B-cell Chronic Lymphocytic Leukemia Cells Research Articles

Regulation of B-Cell Chronic Lymphocytic Leukaemia Cell Survival and Migration by the VEGF/SEMA3A Axis.

B-cell Chronic Lymphocytic Leukaemia (B-CLL) is characterized by the accumulation of B-CLL lymphocytes in the blood, marrow and secondary lymphoid tissues. B-CLL cells have a long survival owing to alterations in the normal pathways of apoptosis. In the marrow and lymphoid tissues CLL cells are in close contact with stromal cells that constitute distinct microenvironments. The secretion of the CXCR4 ligand, CXCL12, by stromal cells attracts B-CLL cells and provides protection from spontaneous or induced apoptosis. Studies in other cell types have shown VEGF signalling is involved in regulating CXCR4 expression levels.The aim of this study was to examine VEGF receptor expression and the role of VEGF signalling in cell survival and CXCR4 expression. Expression levels of CXCR4 and VEGF receptors, VEGFR-1 and -2, in CLL samples were determined by flow cytometry. Expression of the VEGFR co-receptor, Neuropilin-1 (NRP1), was examined by Western blot. Cell migration towards CXCL12 was assessed using CoStar Transwell chambers (5mm pore size). Informed consent was received from all patients.VEGFR1 and VEGFR2 positive cells in 22 patient samples ranged from 0–38% and 1.5–83% respectively. NRP1 expression was detected in all samples analysed thus far (n=6). The NRP1 ligand, SEMA3A, a competitive inhibitor of VEGF binding to NRP1, decreased CXCR4 expression in patient CLL cells (n=8, p<0.05). This decrease in CXCR4 levels correlated with reduced migration of SEMA3A treated CLL cells towards CXCL12 (88 +/− 12.7% of control levels, n=8, p<0.05). Treatment of CLL cells with the VEGFR signalling inhibitor SU5416 decreased CLL cell survival which correlated with VEGFR1 expression levels (n=16, R=0.745, p<0.01) but not with VEGFR2 expression levels.These results show that signalling through the VEGF co-receptor NRP1 plays an important role in regulating CXCR4 levels in CLL cells, as well as CLL cell migration along a CXCL12 gradient. Our results also suggest that VEGF signalling through VEGFR1 may be particularly important in regulating CLL cell survival. Thus, the VEGFR/NRP1 signalling pathway may represent an important therapeutic target in B-CLL.

Prognostic Stratification of Patients with Chronic Lymphocytic Leukemia Based on Quantitative Flow Cytometry Evaluation of ZAP-70 Versus Homology of IgVH Plot.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in the adult population. It has a heterogeneous behavior and variable prognosis. While some patients experience indolent disease requiring no therapy for many years, others demonstrate a more aggressive type unresponsive to therapy. An accurate prognostic stratification is essential for optimizing the therapeutic strategy. Many prognostic factors are presently known, but their relationships and significance are often not clearly understood. Several markers have been identified, including IgVH mutation status and ZAP-70. Mutation of IgVH (less than 98% homology with the germline sequence) is correlated with better prognosis. Expression of ZAP-70, which is a cytoplasmic ζ-associated tyrosine-kinase essential for T-cell receptor signal transduction, is associated with more rapid disease progression and shorter survival. However, its detection by flow cytometry has many technical difficulties, resulting in high interlaboratory variability. The expression of intracellular ZAP-70 in 217 patients with diagnosis of B-CLL was determined using a new approach: quantitative flow cytometry on CD5+19+ tumor cells. Other laboratory and clinical parameters were evaluated, including gender, age, type and number of therapies, CD38 expression, cytogenetics and mutation status of IgVH. The expressions of ZAP-70 and CD38 were measured in molecules of equivalent soluble fluorochrome (MESF units). The results were correlated with mutation status of IgVH. Patients were divided into two groups by cluster analysis in a plot of IgVH homology versus ZAP-70 MESF (left panel). Overall survival curves for these groups are shown (right panel). It could follow that patients above the diagonal (triangles, group B) in the IgHV versus MESF plot have significantly poorer prognoses (p=0.0001) than do patients below this diagonal (circles, group A). Expression of CD38 above 15000 MESF showed worse prognosis for patients in the group B (p=0.035). Only three of 61 patients with chromosomal aberrations associated with poor prognosis (del11q and del17p) were found in the group A, but all of them had del13q present, as well. The authors introduced a new approach for evaluating ZAP-70 expression in B-CLL cells using quantitative flow cytometry that is easily standardized and not burdened with technical problems. Plotting IgVH homology (%) versus ZAP-70 quantitative expression (MESF) could clearly divide patients according to their prognoses. [Display omitted]

Changes in Immunogenicity of Chronic Lymphocytic Leukemia Cells Mediated by Epigenetic Modifiers

The success of immunotherapeutic treatments for B-cell chronic lymphocytic leukemia (B-CLL) hinges upon the circumvention of tolerogenic mechanisms which inhibit the formation of a robust anti-cancer immune response. Recent evidence suggests that DNA methylation inhibitors and histone deacetylase inhibitors can have lasting cancer-specific effects on the expression of highly immunogenic cancer-testis antigens (CTAs) as well as modulating costimulatory and major histocompatability molecule (MHC) surface expression. To investigate the potential efficacy of a combined therapy of methylation (5-aza-2′ytidine) and histone deacetylase (LAQ) inhibitors in human B-CLL we evaluated the expression of CTAs, costimulatory molecules (CD40, CD80, CD86), and MHC molecules (HLA-A, B, C, and HLA-DR) in both cell lines and primary cells from patients with CLL. In addition, we investigated the overall functional immunogenicity of treated B-CLL cells using mixed leukocyte reactions. Results showed that many highly immunogenic CLL-specific CTAs (SSX family, MAGE family, NY-ESO-1, GAGE-2, and NXF2) are significantly upregulated after treatment with both inhibitors. Our data also indicates that the combined effect of both inhibitors was capable of inducing a more immunogenic phenotype (increased CD86, CD80, CD40, HLA-A,B,C, and HLA-DR) in CLL cells consistently, while either drug alone was incapable of producing this effect. In addition treatment with both inhibitors was capable of increasing IFNγ release mediated by allogenic T-cells. Taken together these data indicate that a combined therapy with histone deacetylase and methylation inhibitors, possibly in combination with antigen-specific vaccine immunotherapy, may effectively induce cancer-specific antigen expression and increase costimulatory and antigen presenting capacity of the B-CLL cell.

High CD79b Expression Predicts a Poor Outcome in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

[Display omitted] The immunoglobulin (Ig) beta protein (CD79b) dimerizes with Ig alpha (CD79a) to form the signaling component of the B-cell antigen receptor complex (BCR). Given the critical role of Ig beta in BCR signaling, it has been suggested that its variable expression in CLL may induce either cell proliferation or apoptosis. High CD79b expression has been associated with atypical morphology, strong expression of surface Ig, advanced clinical stage and short overall survival in B-CLL (Garcia Vela, 1999; Zucchetto, 2006). Moreover, high CD38 expression was correlated with high CD40, CD69 and CD79b which are consistent with an activation phenotype (Damle, 2002). Furthermore, we have demonstrated that the ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). Since it has been described that BCR engagement has significant effects on B-CLL cell survival, activation and cell cycle progression (Deglesne, 2006), we decided to evaluate the real impact of CD79b expression on B-CLL prognosis. The primary aims of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD79b expression; 2) whether CD79b could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally 3) whether CD79b was an independent prognostic factor. Therefore, we investigated 401 patients (pts), median age 65 years (range 33–89), 213 males and 188 females. With regard to modified Rai stages, 123 pts had a low stage, 261 an intermediate stage and 17 a high stage. CD79b was determined by multicolor flow cytometry, using the monoclonal antibody anti-CD79 beta-FITC (clone SN8, Dako) and fixing a cut-off value of 30%. CD79b+ B-CLL pts were 207/401 (52%). CD79b>30% was significantly associated with the intermediate/high Rai stage (p=0.00001), beta-2 microglobulin >2.2 mg/dl (p<0.0001) and with multiple intrathoracic/abdominal lymphadenopathies and/or splenomegaly (p<0.0001). Low CD79b was significantly correlated either with IgVH mutated status (>2%) (86/105; p<0.0001) or lower soluble CD23 levels (125/161; p<0.00001). Significant associations were found either between CD38<30% and lower CD79b (172/194; p<0.0001) or ZAP- 70<20% and CD79b<30% (145/193; p<0.0001). With regard to cytogenetics, there were strict correlations either between high CD79b and trisomy 12 (25/33; p=0.001) or high CD79b and del17p (16/20; p=0.001). Median follow up duration from diagnosis was 68 months (range 6–230). Concerning clinical outcome, both a shorter PFS (Figure) and OS were observed in CD79b+ pts (12% vs 58% at 16 years; p<0.0001 and 49% vs 85% at 16 years; p<0.0001) as well as in ZAP-70+ pts (5% vs 52% at 11 years; p<0.0001 and 33% vs 87% at 18 years; p<0.0001). To further explore the prognostic value of CD79b, we investigated its expression within ZAP-70+ (159 pts) and ZAP-70 negative (242 pts) subsets. As a matter of fact, CD79b+ pts showed a shorter PFS and OS both within the ZAP-70+ subset (7% vs 19% at 10 years; p=0.00006 and 25% vs 65% at 16 years; p=0.006) and within the ZAP-70 negative subset (29% vs 67% at 14 years, p<0.00001 and 86% vs 100% at 14 years; p=0.01). In multivariate analysis of PFS and OS, in which age, Rai stages, CD38, CD69, CD79b and ZAP-70 entered, ZAP-70 (p=0.001 and p=0.001), CD69 (p=0.001 and p=0.01) and CD79b (p=0.0007 and p=0.03) resulted to be independent prognostic factors. Therefore CD79b, easily determined by flow cytometry, should be considered another important prognostic parameter and could be used to early identify and stratify B-CLL progressive pts.

Frequently Occurring B-CLL Antibodies Recognize Apoptotic Cells That Expose Non-Muscle Myosin Heavy Chain IIA

B-cell chronic lymphocytic leukemia (B-CLL) is a disease caused by the clonal expansion of a B lymphocyte expressing a unique monoclonal antibody (mAb) of a defined amino acid sequence. At least 27% of B-CLL patients share mAbs with very similar sequences, suggesting common antigen reactivity. A frequently occurring subset of B-CLL patients (at least 49 worldwide) carries an unmutated mAb with a nearly identical or stereotypic sequence that is characterized by a heavy (H) chain encoded by IGHV1-69, IGHD3-16, and IGHJ3 and a light (L) chain generally encoded by IGKV3-20. mAbs from this stereotypic subset (called subset 6) bind a characteristic cytoplasmic protein in human cells. We have recently shown that this protein is non-muscle myosin heavy chain IIA (MYHIIA).Because MYHIIA is an intracellular protein, we hypothesized that MYHIIA becomes exposed on the cell surface during apoptosis, allowing the mAb of the B-CLL cell to interact with MYHIIA. In this report, we provide evidence that subset 6 mAbs recognize MYHIIA-containing apoptotic cell structures.Apoptosis of the human T cell line (Jurkat), either spontaneous, or induced by treatment with camptothecin was assessed phenotypically by nuclear DNA rearrangement revealed by immunohistochemical staining with propidium iodide (PI). Costaining with rabbit anti-human MYHIIA followed by fluorescein isothiocyanate (FITC) labeled donkey anti-rabbit IgG revealed that MYHIIA becomes exposed during apoptosis in structures distinct from those containing fragmented nuclear DNA. PI costaining with recombinant subset 6 B-CLL IgG1 mAb and FITC labeled goat anti-human-IgG antibody shows that this B-CLL mAb binds similar apoptotic structures. Finally, Jurkat cells stained with subset 6 B-CLL mAb as before and rabbit anti-human MYHIIA followed by Rhodamine Red labeled donkey anti-rabbit IgG demonstrate that the B-CLL mAb colocalizes with MYHIIA containing structures.To further study this observation, Jurkat cell apoptosis was assessed by flow cytometry after staining with 7-AAD and Annexin V-Phycoerytherin (AV-PE). Costaining with anti-MYHIIA shows that anti-MYHIIA is exposed in both early (7-AAD+ AV-PE−) and late (7-AAD+ AV-PE+) apoptotic cells, but not in live (7-AAD−, AV-PE−) cells. Similarly, costaining with subset 6 B-CLL mAb demonstrates binding in both early and late apoptotic cells, but not live cells. Intriguingly, not all apoptotic Jurkat cells are MYHIIA+ or B-CLL mAb+.In summary, subset 6 B-CLL mAbs react with early and late apoptotic cells exposing MYHIIA. Reactivity with a specific apoptotic cell structure(s) may result in the maintenance and expansion of the leukemic clone.

A Highly Efficient, Clinically Applicable Transfection Method to Redirect the Specificity of Immune Cells and Enhance Their Anti-Tumor Capacity

Effective immunotherapies rely on triggering specific cytotoxic responses against target cells. Chimeric Antigen Receptors (CAR) composed of antigen recognition domains in the form of scFv portions of monoclonal antibodies and immune cell activation domains provide non-MHC restricted, KIR-independent, immune cellmediated cytotoxicity directed toward target cells expressing specific antigens. It is well demonstrated that CAR transfection into immune cells using viral vectors redirects their specificity and enhances their cytotoxicity. Translation of these findings into clinically feasible treatment strategies is challenged by the labor/costs associated with extended cell processing, and by safety concerns associated with viral vectors. We describe here the use of non-viral methods to deliver a nucleotide sequence encoding CAR into freshly isolated immune cells obtained from peripheral blood leukapheresis collection. Protocols for loading mRNA encoding eGFP (irrelevant control) and a previously described anti-CD19 CAR (Imai et al. Blood 2005) into peripheral blood mononuclear cells (PBMC) and cell subsets (T cells, NK cells, and monocyte-depleted PBMC) were developed and optimized using a clinically validated, automated, closed-system, flow electroporation technology. PBMC were obtained from 5 normal healthy donors and 1 patient with B-cell chronic lymphocytic leukemia (B-CLL). Expression levels of eGFP or anti-CD19-BB-z were analyzed by flow cytometry. Specific cytotoxicity of target cells after 4 hours was measured by flow cytometry using acetoxymethyl-calcein labeled CD19+ target cells which included acute lymphoblastic leukemia (ALL) cell lines, primary B-CLL cells and autologous B cells. Following loading of mRNA encoding eGFP, cell viability was consistently ≥80% for all cell populations. Most cells expressed eGFP: 97±1% (day 1) and 83±2% (day 7). Following loading of mRNA encoding anti-CD19-BB-z, cell viability was 96%±1% for resting T cells (n=2), 93%±10% for PBL (n=2), 94%±8% for PBMC (n=3), 80%±15% for NK cells (n=4). Expression of anti-CD19-BB-z protein receptor was 54%±11% (day 1) in PBMC (n=6). CAR expression decreased over time and was essentially similar to baseline on day 7 post-loading. Cells expressing anti-CD19-BB-z protein (but not those expressing eGFP only), lysed 60%–80% ALL or B-CLL target cells at an effector-to-target ratio (E:T) of 8:1. A shigher E:T ratio of 20:1 was required for 60%–80% lysis of autologous B-cell targets. There was no change in cytolytic activity of CAR mRNA loaded-PBMC during the first 3 days post-loading. Subsequently, cytolytic activity dropped and on day 7 (post-loading) was approximately half of the activity measured on day 3 (post-loading).In vivo testing was performed in a sub-cutaneous tumor grafting model in Beige SCID mice. The mice (N = 5 per group) were transplanted subcutaneously with CD19+ tumor (HS Sultan) cells (1 × 106) co-mixed with PBMC loaded with anti-CD19-BB-z mRNA (doses = 0.74, 2.22, 6.67 and 20 ×106). Controls were tumor cells only or tumor cells co-mixed with unmodified PBMC. Tumors developed and grew in 3/5 animals in each control group and in 2/5 in lowest anti-CD19-BB-z mRNA-loaded dose. No tumor formation was observed in any animal in each of the 3 higher cell doses. More comprehensive animal studies and investigation of engineering additional constructs for chimeric signal receptor with tumor affinities other than anti-CD19 are in progress. These results indicate that CAR can be effectively transfected into immune cells with the methodology that we developed. Because of its simplicity and effectiveness, this methodology is well suited for clinical application and holds promise for improving the specificity and widening the feasibility of cell therapy.

Vitamin D3 Induces Autophagy of Human Myeloid Leukemia Cells

Vitamin D3 causes potent suppression of various cancer cells; however, significant supraphysiological concentrations of this compound are required for antineoplastic effects. Current combinatorial therapies with vitamin D3 are restricted to differentiation effects. It remains uncertain if autophagy is involved in vitamin D3 inhibition on leukemia cells. Here we show that besides triggering differentiation and inhibiting apoptosis, which was previously known, vitamin D3 triggers autophagic death in human myeloid leukemia cells. Inhibiting differentiation does not efficiently diminish vitamin D3 suppression on leukemia cells. Vitamin D3 up-regulates Beclin1, which binds to class III phosphatidylinositol 3-kinase to trigger autophagy. Vitamin D3 phosphorylates Bad in its BH3 domain, resulting in disassociation of the apoptotic Bad-Bcl-xL complex and association of Bcl-xL with Beclin1 and ultimate suppression of apoptotic signaling. Knockdown of Beclin1 eliminates vitamin D3-induced autophagy and inhibits differentiation but activates apoptosis, suggesting that Beclin1 is required for both autophagy and differentiation, and autophagy cooperates with differentiation but excludes apoptosis, in which Beclin1 acts as an interface for these three different cascades. Moreover, additional up-regulation of autophagy, but not apoptosis, dramatically improves vitamin D3 inhibition on leukemia cells. These findings extend our understanding of the action of vitamin D3 in antineoplastic effects and the role of Beclin1 in regulating multiple cellular cascades and suggest a potentially promising strategy with a significantly better antileukemia effect.

Calorimetric study as a potential test for choosing treatment of B-cell chronic lymphocytic leukemia

Differential scanning calorimetry (DSC) and complementary techniques were utilized to evaluate the sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) cell samples in vitro exposed to cladribine or fludarabine in combination with mafosfamide. Mafosfamide, the active in vitro form of cyclophosphamide with both purine analogs produced the cytotoxic effect on mononuclear cell probes, however, to a different degree. Our results indicated that higher sensitivity of examined leukemic cell samples to the used drug combinations was usually accompanied by a marked decrease or even a complete loss of thermal transition at 95 ± 3 °C in DSC scans of nuclear preparations as well as by more significant reduction of cell viability, higher extent of DNA damage estimated by the comet assay and by dropping/disappearance of anti-apoptotic protein Mcl-1 in comparison with untreated cells. We have also observed that the reduction of transition at 95 ± 3 °C in thermal scans of nuclear preparations isolated from blood of B-CLL randomized patients who showed response to cladribine or fludarabine combined with cyclophosphamide, i.e., CC and FC, respectively, corresponded with the decrease or disappearance of anti-apoptotic proteins Bcl-2 and/or Mcl 1. In conclusion, these in vitro and in vivo studies revealed that quick DSC technique, usually supplemented by other methods, is a potent tool to distinguish efficacy of B-CLL treatment and could be helpful in choosing the most effective manner of treatment for this type of leukemia.

Apoptotic effects on B-cell chronic lymphocytic leukemia (B-CLL) cells of heterocyclic compounds isolated from Guttiferaes

A series of 10 heterocyclic compounds purified from Allanblackia were tested on two B cell lines, ESKOL and EHEB, and on cells from B-CLL patients. Several molecules inhibited the proliferation of both cell lines and promoted apoptosis of B-CLL cells through different mechanisms, some of them elicited a dissipation of the mitochondrial transmembrane potential, other triggered caspase-3 activation and cleavage of the inducible nitric oxide synthase. Blood mononuclear cells and B-lymphocytes from healthy donors appeared less sensitive than B-CLL cells. These results indicate that these molecules may be of interest in the development of new therapies for B-CLL.

Lenalidomide Enhances Natural Killer Cell and Monocyte-Mediated Antibody-Dependent Cellular Cytotoxicity of Rituximab-Treated CD20+ Tumor Cells

Lenalidomide has significant activity in myelodysplastic syndromes, multiple myeloma, and non-Hodgkin's lymphoma (NHL). In previous studies, natural killer (NK) cell expansion by lenalidomide was shown to enhance the cytotoxic effect of rituximab. This study assessed the ability of lenalidomide to enhance antibody-dependent cellular cytotoxicity (ADCC) in rituximab-treated NHL cell lines and primary tumor cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) in vitro. An in vitro ADCC system was used to assess the ability of lenalidomide to enhance human NK cell and monocyte function in response to rituximab. Lenalidomide directly enhanced IFN-gamma production via Fc-gamma receptor-mediated signaling in response to IgG. It was also a potent enhancer of NK cell-mediated and monocyte-mediated tumor cell ADCC for a variety of rituximab-treated NHL cell lines in vitro, an effect that was dependent on the presence of antibody and either interleukin-2 or interleukin-12. Lenalidomide also enhanced the ability of NK cells to kill primary tumor cells derived from three patients with B-CLL who have been treated previously with fludarabine plus cyclophosphamide. Enhanced NK cell ADCC was associated with enhanced granzyme B and Fas ligand expression and could be inhibited by a granzyme B inhibitor and partially inhibited by antibody to FasL. Enhanced NK cell Fc-gamma receptor signaling is associated with enhanced phosphorylated extracellular signal-related kinase levels leading to enhanced effector function. These findings suggest that lenalidomide has the potential to enhance the rituximab-induced killing of NHL cell lines and primary B-cell chronic lymphocytic leukemia cells via a NK cell-mediated and monocyte-mediated ADCC mechanism in vitro, providing a strong rationale for the combination of lenalidomide with IgG1 antibodies to target tumor-specific antigens in patients with cancer.

α4β1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells

AbstractAs B-cell chronic lymphocytic leukemia (B-CLL) progresses, malignant cells extravasate and infiltrate lymphoid tissues. Several molecules, including gelatinase B/MMP-9, contribute to these processes. Although mainly a secreted protease, some MMP-9 is present at the B-CLL cell surface and the function, mode of anchoring, and interactions of this MMP-9 are unknown. Here we show that anti–MMP-9 antibodies immunoprecipitated a 190-kDa CD44v isoform and α4β1 integrin from B-CLL cells, but not from normal B cells. Function-blocking antibodies to α4β1 or CD44, or transfection with specific siRNAs, decreased cell-associated proMMP-9 and increased the secreted form. B-CLL cells attached to and bound proMMP-9 and active MMP-9, and this was inhibited by blocking the expression or function of α4β1 or CD44. The MMP-9 hemopexin domain was critical in these interactions. α4β1 and 190-kDa CD44v (but not CD44H) formed a complex at the cell surface, since they both coimmunoprecipitated with anti-α4, anti-β1, or anti-CD44 antibodies. Immunofluorescence analyses confirmed that α4β1 and CD44v colocalized with MMP-9. Binding of proMMP-9 inhibited B-CLL cell migration, and this required MMP-9 proteolytic activity. Thus, we have identified α4β1 and CD44v as a novel proMMP-9 cell surface docking complex and show that cell-associated MMP-9 may regulate B-CLL cell migration and arrest.

Decreased expression of complement receptor type 2 (CR2) on neoplastic B cells of chronic lymphocytic leukaemia

Neoplastic cells from 49 patients with B cell chronic lymphocytic leukaemia (B-CLL) were studied and compared with normal peripheral and tonsillar B cells using CD21 monoclonal antibodies. Membrane expression of CR2 was quantified by calibrated flow cytometry and by binding analysis with radiolabelled antibody. Both assays indicate that B-CLL cells express only 30% of the CR2 found on normal B cells. These findings are further evidence of the aberrant phenotype of B-CLL cells.

Correlation of clinical activity of lumiliximab in combination with FCR and ZAP 70 expression on B-cell chronic lymphocytic leukemia cells

7073 Background: Lumiliximab is an anti-CD23 monoclonal antibody under investigation for the treatment of patients with relapsed B-cell chronic lymphocytic leukemia (BCLL). Lumiliximab binds specifically to human CD23, a glycoprotein expressed on the majority of BCLL cells. As ZAP70 is a poor prognostic factor of CLL, expression of the ZAP70 antigen by malignant B cells was analyzed on samples taken pre-treatment and day 2 post-treatment in a subset of patients with relapsed BCLL enrolled in a clinical study evaluating lumiliximab as monotherapy. The ability of lumiliximab to induce ex-vivo apoptosis was also evaluated in BCLL samples collected in this subset. Subsequently, pre-treatment ZAP70 expression was analyzed in patients treated lumiliximab in combination with fludarabine, cyclophosphamide, and rituximab (L + FCR). Methods: In the lumiliximab monotherapy study, ZAP70 expression was quantified by Western blot assay using lysates from purified BCLL cells and apoptosis was evaluated by flow cytometric assessment of Caspase-3 activation on pre- treatment and day 2 treated samples (ex-vivo). In the L + FCR study, frequencies of BCLL cells and ZAP70 positive cells were estimated by four color flow cytometry using standard commercially available antibodies. BCLL cells were identified by first gating on viable CD45+ cells and subselecting with antibodies to CD5 and CD19 receptors (CD5+CD19+ phenotype). ZAP70 expression was reported as % BCLL cells. Results: Analysis of data from the monotherapy study suggested induction of apoptosis and decrease in CD5+CD19+ cells in patients expressing high levels of ZAP70 on purified B cells. Moreover, apoptosis was also observed in a patient that expressed activated ZAP70. Results from the L + FCR study demonstrated that clinical activity in this combination trial is observed in patients having high or low levels of ZAP70 expression. Conclusions: Lumiliximab potentially provides clinical benefit in BCLL patients with both low and high levels of ZAP70, whose expression is associated with aggressive disease and poor prognosis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Biogen Idec Biogen Idec Biogen Idec

Immunophenotypic characterization of relapsed chronic lymphocytic leukemia patients treated with lumiliximab in combination with FCR

7067 Background: Lumiliximab is an anti-CD23 monoclonal antibody under investigation for the treatment of patients with relapsed B-cell chronic lymphocytic leukemia (BCLL). A comprehensive immunophenotypic characterization of CLL cells obtained from patients treated with lumiliximab in combination with fludarabine, cyclophosphamide, and rituximab (L + FCR) was conducted. This included an analysis of the frequency of BCLL cells (CD5+CD19+), expression levels of CD38 (a negative prognostic factor), CD55 and CD59 (potential resistance markers of rituximab therapy). Methods: Immunophenotypic characterization was performed by four color flow cytometry analysis. BCLL cells were first identified by gating on viable CD45+ cells and subselecting with antibodies to CD5 and CD19 receptors. Antigen expression levels and their frequencies were reported in terms of mean fluorescence intensities (MFI) and fraction (%) of positive BCLL cells. Results: The analysis of peripheral blood samples from L + FCR treated patients indicated a substantial decrease in CD5+CD19+ cells in 30 of the 31 evaluable patients, although, the time to CLL count reduction following therapy was variable, ranging from week 1 to week 13. Clinical activity was observed in patients with both high (>30%) and low (<30%) levels of CD38 expression (MFI 6 - 230). Finally, clinical activity of L +FCR was independent of pre-treatment expression levels of CD55 (MFI 45–272) and CD59 antigen (MFI 20–191) and was also observed in a few patients showing increased expression of CD59 receptor, compared with their individual pre-treatment values, which has been associated with resistance to single agent rituximab. Conclusions: Clinically, L + FCR is an effective regimen in decreasing tumor burden in the peripheral blood of patients with BCLL. Importantly, the clinical activity of L + FCR appears to be independent of CD38 expression (a poor prognostic factor) and thereby may provide clinical benefit to a subset of patients with unfavorable clinical outcomes. Clinical activity was also observed in patients with BCLL expressing high levels of CD59 antigen, thus, suggesting a different MOA than other mAbs active in this indication such as Rituxan. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Biogen Idec Biogen Idec Biogen Idec

Lumiliximab in combination with FCR for the treatment of relapsed chronic lymphocytic leukemia (CLL): results from a phase I/II multicenter study

7003 Background: Lumiliximab is an anti-CD23 monoclonal antibody that is being investigated for the treatment of relapsed B-cell CLL (BCLL). CD23 is a glycoprotein expressed on the majority of CLL B cells. In a previous study, lumiliximab monotherapy given weekly was well tolerated, achieved sustainable CD23 receptor occupancy and showed clinical activity. A Phase I/II, multicenter study was conducted to evaluate the safety and efficacy of lumiliximab in combination with fludarabine, cyclophosphamide, and rituximab (L + FCR) for patients (pts) with relapsed CD23+ BCLL. In addition, CD23 receptor occupancy on BCLL cells with L + FCR and possible effects of elevated serum CD23 were evaluated. Methods: Thirty-one pts with relapsed BCLL received either 375 mg/m2 (n=3) or 500 mg/m2 (n=28) of L+ FCR for up to six 28-day cycles. All pts completed treatment and follow-up is ongoing. A semi-quantitative flow cytometry method was used to measure CD23 receptor occupancy and serum CD23 levels were measured using an enzyme-linked immunosorbent assay. Results: Median age at study entry was 58 yrs, 71% had Rai Stage I/II, and median # of prior regimens was 2 (1- 10). Using NCI-WG criteria, overall response rate was 65%: complete response (CR) 52% and partial response13%. Five of the 8 pts with del(11q22.3) achieved CR. Based on median follow-up of 16.8 mos (1.5 - 37.6), KM estimated median progression-free survival (PFS) for all pts was 19.3 mos. Median PFS for all responders and CR pts were 23.4 mos and 30.4 mos, respectively. Twenty-three pts (74%) reported a Grade 3 or 4 event, similar to what has been previously reported with FCR. L + FCR has a comparable safety profile with no additional toxicity. At lumiliximab schedule used, CD23 receptor occupancy was sustained with no effects by elevated levels of serum CD23. Conclusions: These results suggest that L +FCR is an effective regimen for pts with relapsed B-CLL. L+FCR produced an impressive CR rate, an encouraging PFS and a similar safety profile to that of FCR. Monthly L + FCR achieved sustainable CD23 receptor occupancy on BCLL, which was not affected by elevated levels of serum CD23. A large, randomized, global study of L+ FCR vs FCR (LUCID) is ongoing to further evaluate the safety and efficacy of this regimen. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Biogen Idec Biogen Idec Biogen Idec

Abstract LB-305: Soluble CD23 levels increase following lumiliximab dosing in relapsed chronic lymphocytic leukemia patients

Abstract Lumiliximab is an anti-CD23 monoclonal antibody under investigation for the treatment of patients with B-cell chronic lymphocytic leukemia (B-CLL). Lumiliximab binds specifically to human CD23, a glycoprotein expressed on the majority of CLL B cells. CD23 is a trimeric molecule with both membrane-bound (mCD23) and soluble (sCD23) forms. mCD23 is a 45 kD glycoprotein expressed on B cells, monocytes/macrophages, eosinophils, platelets, and dendritic cells, with increased levels observed on chronic lymphocytic leukemia (CLL) cells. Soluble CD23 is produced by the cleavage of mCD23 by endogenous proteases into various fragments, with the 25 kD fragment found in normal human serum at concentrations around 2 ng/ml. Increased levels of sCD23 are found in the serum of patients with several disease states including CLL. Data from a Phase 1 lumiliximab monotherapy trial in relapsed CLL showed that levels of sCD23 rapidly increased in the serum of some patients after dosing with lumiliximab. To confirm this phenomenon, serum sCD23 levels and their relationship with treatment response were evaluated in a Phase 1/2 study of lumiliximab in combination with fludarabine, cyclophosphamide, rituximab (FCR) in patients with relapsed CLL. The pharmacokinetic (PK) profiles of both lumiliximab and rituximab were also compared to sCD23 profiles. Methods: Thirty-one patients with relapsed CLL were enrolled to receive up to 6 cycles treatment with lumiliximab plus FCR. Lumiliximab PK, Rituxan PK and sCD23 levels were each measured in 31 patients using validated enzyme-linked immunosorbant assays. PK data was analyzed with WinNonlin. Results and Conclusions: Our analysis shows that sCD23 increases from the ng/ml range to the ug/ml range immediately after each dosing with lumiliximab/FCR, then declines throughout the remainder of the treatment cycle. The levels of sCD23 increase again with subsequent lumiliximab/FCR doses, until the there are little to no B-CLL cells remaining in the circulation. The pattern of increase parallels the PK profile of lumiliximab. The intensity of sCD23 increase varied from patient to patient, and did not appear to correlate with treatment outcome. These data demonstrate that although lumiliximab/FCR dosing increases serum levels of sCD23, there is no apparent impact on patient response. This will be evaluated further in upcoming clinical trials. The mechanism of sCD23 release is also currently being investigated.

MDM2 SNP309 Is Associated With Poor Outcome in B-Cell Chronic Lymphocytic Leukemia

A single nucleotide polymorphism (SNP) at position 309 in the promoter region of MDM2 leading to increased expression of MDM2 and attenuated function of p53 has been negatively associated with onset and outcome of disease in solid tumors. Because inactivation of p53 by deletion and/or mutations also impacts on the clinical course of B-cell chronic lymphocytic leukemia (B-CLL), we assessed the role of the SNP309 genotype in B-CLL. The frequency of SNP309 T/T, T/G, or G/G genotypes and the p53 status (wild type, mutated, or deleted) were assessed and correlated with clinical outcome in 140 B-CLL patients and a second independent cohort. In addition, the correlation of the MDM2 SNP309 genotype with the MDM2 protein expression level in B-CLL cells was evaluated by immunoblotting. A significant negative association of the SNP309 T/G and G/G genotypes with overall survival was seen (T/G genotype, relative risk = 3.7; 95% CI, 1.2 to 11.5; P = .02; G/G genotype, relative risk = 9.1; 95% CI, 2.4 to 35.1; P = .001), but no correlation with incidence or onset of B-CLL was observed. The influence of the heterozygous SNP309 T/G genotype on treatment-free survival depended on the p53 status but not on the CD38, Zap-70, or IgVH mutational status or Rai stage of B-CLL patients. The unfavorable SNP309 T/G and G/G genotypes were associated with a gene-dosage-dependent increase of MDM2 expression. The MDM2 SNP309 genotype influencing MDM2 expression levels was identified as an additional independent risk factor in B-CLL. Targeting MDM2-p53 interactions might emerge as a successful treatment strategy for B-CLL.

Stimulation of iNOS expression and apoptosis resistance in B-cell chronic lymphocytic leukemia (B-CLL) cells through engagement of Toll-like receptor 7 (TLR-7) and NF-κB activation

B-CLL cells are characterized by in vivo resistance to apoptosis due, in part, to the presence of an inducible nitric oxide synthase, iNOS, as the NO released plays anti-apoptotic role, notably by inhibiting caspases. The mechanisms leading to spontaneous expression of iNOS in these cells are presently unknown. The restricted use of some V H sub-groups and the sequences of the monoclonal immunoglobulins of the B-cell receptor expressed by the leukemia cells suggested that the latter have encountered specific auto-antigens and/or microbial derived antigens. Their binding to the BCR provides an activation signal resulting in enhanced survival, hence could be involved in the aetiology of the disease. At the interface of innate and cognate immunity, Toll-like receptors, TLR, recognize PAMPs (pathogen-associated molecular patterns) expressed by various bacteria and virus as well as some self-antigens. We thus hypothesized that TLR were involved in the early steps of B-CLL oncogenesis, notably apoptosis resistance through the induction of iNOS expression and the production of NO. Our results show that B-CLL cells express TLR-7 and TLR-9. Incubation of B-CLL cells with TLR-7 agonists effectively resulted in an increased resistance to apoptosis that was reverted with the NOS inhibitor l-NMMA. This resistance was associated with enhanced iNOS expression (protein and mRNA) and NO release, stimulation of NF-κB activation, phosphorylation of IκBα, all these events being suppressed with wedelolactone or Bay 11-7085, two inhibitors of IκBα phosphorylation. Our present data thus suggest that TLR-7 signaling stimulates apoptosis resistance, notably through an NF-κB-dependent activation of the NO pathway.

Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response

Recently, we described that vaccination with allogeneic dendritic cells (DCs) pulsed with tumor cell lysate generated specific CD8+ T cell response in patients with B-cell chronic lymphocytic leukemia (B-CLL). In the present study, the potential of autologous DCs pulsed ex vivo with tumor cell lysates to stimulate antitumor immunity in patients with B-CLL in early stages was evaluated. Twelve patients at clinical stage 0-2 as per Rai were vaccinated intradermally up to eight times with a mean number of 7.4 x 10(6) DCs pulsed with B-CLL cell lysate. We observed a decrease of peripheral blood leukocytes and CD19+/CD5+ leukemic cells in five patients, three patients showed a stable disease and four patients progressed despite DC vaccination. A significant increase of specific cytotoxic CD8+ T lymphocytes against the leukemia-associated antigens RHAMM or fibromodulin was detected in four patients after DC vaccination. In patients with a clinical response, an increase of interleukin 12 (IL-12) serum levels and a decrease of the frequency of CD4+CD25(+)FOXP3+ T regulatory cells were observed. Taken together, the study demonstrated that vaccination with autologous DC in CLL patients is feasible and safe. Immunological and to some extend hematological responses could be noted, justifying further investigation on this immunotherapeutical approach.

Arsenic trioxide induces apoptosis preferentially in B-CLL cells of patients with unfavourable prognostic factors including del17p13

In the last decade, arsenic trioxide (As2O3) has been used very successfully to treat acute promyelocytic leukaemia (APL). Much less is known about the effectiveness of As2O3 in other neoplastic disorders. In this paper, we report that after 18 h in vitro treatment with 4 microM As2O3, 75+/-18% of B cell chronic lymphocytic leukaemia (B-CLL) cells (n=52) underwent apoptosis. It is important to note that B-CLL cells harboring a deletion of chromosome 17p13, which predisposes to fludarabine resistance and has been identified as an important negative predictor of clinical outcome, were more susceptible to As2O3 toxicity than cells lacking this aberration. Furthermore, unfavourable risk profiles such as unmutated IgVH status, high CD38 expression and prior treatment were associated with significantly higher sensitivity of B-CLL cells to As2O3. As2O3 also preferentially killed B-CLL cells compared to B cells from healthy age-matched controls. Molecular analysis revealed that basal superoxide dismutase activity was positively correlated with the pro-apoptotic activity of As2O3 pointing to a role of reactive oxygen species in cell death induction. The high activity of As2O3 in B-CLL cells from high-risk patients makes it a promising drug for high-risk and/or fludarabine-refractory B-CLL patients.