Physical contact with endothelial cells through ß1- and ß2- integrins rescues chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis and induces a peculiar gene expression profile in leukemic cells.

BACKGROUND: Chronic lymphocytic leukemia B cells display prolonged survival in vivo, but when cultured in vitro rapidly undergo spontaneous apoptosis. We hypothesize that interactions with endothelial cells in infiltrated tissues and during recirculation may have a pathogenic role in chronic lymphocytic leukemia. DESIGN AND METHODS: We evaluated apoptosis of leukemic cells after co-culture on a monolayer of human umbilical vein endothelial cells with addition of fludarabine and antibodies that block adhesion. Then, we compared microarray-based gene expression profiles between leukemic cells at baseline and after co-culture. RESULTS: We found that the endothelial layer protected leukemic cells from apoptosis inducing a 2-fold mean decrement in apoptotic cells after 2 days of co-culture. Moreover, the endothelial layer decreased the sensitivity of chronic lymphocytic leukemia B cells to fludarabine-induced apoptosis. Physical contact with endothelium mediated by both ß(1)- and ß(2)- integrins is essential for the survival advantage of leukemic cells. In particular, blocking CD106 on endothelial cells or CD18 on leukemic B cells led to the almost complete abrogation of the survival advantage (>70% inhibition of viability). However, a reduction of apoptosis was also measured in leukemic cells cultured in conditioned medium collected after 2 days of co-culture, implying that survival is partially mediated by soluble factors. Overall, the contact with endothelial cells modulated 1,944 genes in chronic lymphocytic leukemia B cells, establishing a peculiar gene expression profile: up-regulation of angiogenesis-related genes, an increase of genes involved in TGFß and Wnt signaling pathways, secretion of cytokines recruiting stromal cells and macrophages and up-regulation of anti-apoptotic molecules such as Bcl2 and Survivin. CONCLUSIONS: Our study supports the notion that endothelial cells are major players in the chronic lymphocytic leukemia microenvironment. Adhesion to endothelium strongly supports survival, protects from drug-induced apoptosis and extensively modifies the gene expression profile of leukemic cells.

Angiopoietin-2 plasma dosage predicts time to first treatment and overall survival in chronic lymphocytic leukemia.

The clinical relevance of angiopoietin-2 (Ang2) in chronic lymphocytic leukemia (CLL) was previously suggested by the association between high Ang2, and shorter progression-free survival reported in small series of patients. Here, we evaluated Ang2 glycoprotein levels in plasma samples collected from a multicentric cohort of CLL patients (n = 316) using an enzyme-linked immunosorbent assay method, and we investigated its prognostic role in relation to time to first treatment (TTFT) and overall survival. Based on a cutoff equal to 2459 pg/mL, we divided our cohort in 2 subsets (high and low Ang2) composing 100 (31.6%) and 216 (68.4%) patients, respectively. High Ang2 was predictive of reduced TTFT (P < .001) and overall survival (P = .002). Multivariate analysis confirmed that high Ang2 was an independent prognosticator for TTFT (hazard ratio = 1.739; 95% confidence interval, 1.059-2.857; P = .029). Significant associations were found between high Ang2 and advanced Binet stages (P < .001), high beta(2)-microglobulin (P < .001), unmutated variable region of immunoglobulin heavy chain gene status (P < .001), high CD38 and zeta-chain-associated protein kinase 70 expression (P < .001 and P = .003), and intermediate/high cytogenetic risk (P = .005). Moreover, Ang2 added prognostic power to other conventional prognosticators and helped to refine prognosis among CLL subsets with both high and low vascular endothelial growth factor plasma levels. Ang2 plasma level may be a useful independent prognosticator for CLL.

Increased angiogenesis induced by chronic lymphocytic leukemia B cells is mediated by leukemia-derived Ang2 and VEGF.

Emerging evidence suggests that angiogenic signalling pathways play important role in the patho-biology of chronic lymphocytic leukemia (CLL). Our goal was to investigate: (i) the spontaneous and hypoxia-induced production of pro-angiogenic factors, VEGF and Ang2, by Real-time PCR and ELISA, (ii) the degree of vascularization in CLL-infiltrated bone marrow (BM) compartment by CD34 immunohistochemical staining of microvessels and (iii) the direct angiogenic effect of CLL-derived VEGF and Ang2 by function-blocking experiments in Matrigel assays. The results demonstrated that CLL cells spontaneously express both VEGF and Ang2 and are able to secrete these factors in surrounding microenvironment. Full-length Ang2 mRNA and truncated form Ang2(443) were detectable. Moreover, CLL cells were shown to enhance secretion of both VEGF and Ang2 proteins when subjected to hypoxic condition. Furthermore, increased in vivo and in vitro angiogenesis was induced by CLL cells. Enhanced BM vascularity correlated with Ig-unmutated CLL subset and increased expression of Ang2. Then, we demonstrated that supernatants obtained from CLL cells significantly increase the HUVEC tube formation in Matrigel assays and that this enhanced angiogenic capacity is mediated by both CLL-derived VEGF and Ang2. Taken together, these results suggest that several simultaneous mechanisms may be involved in the CLL capacity to induce the disruption of pre-existing vessel structures to give rise to tumor neoangiogenesis. The preliminary studies in solid tumors, showing that the disruption of Ang2 function can inhibit tumor vessel density and growth, are encouraging and suggest the possibility of new future therapeutic options targeting CLL microenvironment.

Increased expression of angiopoietin-2 characterizes early B-cell chronic lymphocytic leukemia with poor prognosis.

We measured the angiopoietin-2 (Ang-2) expression in early chronic lymphocytic leukemia (CLL) patients, pointing our attention on the association with immunoglobulin (IgV(H)) mutational status, CD38 expression and clinical outcome. Our results indicate that Ang-2 expression is heterogeneous among Binet stage A CLL patients. CLL patients can be divided into two subgroups (Ang-2 positive and Ang-2 negative CLL) with 30% of them displaying Ang-2 RNA levels above the cut off. A shorter progression-free survival was observed in Ang-2 positive CLL subset (p=0.032). Abnormal Ang-2 expression was also associated with unmutated IgV(H) genes (p<0.0001) and increased bone marrow angiogenesis (p=0.028), suggesting a role of Ang-2 in disease-progression of early CLL patients.

Immunoglobulin mutational status detected through single-round amplification of partial V(H) region represents a good prognostic marker for clinical outcome in chronic lymphocytic leukemia.

The immunoglobulin (Ig) mutational status in B-cell chronic lymphocytic leukemia (CLL) distinguishes two subsets of patients with different prognosis. Ig status detection is commonly performed with a panel of V(H) family-specific primers. Although this method detects clonal VDJ rearrangement in virtually all cases, it is technically cumbersome and therefore not widely used clinically. Here, we describe a simple and rapid method to establish the mutational status of IgV(H) in CLL. The method is based on a consensus V(H) FR2 primer, used in both polymerase chain reaction (PCR) and sequencing reactions. Overall, monoclonal B-cell populations were detected in 163 of 189 CLL patients (86%). The prognostic value of IgV(H) mutational status was then evaluated by analyzing survival in 146 CLL cases using different V(H) homology cutoffs. CLL prognostic groups were best separated by the classical 98% cutoff: median survival was 127 and 206 months in unmutated and mutated CLL cases, respectively (P = 0.0023). V(H) FR2 consensus and V(H) family PCR were compared in 41 cases, correctly assigning all cases by both methods. Therefore, we suggest a sequential strategy to detect immunoglobulin mutational status in CLL patients by first using the approach described in this study followed by alternative V(H) family-specific PCRs for negative cases.