Abstract
Introduction: Chronic lymphocytic leukemia B (CLL-B) is the most frequent chronic lymphoproliferative disorder in western countries. The majority of patients are diagnosed in incipient Binet stage A. Some prognostic factors have been identified, as mutations in the genes coding for immunoglobulin variable regions (IgVH). Complementary somatic mutations in the BCL6 gene have been observed in 25% of CLL-B patients, although its clinical relevance remains unclear.
Objective: To identify molecular markers of different patient groups of lymphoproliferative disorder through analysis of their proteomic profiles.
Material and Methods: 15 samples of peripheral blood lymphocytes B from Binet stage A CLL-B patients were analyzed. Among them, 5 carried mutations in IgVH and BCL6 (M) genes, 5 had no mutations (IgVH, BCL6 (U)), and 5 with mutations in IgVH(M) but without mutations in BCL6(U). 100 μg of total proteins were isoelectrofocussed using DryStrips (13 cm, pI 4–7), followed by 15% SDS-PAGE, and Coomasie Blue staining. Image acquisition and analysis were performed with the Image Master Platinum software (Amersham Biosciences). Spots showing differential expression were subjected to automatic tryptic digestion. Proteins were identified by MALDI-TOF mass fingerprinting using the Protein Prospector software. Selected peptide ions were sequenced by collision-induced dissociation (CID) using an ESI-QTRAP instrument.
Results: The mean number of spots per gel was 214, which showed a mean percentage matching among the three patient groups of 77%.
Statistics of the 2D-IEF/SDS-PAGE separations of the total lymphocyte B protein from patients with different mutational status of their IgVH and BCL6 genes.
| . | IgVH, BCL6 (M) vs. IgVH(M), BCL6(U) . | IgVH (M), BCL6 (U) vs. IgVH, BCL6(U) . | IgVH, BCL6 (M) vs. IgVH, BCL6(U) . |
|---|---|---|---|
| (*) Test t of student. | |||
| Spot media per gel | 228/180 | 180/235 | 228/235 |
| Matching percentage | 80.2% | 79.3% | 70.5% |
| Number of spots with p<0.05(*) | 6 | 2 | 2 |
| . | IgVH, BCL6 (M) vs. IgVH(M), BCL6(U) . | IgVH (M), BCL6 (U) vs. IgVH, BCL6(U) . | IgVH, BCL6 (M) vs. IgVH, BCL6(U) . |
|---|---|---|---|
| (*) Test t of student. | |||
| Spot media per gel | 228/180 | 180/235 | 228/235 |
| Matching percentage | 80.2% | 79.3% | 70.5% |
| Number of spots with p<0.05(*) | 6 | 2 | 2 |
MALDI-TOF mass fingerprint and/or CID MS/MS identification of spots with differential expresion among the 3 patient groups, and their level of expression in each mutational situation (Center value of volume % in each of the gels).
| Protein . | IgVH, BCL6(M) . | IgVH(M), BCL6(U) . | IgVH, BCL6(U) . |
|---|---|---|---|
| ns: no statistical significance | |||
| 14-3-3 Protein Z (*) | 0.82 | 0.30 | ns |
| Heterochromatin-specific nonhistone protein (*) | 0.29 | 0.02 | ns |
| NADH-Ubiquinone reductase (*) | 0.12 | 0.02 | ns |
| Tubulin α (*) | 0.54 | 1.28 | ns |
| Vimentin (*) | 0.27 | 0.98 | ns |
| Similar Beta Tubulin (*) | 0.40 | 1.85 | ns |
| Cytoskeleton associated protein 1 (**) | ns | 0.03 | 0.26 |
| Endoplasmatic-reticulum-lumenal protein 28 (**) | ns | 0.55 | 0.21 |
| Chaperonin TCP-1 | 0.005 | 0.05 | 0.18 |
| Nucleophosmin | 0.17 | 1.51 | 1.22 |
| Protein . | IgVH, BCL6(M) . | IgVH(M), BCL6(U) . | IgVH, BCL6(U) . |
|---|---|---|---|
| ns: no statistical significance | |||
| 14-3-3 Protein Z (*) | 0.82 | 0.30 | ns |
| Heterochromatin-specific nonhistone protein (*) | 0.29 | 0.02 | ns |
| NADH-Ubiquinone reductase (*) | 0.12 | 0.02 | ns |
| Tubulin α (*) | 0.54 | 1.28 | ns |
| Vimentin (*) | 0.27 | 0.98 | ns |
| Similar Beta Tubulin (*) | 0.40 | 1.85 | ns |
| Cytoskeleton associated protein 1 (**) | ns | 0.03 | 0.26 |
| Endoplasmatic-reticulum-lumenal protein 28 (**) | ns | 0.55 | 0.21 |
| Chaperonin TCP-1 | 0.005 | 0.05 | 0.18 |
| Nucleophosmin | 0.17 | 1.51 | 1.22 |
Conclusions: Proteomic profiling of lymphocyte proteins of the three patient groups revealed 10 proteins with differential expression, which can be divided into the following categories:
The expression of six proteins (*), changed with the mutational status of BCL6 gene.
The expression of two proteins (**), correlated with the mutational status of the IgVH gene.
Nucleophosmin exhibited increased expression in patients with unmutated BCL6 gene.
Chaperonin TCP-1 expression was higher in patients with unmutated IgVH gene.
Work supported by Redes Tematicas de Grupo Grant G03/179 of the Instituto de Salud Carlos III, Madrid, Spain
Author notes
Corresponding author
