Present clinical assays that detect tumor amplification or overexpression of HER2 cannot discriminate between HER2D16 and wild type HER2 expression. Cells were lysed in Laemmli sample buffer, and samples were separated by 1368-1644 sodium dodecyl sulfate polyacrylamide gel electrophoresis Patient data for Cediranib structure the lymph node samples used in this study can be found in Smit et al. ND indicates maybe not determined. Percent of cells positive for CD5 and CD19 surface expression. mutated IgVH gene indicates over 25 variations compared with germline sequence. p53 functional status was assessed via radiation induced RNA expression of p53 target genes Puma and Bax, or by p53 and p21 protein up-regulation via Western blot, as decribed in Mackus et al16 and Pettitt et al. 25 Patient 25 had a so called type A dysfunction. ?As based on FISH. Probes for 11q22. 3, 13q14, and 17p13 were received from Abbott Vysis. Samples with increased than10% aberrant indicators were considered abnormal. 5142 HALLAERT et al BLOOD, 15 DECEMBER Neuroblastoma 2008 VOLUME 112, NUMBER 13 for ERK. Cells were irradiated and after overnight incubation examined for the expression of p21 and p53 by Western blot analysis as described before, to screen for p53 functionality. 25 Blots were probed with polyclonal anti Mcl 1, monoclonal anti Noxa, monoclonal anti Bim, antiserum to actin, polyclonal anti Bcl XL, polyclonal anti Bcl 2, polyclonal anti phospho Erk, and polyclonal anti Erk. Polyclonal antibodies against A1/Bfl 1 and Bid were a kind gift of Prof Doctor T. Borst. In cell lines and vitro CD40 activation BCR Abl positive K562 cells and NIH3T3 fibroblasts were cultured in IMDM as described above for CLL cells. CD40 ligand was expressed on NIH3T3 fibroblasts, stably transfected with a plasmid encoding human CD40L. Fibroblasts were irradiated and plated in culture treated 6 well plates. CLL heat shock protein inhibitor cells were thawed and 5 106 cells per well were added to the fibroblasts in 3 mL IMDM containing 10% FCS and incubated for 48 hours at 37 C. To check the effect of c Abl kinase inhibitors imatinib and dasatinib, and the effect of Erk inhibitor PD 58 059, CLL cells were pretreated with 80 Mimatinib or dasatinib, or 50 M PD 58 059 for half an hour. In the case of dasatinib, also other sessions and concentrations were used, where CLL cells were first cocultured for 48 hours with CD40L showing or get a grip on 3T3 fibroblasts, detached and washed, and subsequently incubated in medium for an additional 48 hours in the presence of varying dasatinib concentrations, followed closely by testing sensitivity to cytotoxic drugs, as described under Analysis of apoptosis,Western mark, and antibodies. In vitro activation via CD40 renders CLL cells resistant to fludarabine and induces expression of various antiapoptotic proteins such as Bcl Xl and A1/Bfl 1 via de novo transcription.