Characterization of the Proteomic and Genomic Profiles of Chronic Lymphocytic Leukemia Patients with Distinct Clinical Prognosis According to the Mutational Status of the IgVH and BCL6 and Expression Level of CD38 and ZAP70.

Abstract

Introduction: In recent years several molecular prognostic factors have been identified in Chronic Lymphocytic Leukemia B (B-CLL). These include mutations in the variable region of the immunoglobulin genes (IgVH), somatic mutations in the BCL6 gene (Leukemia ( 2004) 18, 743–746) and the expression level of CD38 and ZAP70. However its biological significance is not clear. In order to identify novel molecular markers with prognostic and therapeutic value we have analyzed the proteomic and genomic profile of 40 B-CLL patients (Binet stage A).Material and methods: 100 μg of total PBMC proteins were used for IEF followed by 2D electrophoresis. Image analysis of scanned gels was used to identify statistically significant differentially expressed proteins. Image acquisition and analysis were performed using the Image Master Platinum software. Selected spots were subjected to automatic digestion and the proteins were identified by MALDI-TOF (Voyager DE-Pro, Applied Biosystems) peptide mass fingerprint using the Protein Prospector software. To confirm the initial identification amino acid sequencing of selected peptide ions was carried out by collision-induced dissociation (CID) with a nESI-QTRAP mass spectrometer from Applied Biosystems. 100 ng of total RNA was used to analyze the expression profile by cDNA microarrays (Whole Human Genome U133 Plus 2.0 Array from Affymetrix). Differential gene expression has been analyzed using the corresponding module (pomelo) from the GEPAS web tools (http://www.gepas.org ).Results and Discussion: We found 126 proteins and 25 genes exhibiting differential expression (p<0.05, Student t-test, FDR-adjusted for multiple testing contrasts). We found 34 proteins and 18 genes with different expression according to the mutational status of IgVH. We also classified patients according to mutations in the BCL6 gene, which we previously showed clinical relevance (Leukemia ( 2004) 18, 743–746), and found 36 proteins and 2 genes with differential expression. Next, we compared samples attending to the mutational status of both genes, results are summarized in the following table:Compared groups attending to the mutational status of IgVH and BCL6 genestargetsIgVH wt/BCL6wt IgVH mut/BCL6 mut16 proteins / 10 genesIgVH mut/BCL6 wt28 proteins / 9 genesOf particular interest is the comparision between BCL6 status in IgVH mutated patients where we found 27 proteins with differential expression but no difference in the gene expression profile. This result suggest that the bad prognostic associated to BCL6 mutation may be due solely to postransductional modification of proteins.CD38 and ZAP70 expression level have also been associated with bad clinical prognosis, however, we found no significant difference in the gene expression profile according to CD38 or ZAP70 expression level.We are currently assessing a wider number of patients in order to obtain stronger conclusions. The study of these proteins and genes may lead to a better understanding of the different clinical behaviour of these B-CLL forms.