| Popis: |
Chronic Lymphocytic Leukaemia (CLL) is the most common adult Leukaemia in the UK, Western Europe and America. It is a malignancy of naive B-cells. The clinical course of patients with CLL is heterogeneous; some patients survive for years without treatment, others die of a chemotherapy resistant disease within two years of presentation. Genomic studies have found little variation in patients showing differing prognosis, suggesting that it is the same disease but with varying outcomes. At present there is no cost effective, reliable and routine clinical test which can distinguish patient prognosis and a "watch and wait" strategy is currently in clinical use. Studies have shown that patients who express mutated IgVH genes on the B-cell receptor (BCR) have a good prognosis, whereas patients who express unmutated IgVH genes have a poor prognosis. However IgVH gene mutational status is an expensive and time consuming test and would not be practical for routine clinical practice. If the B-cell has not been sensitised to a specific antigen (i.e. unmutated IgVH genes on BCR) it is hyper-responsive to stimulation through the BCR by antigen. Stimulation of the BCR may prevent apoptosis of malignant cells; therefore a hyper-responsive BCR is linked to poor prognosis. By artificially stimulating cells and using proteomic techniques we have investigated signaling pathways activated by the BCR to gain a greater understanding of the anti-apoptotic nature of the malignant B-cells and to find potential prognostic biomarkers related to a hyper-active BCR. Protein was extracted from stimulated and unstimulated cells from CLL patients categorised as having a poor prognosis (unmutated IgVH genes and hyper-responsive BCR). The extracts were separated using conventional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The gels were stained with coomassie blue total protein stain and analysed with statistical software. Proteins with a two-fold (p less than 0.05) change in expression between stimulated and unstimulated samples were excised from the gels and analysed by matrix assisted laser desorption/ionisation with time of flight mass spectrometry (MALDI-TOF-MS). Antibody microarrays were used as a complimentary method to 2D- PAGE and immunoblotting was applied as a verification technique. Changes in protein expression were detected in response to prolonged BCR stimulation. Many of the proteins have had no previous connection with BCR signaling or leukaemia and give a greater insight to the mechanisms of the BCR. Targets found include ones which are associated with the activation of anaplastic lymphoma kinase (ALK), the plasma kallikrein-kinin system (KKS), the AKT-1 pathway, the MAPK pathways, the adenylate kinase system and involvement in the CD40-dependant activation of B-CLL cells. One of the protein targets found (kininogen) was increased by over two-fold in three independent clinical samples after sustained BCR stimulation. If we understand more about the BCR signaling pathways then we may be able to identify potential prognostic biomarkers and novel targets for therapeutic intervention that may inhibit survival of the malignant B-cells. |
