in MCL associated with tetraploid clones. Inactivation of ATM and CHK2 genes seem to be associated with increasing number of chromosomal imbalances in these tumors. These results indicate that genetic alterations in all these elements are probably the workforce driving the aggressive behaviour of these tumors, provide a new framework to establish more accurate prognostic parameters, and define new potential therapeutic targets to improve the outcome of the patients.

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