Enhanced Apoptosis and Reduced Angiogenesis in DLBCL.

Introduction: Angiogenesis is a necessary step in tumors progression correlating and beeing indicative of an unfavourable prognosis. Preliminary reports indicated that microvessel quantification may be useful in predicting tumors outcome. There is scant data regarding caspase-3 and vascular endothelial growth factor(VEGF) as well as the appraisal of vessel density in diffuse large B-cell lymphomas (DLBCL). These aforementioned variables are of significance in the surveillance and prognostic assessment of patients diagnosed and treated for DLBCL. The purpose of the abstract is to report our findings concerning the different biological characteristics between follicular lymphoma (FL) and DLBCL regarding angiogenesis and apoptosis.

MATERIALS AND METHODS: Nodal biopsies from 40 patients suffered from DLBCL were analysed. Control group included nodal biopsies from FL(5 patients) and benign nodal hyperplasia (5 patients). Microvessel quantification was performed by immunohistochemical staining, using monoclonal antibodies against factor VIII related antigen (F8RA) and against CD34. The assessment of caspase 3 level of expression was done by using monoclonal specific antibody. We also assessed the level of expression of VEGF and Ki-67 (by using specific monoclonal antibodies, qualitatively, was done by two separate trained pathologists). The evaluation of the microvessel density (MVD) and the caspase 3 was done by using the Image analyzer" Provision".

Results: All the biopsies that were included had proliferative index between 40–60%. VEGF was expressed in all DLBCL, follicular lymphoma and benign nodal hyperplasia specimens, mainly extracellulary. There was no qualitative difference in the level of VEGF expression in the various biopsies. By using the Provision analysis of the MVD, we demonstrated low level of angiogenesis in nodal DLBCL as opposed to the marked angiogenesis shown in FL and benign nodal hyperplasia. The average MVD in DLBCL specimens was 17±5, in the tumor margins 48±10, FL 55 ±10 and benign nodal hyperplasia 63±5. The morphology of the blood vessels showed diversity: while in the DLBCL the blood vessels were small and almost undeveloped, in the DLBCL tumor margins, in FL and benign nodal hyperplasia the blood vessels are fully developed, large and numerous. We assessed the apoptotic level by using anti caspase 3 antibody. Remarkable difference was demonstrated: the positive caspase-3 expressing DLBCL cells was 60±12, in the FL cells it was 6± 3, and in the benign nodal hyperplasia cells it was 2±2.

Discussion: In this preliminary study different biological characteristics were observed in DLBCL as opposed to FL: as in DLBCL there is low angiogenesis and high apoptotic activity, in FL and in benign nodal hyperplasia there are high angiogenic activity and low apoptotic rate. We postulate that the high apoptotic rate in DLBCL may be linked to the low angiogenesis: probably the high rate of the cellular turnover in the tumor does not permit blood vessels formation. On the other hand, the low angiogenic activity in the tumor, may, not allow sufficient nutrients and cytokins supply to the tumor cells and thereby cause to the high apoptotic rate of the tumor cells. Anti angiogenic therapy has been introduced in the last years in hematologic malignancies. Taking into consideration the above preliminary results, it is uncertain that patients suffered from DLBCL will benefit from the anti-angiogenic compounds. Further studies and clinical trials are warranted.