B-cell Chronic Lymphocytic Leukemia Patients Research Articles

B-Cell Chronic Lymphocytic Leukemia (B-CLL) Cells Unresponsive to CD180 Ligation Fail to Respond to Anti-IgM Stimulation as Well

AbstractAbstract 3582 Introduction:We have demonstrated that CD180, an orphan receptor of the Toll-like receptor family, is expressed heterogeneously on B-CLL cells, mainly on those with mutated IGVH genes. We further showed that specific ligation of CD180 with mAbs induced activation and cycling of only ~50% CD180+ B-CLL clones (“R”: responders), while CD180+ B-CLL cells unresponsive to CD180 ligation (“NR”: non-responders) or CD180− B-CLL cells could not be activated through either CD40 or IL-4 suggesting anergy. Because CD180 has a short intracellular domain, it presumably, signals through pathways associated with other receptors, such as smIgM. Indeed, engagement of smIgM or CD180 induces Lyn and Syk phosphorylation. Here we compare activation, cycling and phosphorylation of intracellular protein kinases in R and NR and CD180− B-CLL clones and B lymphocytes from healthy subjects upon ligation of smIgM. Methods:B-CLL cells were analyzed for smCD180 and smIgM, and sm CD180+IgM+ B-CLL clones were categorized as R and NR by responsiveness to CD180 ligation. Leukemic clones from 15 smCD180+IgM+R, 14 smCD180+IgM+NR, 12 smCD180−IgM+ untreated B-CLL patients and 14 healthy age-matched individuals were stimulated with goat F(ab')2 anti-human IgM pAbs for 72h, and stained with PE~anti-CD86 mAbs, or fixed, permeabilized and stained with PE~anti-Ki-67 to assess B-cell activation and cycling, respectively. In order to study early intracellular signalling events, cells were stimulated with the same antibodies for 20 min, fixed, permeabilized and stained with Alexa Fluor~rabbit/mouse antibodies to phospho-Akt, phospho-ERK, phospho-p38MAPK, and phospho-ZAP70/Syk. Unstimulated cells in medium were used as controls. Results were assessed by flow cytometry and analyzed with the Mann-Whitney U test and paired t-test where appropriate. Results:ligation of sIgM on smCD180+IgM+R B-CLL cells resulted in a significant increase in CD86+ cells (66.3±21.7% vs 18.7±12.0%, p=0.00004) and Ki-67+ cells (38.9±10.5% vs 11.1±5.9%, p=0.0001) compared to medium controls; this was not different from the increase in activation and cycling of normal B cells (not shown). In contrast, smCD180+IgM+NR B-CLL cells failed to significantly upregulate CD86 in response to anti-IgM pAbs (20.6±13.8% vs 17.6±13.7%, p=0.334) and Ki-67 (8.4±4.6% vs 5.3±1.4%, p=0.063). Interestingly, smCD180−IgM+ B-CLL cells demonstrated diminished CD86 upregulation following sIgM ligation: 36.9±21.7% vs 11.0±4.7% in medium, p=0.058 (difference with smCD180+IgM+R B-CLL, p=0.0069). Cell cycling was also decreased: 9.7±4.1% vs 5.4±3.6% in medium, p=0.015 (difference with smCD180+IgM+R, p=0.0022). The proximal stages of anti-smIgM responses were further studied by intracellular signalling of protein kinases associated with the IgM-signalling pathway. While ligation of sIgM on control B cells and smCD180+IgM+R B-CLL cells resulted in phosphorylation of all four enzymes studied, smCD180+IgM+NR cells failed to signal downstream from ZAP70/Syk following sIgM ligation (Table 1), although there was a greater heterogeneity in smCD180+IgM+R B-CLL responses, compared to normal B cells. Importantly, smIgM ligation of smCD180−IgM+ B-CLL cells did not increase phosphorylation of Erk or p38MAPK, although some such clones responded to smIgM ligation by phosphorylation of ZAP70/Syk and Akt (data not shown).PercentagesControl B cellsR CD180+ B-CLL cellsNR CD180+B-CLL cellsmediumanti-sIgMmediumanti-sIgMmediumanti-sIgMpZAP 70/Syk29.4 ± 7.690.2 ± 2.6 p<0.000155.5 ± 25.872.8 ± 18.1p=0.04857.1 ± 23.774.7 ± 19.9p=0.04pERK23.9 ± 12.152.2 ± 7.0 p<0.000113.9 ± 7.539.8 ± 30.3p=0.006911.6 ± 7.117.1 ± 11.2p=NSp38 MAPK28.3 ± 11.954.9 ± 5.8 p=0.000133.4 ± 19.050.1 ± 20.8p=0.04532.5 ± 20.735.8 ± 13.2p=NSpAkt19.2 ± 10.347.3 ± 5.4 p<0.000123.4 ± 12.147.2 ± 21.1p=0.001917.2 ± 8.122.8 ± 11.5p=NS Conclusions:B-CLL clones that are smCD180+IgM+ but unresponsive to CD180 ligation (~30% of all B-CLL cases) are also unresponsive (anergic) to smIgM ligation measured by intracellular signalling, cell activation and cycling. Meanwhile, smCD180−IgM+ B-CLL clones respond heterogeneously to IgM crosslinking. Disclosures:No relevant conflicts of interest to declare.

B-chronic lymphocytic leukemia chemoresistance involves innate and acquired leukemic side population cells

B-cell chronic lymphocytic leukemia (B-CLL) therapy remains unsatisfactory due to repeated resurgences of the chemoresistant disease. In this study, we investigated the basis of this chemoresistance by applying the 'side population' (SP) analysis to blood samples from B-CLL patients. We report the existence of few natural SP cells, which harbors phenotypic and cytogenetic hallmarks of B-CLL in most patients with this disease (n=22). SP cells appeared resistant to conventional B-CLL treatments, such as Fludarabine, Bendamustin or Rituximab. Indeed, treatment with Fludarabine (16/18 cases) or Bendamustin (5/7 cases) resulted in complete elimination of non-SP, whereas cells displaying the SP phenotype were the only surviving. Although some B-CLL SP cells were innately chemoresistant, chemotherapy by Fludarabine selected not only innate SP cells but also induced some acquired SP cells, which arose from non-SP by drug-driven evolution. This SP selection by chemotherapeutic treatments is further supported by the overall increase of the SP percentage in patients who experienced chemotherapy in the preceding year. Functionally, proliferative stimulation of SP cells was able to partially replenish in vitro the non-SP cell compartment of the B-CLL disease. The chemoresistance of B-CLL relies, in our model, on the cellular heterogeneity of B-CLL SP cells and on their regenerating dynamics.

NOTCH1 PEST domain mutation is an adverse prognostic factor in B‐CLL

<i>NOTCH1</i> PEST domain mutation is an adverse prognostic factor in B‐CLL

Selection of reference genes for quantitative polymerase chain reaction studies in purified B cells from B cell chronic lymphocytic leukaemia patients

The clinical heterogeneity of B-cell chronic lymphocytic leukaemia (B-CLL) makes it necessary to identify potent prognostic indicators to predict individual clinical course and select risk-adapted therapy. In recent years, numerous gene expression models have been suggested as prognostic factors of B-CLL. Today, quantitative polymerase chain reaction (qPCR) is a preferred method for rapid quantification of gene expression and validation of microarray data. The reliability of qPCR data is highly dependent on the use of appropriate reference genes for normalization. To date, no validated reference genes have been reported for the normalization of gene expression in B-CLL. Therefore, the present study was conducted to identify suitable reference genes for gene expression studies in CD19(+) B cells isolated from B-CLL patients' peripheral blood. The stability of ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, MRPL19, TBP and UBC genes was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper-1, which produced highly comparable results. Based on our results, B2M, HPRT1, and GUSB were found to be the most suitable reference genes for qPCR studies in B-CLL patients' peripheral blood B cells.

MDM2 SNP309 promoter polymorphism, an independent prognostic factor in chronic lymphocytic leukemia

The single nucleotide polymorphism SNP309 with a change from T to G in the promoter region of the MDM2 gene is shown to increase the MDM2 protein levels and attenuate the p53 levels and associates with disease progression in several tumors. In this study, the role of the polymorphism was investigated with regard to the clinical outcome in B-cell chronic lymphocytic leukemia (B-CLL). A total of 210 patients with B-CLL were followed for up to 19 yr. The overall survival (OS) of patients with at least one G-allele was significantly shorter when compared with those with two T-alleles (P = 0.024) with a more pronounced difference in patients below the median age. Age at onset of B-CLL was similar irrespective of MDM2 status. The presence of a G-allele in combination with TP53 mutations or unmutated IgVH gene status resulted in an additive risk of death. In this report, with a high proportion of B-CLL patients with an advanced Binet stage and with an unmutated IgVH gene, MDM2 SNP309 was found to be independently associated with OS. The survival difference was more pronounced in younger patients.

Dequalinium induces apoptosis in peripheral blood mononuclear cells isolated from human chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) is an abnormal neoplasic proliferation of B cells, which accumulate mainly in the bone marrow and blood preventing both B cells development in the lymph nodes and the ability to fight against infection. The antitumor agents used in chemotherapy are aimed at inducing malignant cell death, thus limiting the growth and spreading of these cells. However, the lack of specificity for tumor cells exhibited by these agents causes undesirable side effects that have led to the investigation of new therapeutic strategies designed to specifically target malignant cells and thus trigger selective cell destruction. Dequalinium (DQA) is an antitumoral agent that selectively accumulates in the mitochondria and has been shown to display anticancer activity in cells from different malignancies. In the present study, the DQA-induced cytotoxicity in B-CLL cells was analyzed by measuring cell viability and cell death, either by necrosis or apoptosis. Our results support the importance of DQA as a selective and potential antileukemic drug with a higher cytotoxic effect on peripheral blood mononuclear cells from B-CLL patients than in those from healthy donors and encourage the performance of further studies in combination with other agents.

Immune recovery after fludarabine–cyclophosphamide–rituximab treatment in B-chronic lymphocytic leukemia: implication for maintenance immunotherapy

Thirty B-cell chronic lymphocytic leukemia patients were treated with fludarabine-cyclophosphamide-rituximab (FCR) and immune cell counts (natural killer (NK) cells, CD4, CD8, Tgammadelta and monocytes) were monitored from the end of treatment (EOT) up to 36 months (M36). Moreover, nonleukemic peripheral blood lymphocyte cytotoxicity (PBL/CTC) as well as rituximab (RTX)-dependent PBL/CTC was also measured at the initiation of therapy, EOT and M12. These parameters were correlated with post-FCR monitoring of the minimal residual disease (MRD) level in blood using a four-color flow cytometry technique. FCR induced a profound and sustained depletion of all T-cell populations, Tgammadelta being the most affected, whereas NK cells were relatively preserved. Both basal and interleukin-2-stimulated nonleukemic PBL/CTC against MEC-2, a CLL cell line, increased during the post-FCR period. There was no correlation between immune recovery parameters and MRD progression profile, except that patients with high post-FCR CD4(+) counts experienced rapid MRD progression. MRD at M12 predicts clinical relapse. The limited data show RTX-mediated LBL/CTC activity against autologous B-cell cells in individuals with <1% residual disease at M12, opening avenues for immunomodulation post-FCR with anti-CD20 antibodies. To conclude, our study suggests that MRD increase at M12 precedes disease evolution post-FCR, and should be assessed as a surrogate marker for proactive management of CLL relapse.

A new method to detect loss of heterozygosity using cohort heterozygosity comparisons

BackgroundLoss of heterozygosity (LOH) is an important marker for one of the 'two-hits' required for tumor suppressor gene inactivation. Traditional methods for mapping LOH regions require the comparison of both tumor and patient-matched normal DNA samples. However, for many archival samples, patient-matched normal DNA is not available leading to the under-utilization of this important resource in LOH studies. Here we describe a new method for LOH analysis that relies on the genome-wide comparison of heterozygosity of single nucleotide polymorphisms (SNPs) between cohorts of cases and un-matched healthy control samples. Regions of LOH are defined by consistent decreases in heterozygosity across a genetic region in the case cohort compared to the control cohort.MethodsDNA was collected from 20 Follicular Lymphoma (FL) tumor samples, 20 Diffuse Large B-cell Lymphoma (DLBCL) tumor samples, neoplastic B-cells of 10 B-cell Chronic Lymphocytic Leukemia (B-CLL) patients and Buccal cell samples matched to 4 of these B-CLL patients. The cohort heterozygosity comparison method was developed and validated using LOH derived in a small cohort of B-CLL by traditional comparisons of tumor and normal DNA samples, and compared to the only alternative method for LOH analysis without patient matched controls. LOH candidate regions were then generated for enlarged cohorts of B-CLL, FL and DLBCL samples using our cohort heterozygosity comparison method in order to evaluate potential LOH candidate regions in these non-Hodgkin's lymphoma tumor subtypes.ResultsUsing a small cohort of B-CLL samples with patient-matched normal DNA we have validated the utility of this method and shown that it displays more accuracy and sensitivity in detecting LOH candidate regions compared to the only alternative method, the Hidden Markov Model (HMM) method. Subsequently, using B-CLL, FL and DLBCL tumor samples we have utilised cohort heterozygosity comparisons to localise LOH candidate regions in these subtypes of non-Hodgkin's lymphoma. Detected LOH regions included both previously described regions of LOH as well as novel genomic candidate regions.ConclusionsWe have proven the efficacy of the use of cohort heterozygosity comparisons for genome-wide mapping of LOH and shown it to be in many ways superior to the HMM method. Additionally, the use of this method to analyse SNP microarray data from 3 common forms of non-Hodgkin's lymphoma yielded interesting tumor suppressor gene candidates, including the ETV3 gene that was highlighted in both B-CLL and FL.

SCID mice model in vivo evaluation of autologous and allogeneic dendritic cells activity on B-cell chronic lymphocytic leukemia.

In the present study we investigated in vivo therapeutic potential of DCs vaccines in B-cell chronic lymphocytic leukemia (B-CLL). On the day 0 the SCID mice were intraperitoneally inoculated with peripheral blood mononuclear cells (PBMC) of B-CLL patients at a dose of 10-30 x 10(6) and left untreated (controls) or i.p. injected on the day 7 with 0.2 - 14.0 x 10(6) dendritic cells. DCs were generated in vitro from peripheral blood monocytes of B-CLL donors (autologous DCs) or healthy donors (allogeneic cells) and pulsed with B-CLL antigens. On the day 14, the effect of implanted cells interactions was evaluated by a counting of CD19+CD5+ human leukemic cells and human T cells in the peritoneal fluid of mice. We found, that mean numbers of CD19+CD5+ leukemic cells as well as human T cells were lowered in peritoneal fluid of mice treated with allogeneic APCs. However, we did not observe similar effects with autologous DCs.

Abstract 181: Biological and clinical relevance of quantitative global methylation in repetitive DNA sequences in B-cell chronic lymphocytic leukemia

Abstract Background: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by highly clinical heterogeneity; the identification of reliable prognostic factors useful in predicting patient outcome and planning therapeutic strategies represent a crucial task. Global DNA hypomethylation affecting repeat sequences, such as long interspersed nuclear elements-1 (LINE-1), Alu and satellite α DNA (SAT-α DNA), has been reported in different cancer types and associated with chromosomal instability. Aim: In order to investigate the role of global DNA methylation in B-CLL we quantified the methylation levels of Alu, LINE-1 and SAT-α repetitive in B-CLL patients and correlated them with the major cytogenetic and molecular features and clinical relevance in predicting therapy-free survival (TFS). Patients and methods: A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, LINE-1, and SAT-α in a panel of highly purified (&amp;gt;90%) peripheral mononuclear CD19+ cells from 7 healthy donors and 77 B-CLL untreated well-characterized patients in early stage disease (Binet stage A). Results: Forty-eight patients had unmutated IgVH genes; ZAP-70 and CD38 were positive in 28 and 35 cases, respectively; 13q14.3 deletion was present as a sole abnormality in 21/33 patients while in the remaining cases it was combined with 17p13.1 (4 pts) or 11q22.3 deletions (7 pts) or both (1 pt). The 11q22.3 and 17p13.1 deletions were detected as sole abnormality in 6 and 7 patients, respectively. Trisomy 12 occurred in 17 patients, as a sole abnormality in all cases. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; and 84.0, vs. 88.2; for Alu, LINE-1 and SAT-α, respectively). A significant association was observed with 17p13.1 deletion (15.4 vs. 22.4; 42.9 vs. 68.5; 32.7 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Notably, lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with a shorter TFS. Conclusion: Our study extended limited evidence in methylation of repetitive sequences in B-CLL suggesting an important biological and clinical relevance in the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 181.

35 BCR-signaling profiles associated with prognosis and progression in B-CLL

Background. B-cell chronic lymphocytic leukemia (B-CLL) patients exhibit a variable clinical course. Several biological parameters have been shown to be associated with clinical outcome in CLL. Among them, the most reliable markers are represented by the absence of somatic mutations within the immunoglobulin variable heavy chain genes (IGHV), the expression of CD38 antigen, or the presence of the ZAP-70 tyrosine kinase. These parameters of poor clinical outcome are structurally and/or functionally linked to B-cell Receptor (BCR) expressed by CLL cells, thereby strengthening the hypothesis that antigenic stimulation mediated by the BCR represents a driving event in the onset and progression of the malignant B cells. Methods. We applied a “network level” analysis of BCR signaling by measuring single-cell profiles of phosphoprotein networks by flow cytometry. We evaluated the response to BCR engagement in primary cells isolated from 27 CLL patients by analyzing the phosphorylation states of 5 phosphoproteins on the route of BCR signaling, including p-Syk, p-NF-kappaB, p-Erk1/2, p-p38 and p-JNK. BCR was cross-linked by incubating cells with anti-IgM antibodies. Results. Unsupervised clustering analysis distinguished BCR response profiles of phosphoproteins that differentiated cases of CLL with mutated IGHV from those with unmutated IGHV (P=0.0003), cases with low levels of CD38 expression from those with high levels (P=0.0004) and cases with ZAP-70negative leukemic cells from cases that were ZAP-70-positive (P=0.001). Furthermore, the same BCR response profiles were also associated with time to progression (P=0.0014) and with overall survival (P=0.049), as assessed by Kaplan–Meier curves and the log-rank test. Independent survival analysis of time to progression via fitting Cox proportional hazards models comprising clinical covariates and/or BCR network response to modulation demonstrated that measuring modulated BCR network signaling can yield improved prognostic information compared to CD38 status alone (likelihood ratio test 5.8 for CD38 versus 10.6 for signaling) and enhance prognostic assessment using IGHV status (likelihood ratio test for IGHV = 14.8 versus for IGHV + signaling = 17.9).

Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression

AbstractMicrovesicles (MVs) released by malignant cancer cells constitute an important part of the tumor microenvironment. They can transfer various messages to target cells and may be critical to disease progression. Here, we demonstrate that MVs circulating in plasma of B-cell chronic lymphocytic leukemia (CLL) patients exhibit a phenotypic shift from predominantly platelet derived in early stage to leukemic B-cell derived at advanced stage. Furthermore, the total MV level in CLL was significantly greater compared with healthy subjects. To understand the functional implication, we examined whether MVs can interact and modulate CLL bone marrow stromal cells (BMSCs) known to provide a “homing and nurturing” environment for CLL B cells. We found that CLL-MV can activate the AKT/mammalian target of rapamycin/p70S6K/hypoxia-inducible factor-1α axis in CLL-BMSCs with production of vascular endothelial growth factor, a survival factor for CLL B cells. Moreover, MV-mediated AKT activation led to modulation of the β-catenin pathway and increased expression of cyclin D1 and c-myc in BMSCs. We found MV delivered phospho-receptor tyrosine kinase Axl directly to the BMSCs in association with AKT activation. This study demonstrates the existence of separate MV phenotypes during leukemic disease progression and underscores the important role of MVs in activation of the tumor microenvironment.

The Bcl-2 Family as a Rational Target for the Treatment of B-Cell Chronic Lymphocytic Leukaemia

B-cell chronic lymphocytic leukaemia (B-CLL) is the most common lymphoid malignancy in the Western world, characterized by clonal growth and accumulation of monoclonal CD5+ B-cells in peripheral blood, bone marrow and peripheral lymphoid organs. Although the clinical course in B-CLL patients is highly variable, the most conserved feature is the prolonged survival of malignant B-cells, which has been associated to defects in the apoptotic machinery. The apoptosis defects are mainly determined by a defective balance among pro- and anti-apoptotic members of the Bcl-2 family, often related to resistance of CLL B-cells to chemotherapy. Purine nucleoside analogs or alkylating agents, alone or in combination, are the first-line treatment for B-CLL patients. Alternative, more specifically tailored therapeutics have been developed in recent years, including humanized monoclonal antibodies and kinase inhibitors. Here we shall review the drugs which are commonly used or are currently being assessed in clinical trials on B-CLL patients, their chemical structure, mechanisms of action, pharmacological properties, molecular targets, clinical efficacy and side effects, with a focus on drugs designed to promote apoptosis of malignant B-cells by targeting the Bcl-2 family.

Possible prognostic value of nucleolar morphology in pathologic cells of B-chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) represents a heterogeneous disease with a very variable outcome. The reliable prognosis of this disease at the time of initial diagnosis is difficult to predict. The purpose of this preliminary study was to utilize the nucleolar morphology and to investigate the incidence of main nucleolar types in leukemic lymphocytes in B-CLL patients to assess their possible predictive value for the disease outcome, in correlation with immunophenotype parameters. The evaluation of nucleolar morphology of pathologic lymphocytes was performed at diagnosis and during the course of disease. Median follow up period of patients was 16.4 months (range from 2 to 32 months) from diagnosis. The nucleoli were visualized by a simple cytochemical demonstration of RNA and the proportion of main nucleolar types in pathologic lymphocyte population infiltrating bone marrow of 84 patients suffering from B-CLL was analyzed. The presence of ring shaped and compact nucleoli in leukemic lymphocytes divided patients into two subgroups with different outcome of the disease. Malignant lymphocytes of the majority of patients (Group 1, 71 patients, 84.5%) mostly contained ring shaped nucleoli. These patients were in stable phase and did not require any treatment during the follow up. The population of leukemic cells of a small group of B-CLL patients (Group 2, 13 patients, 15.4%) was characterized by the presence of various proportions of pathologic lymphocytes with one large compact nucleolus.Different response to the therapy discriminated the B-CLL patients whose leukemic lymphocytes revealed evident compact nucleoli at presentation, to next two subsets. Four of these patients (Group 2, 4/13, 31%) appeared to be resistant to chemotherapy, others (9/13, 69%) showed response to therapy, though the response time was variable. Leukemic cells with compact nucleolus morphologically resembled prolymphocytes, but hematologically and immunophenotypically did not fulfill the diagnostic criteria for prolymphocyte population. None of our B-CLL patients had the signs of transformation to prolymphocytic or other type of B cell neoplasms during the follow up. Our results indicate the possibility of relationship between the presence of malignant lymphocytes with compact nucleoli and unfavorable outcome in patients with B-CLL. The simplicity and utility of the nucleolar test as a possible prognostic parameter may help to identify the subset of patients with early B-CLL disease that will run a more progressive course.

Patients with B cell chronic lymphocytic leukaemia have an expanded population of CD4+ perforin expressing T cells enriched for human cytomegalovirus specificity and an effector‐memory phenotype

We have previously shown an expansion of cytotoxic antigen-experienced CD4(+)T cells (CTLs) that express perforin (PF) in the peripheral blood of patients with B cell chronic lymphocytic leukaemia (B-CLL). Increased frequencies of CD4(+)CTLs have since been attributed to chronic viral infections, particularly, human cytomegalovirus (HCMV). The present study examined the involvement of CD4(+)CTLs in responses to HCMV in B-CLL, and characterized their differentiation. We studied 36 HCMV seropositive (SP) and seronegative B-CLL patients and 20 healthy age-matched individuals. The HCMV reactivity of CD4(+)PF(+) and CD4(+)PF(-) cells was determined by interferon-gamma expression, and expression of CD45RA and CCR7 was assessed by flow cytometry. Fluorescence in-situ hybridization was used to measure relative telomere lengths. CD4(+)PF(+)T cell expansion in B-CLL patients and controls was strongly associated with HCMV seropositivity. CD4(+)PF(+) compared to CD4(+)PF(-) cells from SP B-CLL patients elicited major histocompatibility complex (MHC) class II-restricted responses to HCMV. CD4(+)PF(+)T cells from patients and controls were enriched with highly differentiated T-effector/memory (CCR7(-)) and revertant (CCR7(-)CD45RA(+)) phenotype. CD4(+)PF(+)T cells from B-CLL patients had shorter telomeres than CD4(+)PF(-)T cells, indicating an extensive replicative history. We conclude that persistent exposure to HCMV antigens in SP B-CLL patients leads to an expansion of the circulating MHC class II-restricted CD4(+)PF(+)T cell population with effector/memory phenotype.

KIR Genes and Their HLA-C Ligands in B-Cell Chronic Lymphocytic Leukemia in a Polish Population.

Abstract 4402 IntroductionDysfunction in cellular and humoral immunity entails an increased risk of B-cell chronic lymphocytic leukemia (B-CLL). It has been suggested that innate immunity, especially natural killers cells play key role in antitumor cytotoxicity regulated by interaction between killer immunoglobulin-like receptors (KIRs) on NK cells and some T-cell subpopulations and their HLA-class I ligands on target cells. KIRs receptors have been divided into two functional groups: with long cytoplasmic tails (DL) - inhibitory and with short cytoplasmic tails (DS) - activating. Two broad haplotypes of KIR genes have been defined: the A haplotype characterized by presence of single activating KIR gene (2DS4) and the B haloptype characterized by two or more activating KIR genes (2DS1, 2DS2, 2DS3, 2DS5 and 3DS1).The 2DL2, 2DL3 and 2DS2 KIRs bind to HLA-C1 allotype (Asp80), while 2DL1 and 2DS1 KIRs bind to HLA-C2 allotype (Lys80). Due to lack of representative data in literature the study was undertaken to determine the association between polymorphism of KIR genes and HLA-C allotypes and susceptibility to B-CLL. Patients and methodsThe following KIR genes were typed: 2DL1, 2DL2, 2DL3, 3DL1, 2DS1, 2DS2, 2DS3, 2DS4fl, 2DS4del, 2DS5, 3DS1, in 200 B-CLL patients and 199 healthy individuals using PCR-SSP method. The HLA-C1 and HLA-C2 were determined using Olerup SSP typing kit in 187 B-CLL patients and 104 controls. ResultsIndividual KIR gene content was similar in B-CLL cases and healthy controls although patients with KIR2DS3 gene were more common among cases compared to controls (36% vs. 27%, p=0.06) (Table1). The frequency of AA haplotype did not differ between studied groups (29% vs. 28%). Moreover no differences were found between incidence of HLA-C allotypes (HLA-C1: 74.3% vs. 76.0%, HLA-C2: 67.4% vs.70.2%), genotypes (C1/C1: 32.6% vs 29.8%, C1/C2: 41.7% vs 46,2% and C2/C2: 25.7% vs 24.1) and KIR genes in combination with genes for their HLA-C ligands in B-CLL patients and controls (Table 2).Table 1Frequency of KIR genes in B-CLL patients and controls.2DL12DL22DL32DS12DS22DS32DS4del2DS4fl2DS53DL13DS1B-CLL (n=200)1951061767510854166565318670[%]97.553.088.037.554.027.083.028.026.593.035.0Controls (n=199)1971061808111171162586118178[%]99.053.390.540.755.835.781.429.130.791.039.2Table.2Frequency of the KIR-HLA gene combinations in B-CLL patients and controls.B-CLL (n = 187)Controls (n = 104)N%N%2DL1+/C2+12265.247370.192DL2/3+/C1+13974.337975.962DS1+/C2+4926.202423.082DS2+/C1+7137.974038.462DL1+/C2-6032.083028.802DL2/3+/C1-4825.692524.042DS1+/C2-2211.761817.312DS2+/C1-1910.371110.58 ConclusionsThe data obtain in this study imply that there may be not direct association between KIR and HLA-C gene content in the genome and the presence of B-CLL. Disclosures:No relevant conflicts of interest to declare.

Intrathecal Liposomal Cytarabine (DepoCyte®) for Treatment of Leptomeningeal Involvement From Transformed (Richter's syndrome) and Non-Transformed B-Cell Chronic Lymphocytic Leukemia (B-CLL) in Spain: Report of 8 Cases.

Abstract 4411 BackgroundLeptomeningeal disease (LD) is an uncommon and severe complication in the course of B-CLL and may occur particularly in the course of transformation into an aggressive lymphoma, so-called Richter's syndrome. Commonly, repeated administrations of intrathecal (IT) chemotherapy with either methotrexate or cytarabine is the mainstay treatment in these B-CLL patients (pts). IT chemotherapy with standard agents is limited by the need for multiple injections via lumbar puncture or an Ommaya reservoir. A depot formulation of liposomal cytarabine (DepoCyte®) has already demonstrated to be well tolerated and effective in the treatment of lymphomatous meningitis. However, there are no studies that have analyzed the efficacy of liposomal cytarabine in the treatment of leptomeningeal involvement from B-CLL and/or Richter′s syndrome. AimA multicenter and retrospective study was performed in pts with meningeal involvement from transformed (Richter's syndrome) and non-transformed B-CLL in which IT depot liposomal cytarabine was employed as compassionate therapy. All the cases were evaluated for cytological negativization of the cerebro-spinal fluid (CSF) from neoplastic cells and improvement of neurological symptoms. Patients and MethodsFrom October 2005 to February 2009, eight patients (median age 66 years; range 44–81; 3 females) treated with liposomal cytarabine at the standard dose of 50 mg every 15 days, for a total of 39 doses (median cycles/pt: 5; range 1–9) were recorded. Six patients had a diagnosis of Richter′s syndrome from B-CLL [ (diffuse large B-cell lymphoma (DLBCL; n = 5); or secondary involvement of Central Nervous System (CNS) from acute myeloid leukaemia (AML) (n = 1)] and the remainder (n = 2) were diagnosed of non-transformed B-CLL. At time of meningeal involvement, the eight patients were also in hematological relapse. The patients had been previously treated by cytotoxic chemotherapy according to the current protocols; intrathecal prophylaxis had not been administered previously. In all patients neurological symptoms were present; lymphomatous cells were identified in the CSF of 7 patients (median, 296/μL; range, 3–1440/μL) and CNS localizations were detected by CT scan and/or MR imaging in 2 patients. The treatment with liposomal cytarabine (50 mg IT every 15 days) was associated with systemic chemotherapy in 7 patients [ R-CHOP (n = 1), CHOP (n = 1), R-FC (n = 1), high or intermediate methotrexate-based regimens in combination with cytarabine and vincristine (n = 3) or only chlorambucil in 1 elderly pt]. The patient with AML meningeal involvement was treated only with DepoCyte®. Six patients received concurrent dexamethasone therapy as prophylaxis for chemical arachnoiditis; oral dexamethasone (4 mg bid for 5 days per cycle) (n = 1), IV dexamethasone at varying dosages (n = 2) or IT dexamethasone (4 mg) (n = 3). ResultsThe patient with AML meningeal relapse was not evaluable for response because he received only 1 administration of liposomal cytarabine before dying for disease progression. In seven evaluable cases, overall neurological and cytological responses were obtained in five cases with Richter′s syndrome (71.4%; 3 complete responses, 2 partial responses) whereas 2 complete responses (100%) was observed in B-CLL pts. Median time from treatment to cytological response was 15 days, (range, 2 - 60). Neurological progression was not subsequently reported in any patients (median follow-up 5 months; range 1 – 12). Two patients were still alive and in CR at the time of reporting, including 1 of 7 patients with Richter's syndrome, whereas the remaining six died from systemic relapse (5) or infection (1), without evidence of neurological progression at hematological disease progression. No adverse effects due to the IT administration of liposomal cytarabine were reported. No dose reduction was required for toxicity. ConclusionsThese preliminary data indicate that IT liposomal cytarabine is effective in clearance of tumoral cells from CSF in transformed and non-transformed B-CLL patients. The association with systemic chemotherapy has been demonstrated to be suitable and well tolerated. Studies with higher number of patients and longer-term follow-up will be warranted to evaluate the efficacy and safety of IT depot liposomal cytarabine in meningeal involvement from B-CLL and Richter′s syndrome. Disclosures:No relevant conflicts of interest to declare.

Prelinical Evaluation of Lyn Kinase Inhibition for the Treatment of B Cell Chronic Lymphocytic Leukemia (B-CLL).

Abstract 3447Poster Board III-335B-CLL is characterized by the accumulation of mature B lymphocytes incapable of undergoing apoptosis. Although several mechanisms have been implicated in the apoptotic defects in B-CLL cells, the signal transduction pathways underlying these defects remain unresolved.Lyn kinase is known to be a negative regulator of apoptosis, and is linked to chemo-resistance. Our preliminary study indicated that Lyn activity was 4- to 7-fold higher in primary B cells from six randomly-selected B-CLL patients than in normal B cells from healthy donors. Treatment of the B-CLL cells for 4 h with 10 μM concentration of a Lyn-specific inhibitor peptide targeting a unique interaction site within Lyn (Cancer Res. 2004;64:1058) resulted in 40 to 50% inhibition of Lyn kinase activity compared to negligible inhibition with control peptide. Further, treatment of the B-CLL cells for 16 to 20 h with the Lyn inhibitor peptide at 5 μM and 10 μM concentrations, decreased the cell viability by ∼25% and ∼50%, respectively, compared with no peptide or the control peptide. Fludarabine is one of the key chemotherapeutic agents for B-CLL and is known to induce cell death by apoptosis. The combined treatment with Lyn inhibitor peptide (5 μM) and fludarabine (2 μg/ml), decreased cell viability by ∼50% compared to ∼25% decrease with fludarabine or Lyn inhibitor peptide alone. In addition, the combined treatment showed ∼75% increase in caspase-3/7 activity compared to ∼25% increase with the Lyn inhibitor peptide or fludarabine alone. Because, overexpression of antiapoptotic proteins is correlated with apoptotic resistance of B-CLL cells, we examined the effect of Lyn inhibitor peptide on the changes in the expression of antiapoptotic genes, myeloid cell leukemia-1 (Mcl-1), x-linked inhibitor of apoptosis (XIAP), and B-cell leukemia/lymphoma-2 (Bcl-2). Treatment of B-CLL cells with 10 μM Lyn inhibitor peptide for 16 to 20 h resulted in more than 50% decreases in the expression of all the three antiapoptotic genes. Further, the Lyn inhibitor peptide markedly inhibited NF-κB activation (∼60%) and VEGF production (∼80%), both strongly implicated in the apoptotic resistance of B-CLL cells. Collectively, our results suggest that targeting Lyn kinase pathway with a clinically relevant Lyn kinase inhibitor may have a therapeutic potential for B-CLL. DisclosuresNo relevant conflicts of interest to declare.

Acadesine (Acadra‘) for Patients with Resistant/Relapsed Chronic B-Cell Lymphocytic Leukemia (B-CLL): A Multicentre Phase I/II Study.

Abstract 4405Acadesine (Acadra‘) is a nucleoside that induces cell death in B-cell chronic lymphocytic leukemia (B-CLL) cells ex vivo in a dose-dependent manner over the concentration range 50 mM to 1 mM. Acadesine enters B-cells and is phosphorylated to its active metabolite, ZMP, which induces apoptosis independently of ATM or p53. It is active against B-CLL cells from patients who have not responded to prior treatment with fludarabine and/or chlorambucil (Campas et al, 2003).Based on this pre-clinical data, a Phase I/II clinical study was designed to determine the safety and tolerability of acadesine given intravenously as a 4-hour infusion to patients with B-CLL. This is an open-label dose escalation study of acadesine with two parts. In Part I, the dose escalation part of the study, patients receive a single dose of acadesine on Day 1 and are followed up to Day 22. In Part II, patients will receive up to 5 doses of acadesine at the maximum tolerated dose (MTD) identified in Part I over a period of up to 20 days starting on Day 1. The patient population includes B-CLL patients with refractory or relapsed disease who have received one or more (≥ 1) prior lines of treatment which must have included either a fludarabine or an alkylator based regimen. Primary endpoints of the study evaluate the safety and tolerability of acadesine. Secondary endpoints evaluate the pharmacokinetics of acadesine in plasma, its metabolite ZMP in whole blood, and changes in peripheral blood B-cell and T-cell counts as efficacy biomarkers. Optimal Biological Dose (OBD) is defined as the minimal acadesine dose that gives a maximum Cmax for ZMP in whole blood with a good safety profile.Twelve patients (6 males and 6 females) have been administered acadesine to date in 3 cohorts in Part I. Acadesine has an excellent safety profile to date. The principal safety finding was asymptomatic acadesine-related hyperuricaemia of short duration in four patients. Prophylactic allopurinol has significantly reduced the incidence of hyperuricaemia.Pharmacokinetic data showed acadesine rapidly disappears from plasma to be converted into ZMP inside blood cells. The OBD has not been reached yet and maximum dose administered to date is 139.5 mg/kg. The next cohort will receive 210 mg/kg. Additional safety, pharmacokinetics and efficacy data will be presented at the meeting. Disclosures:Off Label Use: Acadesine (AcadraTM): nucleoside that induces cell death in B-cell chronic lymphocytic leukemia.. Auton:BTG Limited: Consultancy; Advancell Technologies Inc.: Consultancy. Saunders:BTG limited: Consultancy; Advancell Technologies: Consultancy. Campas:Advancecell Technologies Inc: Employment, Equity Ownership.

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display

AbstractAllogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.