ALBERT

Description: In B-cell chronic lymphocytic leukemia (B-CLL) the mutation status of the immunoglobulin variable heavy chain gene (VH) is known as a prognostic factor. We have investigated if specific VH-gene usage is of additional prognostic importance regarding survival. Peripheral blood samples from 147 B-CLL patients were analysed for VH usage. Recombinations occurring in at least 5% of cases were studied in depth. The most frequently used VH-gene segments were VH1-69 (10.9%), VH3-7 (7.5%), VH3-30 (6.8%), VH4-34 (6.8%), VH3-21 (6.1%), VH3-23 (6.1%), and VH3-33 (5.4%). The VH gene usage was compared with mutation status, cytogenetic abnormalities and survival. Comparison with age matched controls reveals the restricted VH gene usage in B-CLL. VH gene usage showed a distinct prognostic value (p=0.01) when the patients using VH genes with bad prognosis were grouped together (VH1-69, VH3-21, VH3-23 and VH3-33; 43% of these patients died) and were compared with the patients using VH genes with a relatively good prognosis (VH3-7, VH3-30 and VH4-34; mortality rate of only 13%). The prognostic significance holds true when all patients were included (p=0.03). Also the mutation status (p=0.04) and cytogenetics (p=0.03) showed a prognostic significance. The VH gene usage did not correlate with mutation status, nor with cytogenetics. On the contrary mutation status and cytogenetics were strongly correlated (p

Description: Introduction Zap70 is superior to mutation status as a prognostic indicator in B-CLL, but whether it is causally implicated or just associated with progressive disease is unclear. Zap-70 transduces the TCR signal through the CD3 zeta-unit in T cells, but although it is important in preB development, it has no known function in mature B-cells. Zap-70 positive B-CLL show enhanced B cell receptor mediated Syk-phosphorylation upon surface IgM (sIgM) crosslinking, simulating antigen driven B cell proliferation. This may increase the proliferative drive in Zap-70+ B-CLL compared to Zap-70- leading to a higher risk of secondary chromosomal damage. Zap-70 was recently implied in Erk activation via CXCL-12/CXCR4 in T-cells and this is enhanced by cell adhesion through beta-1 integrins that may influence P-Erk phosphatases. Since both CXCL12/CXCR-4 and integrins influence B-CLL survival in vitro, we investigated the duration of downstream Erk activation in Zap-70 + versus Zap-70 B-CLL after CXCL-12 stimulation in the presence and absence of integrin binding by fibronectin. Materials/methods Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-gradient. Zap-70 was measured by flow cytometry (Upstate Ab), and PCR. B-CLL cells from 6 patients were stimulated with 100 ng/ml CXCL-12 after preincubation in serum free RPMI for 2 hrs at 37°in fibronectin or BSA coated plates and in suspension culture. After CXCL-12 addition supernatant and adherent cells were separately lysed at 0, 2 and 10 minutes and studied by Western blot for total Erk, Akt, P-Erk and P-Akt. Parallel cultures were extended for 24 hours and apoptosis of supernatant versus adherent cells was performed by AnnexinV-PI staining and flow cytometry. Results Erk-phosphorylation was markedly stimulated by CXCL-12 after 2 minutes, both in supernatant and adherent fractions of fibronectin and BSA coated plates compared to suspension cultures. This stimulation decreased in Zap-70- B-CLL after 10 minutes but remained high in Zap-70+ B-CLL particularly in the fibronectin adherent fraction. These cells were also protected from spontaneous apoptosis at 24 hours when compared to supernatant Zap70+ and Zap-70- cells. Conclusion CXCL-12/CXCR4 ligation leads to prolonged Erk-phosphorylation in Zap-70+ but not in Zap70- B-CLL cells. Prolonged Erk-activation is required for nuclear translocation and effective signal transduction along this way. This effect is enhanced by concomitant fibronectin engagement of integrins, which may give Zap-70+ B-CLL cells a proliferative advantage upon homing in lymphoid tissues.

Description: Radioimmunotherapy specifically targets malignant cells using radionuclides conjugated with monoclonal antibodies. Alpha-particle emittors such as 213Bi have high linear energy transfer and short path length compared with gamma-rays, which allows them to kill cells by only few nuclear hits. As 213Bi-based targeted alpha-immunotherapy evolves as a new treatment modality currently proved in clinical trials for AML, NHL and preclinically for CLL and multiple myeloma, understanding the molecular effects of 213Bi-immunoconjugates is of relevance. We assessed gene expression profiles of PB lymphocytes from 12 CLL pts using cDNA arrays comprising 1,185 genes. Cells treated in vitro with 213Bi-CD20 or an equivalent dose of gamma radiation (60Co) were intra-individually compared with non-treated cells. According to the two variants of B-CLL, with mutated (6 pts), or unmutated (6 pts) rearranged VH, we also aimed to verify whether radiation effects are mutation status specific. Globally, alpha radiation affected two-fold more genes than gamma. This was true for both cell types. Interestingly, there was little overlap between genes affected by both alpha and gamma radiation: 17% of genes in cells with mutated and 14% in cells with unmutated VH. Common genes mainly were involved in DNA repair: PCNA and CDKN1A acting in the p53-mediated pathway, CDC25B, CLK1 and CRY1 known to mediate repair after UV-induced DNA damage. Compared with alpha, effects of gamma radiation were of narrower range, with 24 genes altered in mutated and 16 in unmutated cells. The 46 genes specifically altered by alpha exposure in mutated cells and the 33 genes in unmutated cells belong to a multitude of functional families, showing that a generalized response was induced including deregulation of cytokines (IL-4, TGF β1, TGF ß2), oncogenes (v-myc, v-ski), cell cycle regulating genes (CDK6, cyclin D3, CDC2-like 10, CDKN2D). A network of intracellular signaling by phospholipases ß2, γ1 and 2, PKCε, CXCR4, extracellular signaling by integrin α7, chondroitin sulfate proteoglycan 5 and transcriptional regulation was involved. According to the divergent clinical behaviour of pts with mutated or unmutated VH, we expected differences at molecular level after radiation exposure. Indeed, 53 genes accounted for differences in response to alpha radiation between the two types of lymphocytes. Affiliation to functional families was mutation status specific. Expression of genes coding for interleukins, oncogenes and DNA synthesis was restricted to mutated cells. Hierarchical cluster analysis after alpha and gamma radiation resulted in two clearly distinguishable array clusters, one consisting of samples from pts with mutated VH, the other of samples from pts with unmutated VH. In conclusion, transcriptional changes induced by alpha and gamma radiation encompass molecularly distinct mechanisms and reflect the higher complexity of DNA damage produced by alpha radiation. The two subtypes of B-CLL respond to cytotoxic damage by different pathways which might explain the divergent clinical behaviour of these subgroups of patients.