Increased SHISA3 expression characterizes chronic lymphocytic leukemia patients sensitive to lenalidomide.

Lenalidomide is a therapeutically effective drug in chronic lymphocytic leukemia (CLL). Twenty-seven CLL patients were treated with lenalidomide in a phase II clinical trial. Ten patients were grouped as responders (R) and 6 as nonresponders (NR). We evaluated T lymphocytes, NK, monocytes and dendritic cells at baseline and after treatment. A gene expression analysis was performed on 16 CLL samples collected before treatment. The levels of immune cells or immune-related cytokines are not different between R and NR patients. However, CLL patients sensitive to lenalidomide clearly show a peculiar gene expression profile in leukemic cells. The most up-regulated gene (fold change = +23 in R vs. NR) is Wnt inhibitor SHISA homolog 3 (SHISA3). SHISA3highCLL are characterized by a restrained activation of Wnt signaling and sensibility to lenalidomide-induced apoptosis. In conclusion, SHISA3 is a candidate gene for the identification of CLL patients who will benefit of lenalidomide treatment as single agent.

Endothelium-mediated survival of leukemic cells and angiogenesis-related factors are affected by lenalidomide treatment in chronic lymphocytic leukemia.

Lenalidomide is an IMID immunomodulatory agent clinically active in patients with chronic lymphocytic leukemia (CLL). We evaluated the activity of lenalidomide inside an in vitro coculture system of endothelial and CLL cells. Lenalidomide was able to inhibit CLL survival advantage mediated by endothelial contact. Moreover, the marked increase of in vitro angiogenesis determined by CLL-derived conditioned media was reduced by lenalidomide. We also analyzed peripheral blood collected from 27 patients with relapsed or refractory CLL being treated with lenalidomide within a phase II trial. Plasma levels of VEGF and THBS-1 decreased, whereas Ang2 and Ang increased during treatment. Patients who respond to lenalidomide showed a more pronounced decrease of VEGF and bFGF than did patients with stable or progressive disease (p = 0.007 and p = 0.005). Furthermore, lenalidomide reduced circulating endothelial cells and endothelial progenitors by increasing the percentage of apoptotic cells. Conversely, for six matched bone marrow biopsies available before and after treatment, we did not detect any modification in vessel density, suggesting a possible mechanism of vessel normalization rather than regression. In conclusion, our study provides further evidence that the anti-CLL effect of lenalidomide is mediated through the alteration of microenvironmental elements, implying the modulation of several angiogenesis-related factors and disruption of CLL crosstalk with endothelial cells.

Genome-wide DNA profiling better defines the prognosis of chronic lymphocytic leukaemia.

The integration of molecular and clinical information to tailor treatments remains an important research challenge in chronic lymphocytic leukaemia (CLL). This study aimed to identify genomic lesions associated with a poor outcome and a higher risk of histological transformation. A mono-institutional cohort of 147 cases was used as the test series, and a multi-institutional cohort of 176 cases as a validation series. Genomic profiles were obtained using Affymetrix SNP 6.0. The impact of the recurrent minimal common regions (MCRs) on overall survival was evaluated by univariate analysis followed by multiple-test correction. The independent prognostic significance was assessed by multivariate analysis. Eight MCRs showed a prognostic impact: gains at 2p25.3-p22.3 (MYCN), 2p22.3, 2p16.2-p14 (REL), 8q23.3-q24.3 (MYC), losses at 8p23.1-p21.2, 8p21.2, and of the TP53 locus. Gains at 2p and 8q and TP53 inactivation maintained prognostic significance in multivariate analysis and a hierarchical model confirmed their relevance. Gains at 2p also determined a higher risk of Richter syndrome transformation. The prediction of outcome for CLL patients might be improved by evaluating the presence of gains at 2p and 8q as novel genomic regions besides those included in the 'standard' fluorescence in situ hybridization panel.

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.

Development of hypogammaglobulinemia in patients treated with imatinib for chronic myeloid leukemia or gastrointestinal stromal tumor.

Imatinib mesylate is a tyrosine kinase inhibitor used as first line treatment in chronic myeloid leukemia and gastrointestinal stromal tumor patients. Although several in vitro and animal studies demonstrated that imatinib affects immune response, few immune alterations are described in humans. We retrospectively studied hematologic and immunological parameters in 72 chronic myeloid leukemia and 15 gastrointestinal stromal tumor patients treated with imatinib at standard dosage and in 20 chronic myeloid leukemia patients treated before the introduction of imatinib in clinical practice. Both chronic myeloid leukemia and gastrointestinal stromal tumor patients developed a significant reduction of gammaglobulin and immunoglobulin serum levels. No significant hypogammaglobulinemia was observed in chronic myeloid leukemia patients in the pre-imatinib era. These data demonstrate that imatinib treatment induces hypogammaglobulinemia that can reach a severe entity in 10% of cases, both in chronic myeloid leukemia and in gastrointestinal stromal tumor patients. Prospective studies are needed to evaluate immune humoral alterations and to define the real incidence of infectious events, including viral reactivations.

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.