4.8 Investigation of Notch1 Mutation in Chinese Patients with B-Cell Chronic Lymphocytic Leukemia

Abstract

ther a stable or progressive disease course. This heterogeneity in clinical behaviour makes it difficult to accurately distinguish potentially aggressive from indolent disease at diagnosis. Disease will progress in approximately 20% to 30% of patients with CLL; these patients may benefit from early treatment. This study was conducted to elucidate novel surface and intracellular proteins that are differentially expressed between stable or progressive CLL disease. Methods: Surface antigen profiling was performed on Ficoll-purified B-cell fractions from 100 patients with CLL of known clinical status. Profiling was carried out using a new array targeting a total of 195 surface antigens as previously documented. Digital images for the dot patterns of the cells captured on the array slides were recorded and analysed for binding intensities using DotScan software. Isobaric tags for relative and absolute quantitation labelling (i-TRAQ), in combination with multidimensional liquid chromatography-mass spectrometry (MS)/MS analysis, was used to identify differentially abundant proteins in CLL patient samples grouped according to clinical status. Whole-cell protein extracts were digested with trypsin and labelled with i-TRAQ tags. The resulting peptides were then fractionated using strong cation exchange, followed by reverse phase chromatography. Each fraction was analysed by tandem mass spectrometry using a QSTAR Elite mass spectrometer. Protein identification was performed using ProteinPilot 3.0. Results: Surface antigen profiling of the 100 patient samples using DotScan showed a definite clustering of samples from patients with stable or progressive disease. Progressive CLL samples showed significant upregulation of CD38, CD52, CD62L, CD84, CD184, CD196 and HLA-G, compared with clinically stable patient samples. Approximately 1000 proteins were identified by analysis of the CLL cell proteome using i-TRAQ. Of these, 41 were found to be significantly differentially expressed between cases defined as having stable or progressive disease. The proteins are largely associated with cell death, cellular assembly, and organisation. In conclusion, this study has identified surface antigen and intracellular protein expression profiles that are indicative of progressive disease. Further work to validate these proteins for use as novel prognostic biomarkers is under way.