ALBERT

Description: After the report of two cases of leukaemia caused by insertional mutagenesis of a retroviral vector in children with SCID, it became clear that safety issues of therapeutic gene transfer must be addressed more thoroughly. We analysed whether gene transfer into mature T cells and haematopoietic stem cells bear the same risk of generating T cell leukaemia through activation of specific T cell oncogenes, such as LMO2, TCL1 and ΔTrkA. To address this issue, we used the Rag-1 mouse model, which allows long term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ΔTrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphomas/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes, although none of the control- vector- transduced mice developed lymphoma/leukaemia. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell transplanted animals showed no signs of leukaemia development so far. However, after several attempts, one immortalized T cell progenitor clone could be generated after transduction with LMO2. Our results so far indicate that mature T cells are less susceptible to transformation by known T cell proto-oncogenes, but the studies are still ongoing.

Description: Mantle cell lymphoma (MCL) is a prime example of a well-defined entity based on morphology, phenotype, genetics and also clinical features. Although, most patients have an adverse clinical course, some have a better survival than others. The most consistently reported adverse histopathological prognostic parameter is a high mitotic rate. Recently, it has been shown that hypermutation in the immunoglobulin heavy chain gene occurs in a subset of mantle cell lymphomas. It is, however, unclear, how hypermutation needs to be defined and whether the mutational status is stable over time within a given case. Also it is not clearwhether hypermutation might be influenced by therapy and how it is related to other relevant biological features of MCL, like genetic imbalances, breakpoint of the cyclin D1 gene, and proliferative and apoptotic rate. In the present study we analyzed a series of typical MCL with respect to mutational status, and compared the results with clinicopathological and genetic data to determine whether the presence of mutation does indicate a sub entity with clinical or pathological relevance. For this study 28 specimens from 24 patients were selected and stained for cyclin D1 and CD5 and were reviewed by four experienced pathologists at a multiheaded microscope. Morphological features were assessed, mitotic figures were counted, clonal IGH-VJ gene rearrangements of the MCL were identified according to the Biomed-2-protocol. All, but one case, were clonal by PCR. The negative case was excluded from further molecular studies. Mutation in the VH gene segment was absent in 7 cases, another 6 cases carried 0.5% mutation frequencies; in 10 cases more than 1.5% mutation frequencies were present, in 6 of these more than 2%. We found a preferential usage of VH3-21 (26%) and VH4-34 (17%) in tumor cells of both, mutated and unmutated cases. Of two cases from our series in which sequential biopsies were available tumor cells harbored mutations in the VH genes (1.5% mutation frequency and 2.2% mutation frequency, respectively), but there was no change of mutation status over time, the duration being as long as 7 years in one case. In our study there was a statistically significant difference between MCL with a Ki-67-positivity =35% in regard to a longer survival time in the former whereas mutation status of both groups did not correlate with Ki-67-positivity. Further, no significant correlations were found between mutation status and the other morphologic and genetic features analyzed. In conclusion, our results provide additional evidence that mutation status does not permit definition of sub entities in MCL that are associated invariably with a certain prognosis. Mutation status in MCL is better interpreted as a feature within the spectrum of disease that seems to have little clinical or pathological relevance. Margit Schraders and Sabine Oeschker contributed equally to this work.