Cells Of CLL Patients Research Articles

CD1d expression is higher in chronic lymphocytic leukemia patients with unfavorable prognosis

Through the analysis of CD1d expression by flow cytometry and qRT-PCR we showed lower CD1d molecule and CD1d mRNA expression in B cells of CLL patients than of healthy controls. The frequency of CD1d(+)/CD19(+) cells, CD1d staining intensity and CD1d transcript levels increased with the disease stage. CD1d expression was positively associated with ZAP-70 and CD38 expressions as well as with unfavourable cytogenetic changes. We established the relationship between high CD1d expression and shorter time to treatment and overall survival. We observed that CD1d expression in individual patients significantly changed over time. The percentage of CD1d(+)/CD19(+) cells inversely correlated with the percentage of iNKT cells.

Metaherin Contributes To The Pathogenesis Of Chronic Lymohcytic Leukemia

IntroductionDysregulation of proliferation and apoptosis is associated the pathogenesis of CLL. More recently, Metadherin (MTDH) involved in aberrant proliferation, survival, and increased migration, invasiveness, and metastasis of tumor cells, has been demonstrated as a potential crucial mediator of various types of huamn malignancies. MTDH promotes tumor progression by modulating multiple oncogenic signaling pathways (NF-kB, PI3K/Akt and Wnt/beta-catenin). However, there is no report about the role of MTDH in CLL. Since Wnt signaling pathway had been proven to be unusual activated in CLL, the objective of this study was to investigate the role of MTDH in CLL and the relationship between MTDH and Wnt/beta-catenin signaling pathway. MethodsPeripheral blood mononuclear cells (PBMCs) came from samples of 31 CLL patients. The characteristics of CLL patients were shown in Table 1. CD19+B cells were selected from peripheral blood of age-matched heathy donor, cord blood, bone marrow and tonsil of normal controls using CD19+ magnetic selection kits and detected the purity with anti-CD19-PE antibody by flow cytometry. Qantitative PCR and Western blot were used to detect the expression of mRNA and protein for MTDH, and the key functional components of Wnt/beta-catenin signaling pathway (beta-catenin and LEF-1). We also measured MTDH level in B cells by flow cytometry after intracellular staining. CLL cell line(MEC-1) were infected by lentivirus to interfer MTDH and the infection efficiencies were determined by fluorescence microscope and flow cytometry. Both primary CLL cells and MEC-1 were exposed to 10ug/ml goat F(ab`)2 anti-human IgM for 48hours to mimic activation of BCR. The proliferation and apoptosis of these cells were evaluated by CCK-8 method and Annexin V kits.Table 1Characteristic of patientsCharacteristicnTotalAge<=6018>601331SexMale25Female631Rai StageI13II12III4IV331CD38Positive1Negative2324ZAP-70Positive2Negative2123TP53Positive2Negative1820 ResultsmRNA of MTDH in PBMCs of 31 CLL patients were overexpression compared with CD19+ B cells coming from 15 age-matched healthy donors (Figure 1A). 27 out of 31 CLL samples were detected MTDH expression in protein level but none in normal controls (Figure 1B). The expression of MTDH was associated with Rai staging of CLL. There were no MTDH detection in CD19+ B cells collected from bone marrow, peripheral blood, tonsil and cord blood, which stand for precursor, mature, germinal center, and lineage B cells, respectively. The transfection efficiency of MEC-1 cells by interfering MTDH expression with Lentivirus was shown in Figure 1C. The level of MTDH knockdown was accompanied with LEF-1 downregulation (Figure 1D, 1E), as well as the downregulation of c-myc and cyclinD1 expression (Figure 1F). siRNA targeting MTDH treatment in MEC-1 decreased the proliferation and increased the apoptosis(Figure 2A, 2B). We further observed that the proliferation and MTDH expression both in CLL cells and MEC-1 were upregulation after stimulation of anti-human IgM (Figure 2C, 2D, 2E). This effect in the proliferation was blocked by MTDH inteference (Figure 2F). [Display omitted] [Display omitted] ConclusionsOur results demonstrated that MTDH is aberrant expression in B cells of CLL patients and correlated with clinical staging of CLL. MTDH was not expression in any subsets of normal B cells. MTDH may exert a preservative role through activation of Wnt signaling pathway. The CLL cell proliferation activation by BCR signaling pathway may be inhibited by MTDH interference. Our findings indicated that MTDH may be a potential therapeutic target of CLL. Disclosures:No relevant conflicts of interest to declare.

91-P: KIR GENOTYPE DISTRIBUTION AMONG PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: IS THERE A ROLE FOR KIR 2DS4 AND KIR 2DS5 GENES?

Aim Study KIR expression of NK cells in CLL patients and in healthy controls, to check for any association between KIR genotypes and CLL. Methods KIR genotype was analysed for 120 healthy Lebanese patients and 56 CLL patients. using the KIR Genotyping SSP kit. Results KIR genotypic profile distribution among the 56 Lebanese patients with CLL is shown in Table 1 .[figure1] Table 2 shows the distribution of different KIR genotypes among the 120 healthy Lebanese controls. The content of KIR genes ranged from 6 to 15 and, as per Table 4, the AA, AB, and BB genotypes frequencies were, respectively, 42.5%, 50%, and 7.5% with an A:B ratio of 2.08:1. Table 3 shows the distribution of different KIR genes among the 120 controls and the 56 CLL patients.[figure2] KIR 2DL4 , KIR 3DL2, and KIR 3DL3 were present in all individuals. KIR 2DS4 ∗ 001/002 and KIR 2DS5 were found to be significantly (with a p-value of 0.02 and 0.04, respectively) more prevalent among CLL patients as compared to controls. Conclusions The interesting observation of the significant presence of KIR 2DS4 and KIR 2DS5 genes more among Chronic Lymphocytic Leukemia than controls is worth further clinical and translational research in the future as well as correlation with outcome of bone marrow transplantation in these cases.

Intracellular cytokine expression in T cells from patients with chronic lymphocytic leukemia

Functional disorders of T lymphocytes play an essential role in abnormal immune response in patients with chronic lymphocytic leukemia. The aim of this study was to assess the profile of cytokines expressed by T cells derived from patients with CLL. We have demonstrated that the intracellular levels of IL-2, IL-4, IFN-γ, TNF, IL-6 and IL-10 were significantly higher in T cells of CLL patients than in healthy donors. Moreover, the percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-γ+, and CD4+/CD3+/IL-2+ cells were significantly higher in ZAP-70-positive patients compared with ZAP-70-negative ones. Likewise, significantly higher percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-γ+ cells were observed in CD38-positive than in CD38-negative cases. What is more, there was a significant difference in median percentage of CD3+/CD4+ cells expressing TNF, IL-4, IFN-γ, IL-2 or IL-6 between patients carrying the 11q22.3 deletion and/or the 17p13.1 deletion and patients without these genetic aberrations. Our results confirm the functional disorders of T cells in CLL and their influence on the clinical course of the disease.

Circulating microenvironment of CLL: Are nurse-like cells related to tumor-associated macrophages?

B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hematologic malignancies in Western countries. Accumulation of leukemic lymphocytes in peripheral blood, bone marrow and secondary lymphatic organs of CLL patients is due to decreased apoptosis rather than to increased proliferation. The former is driven by signals from a specific microenvironment, created by stromal cells of mesenchymal origin, follicular dendritic cells, T lymphocytes and others. Nurse-like cells (NLCs) were first described to differentiate from peripheral blood mononuclear cells of CLL patients in vitro, then they have been also found in proliferation centers of their lymphatic tissues. Like tumor-associated macrophages (TAMs) in solid tumors, nurse-like cells promote survival of CLL lymphocytes. NLC gene expression patterns suggest their similarity to TAMs and differ between patients depending on ZAP70 protein expression status. NLC number in vitro corresponds with CD14 expressing cell count and beta-2-microglobulin serum level, and positively correlates with leukemic lymphocyte viability. As NLCs strongly express genes for adhesion molecules and secrete chemokines of antiapoptotic activity, they should be considered as a target for anti-microenvironment therapy of this incurable disease.

T Cell Receptor Gene Repertoire Restriction in Chronic Lymphocytic Leukemia with Stereotyped IGHV4–34/IGKV2–30 Antigen Receptors

AbstractAbstract 3908Chronic lymphocytic leukemia (CLL) exhibits a remarkably skewed immunoglobulin (IG) gene repertoire mainly evident in the existence of subsets of patients with quasi-identical IGs in their B cell receptors (BcRs), collectively accounting for one-third of CLL patients. BcR stereotypy is strongly suggestive of clonal selection by a restricted set of antigens. However, it is not yet clear at which phase of clonal evolution these antigens act, or whether the stimulation is persistent. Furthermore, the possible role of antigens in the selection and activation of cognate T lymphocytes remains obscure yet highly relevant, given recent data about T cell interactions with CLL B cells and their tolerized behavior. Here, we analyzed the repertoire of T cell receptor β chain genes (TRB) in CLL expressing stereotyped IGHV4–34/IGKV2–30 BcR IGs (subset #4), which exhibit a series of immunogenetic features, such as pronounced intraclonal diversification of IG genes, suggestive of ongoing interactions with (auto)antigens. Furthermore, subset #4 CLL cells have distinctive functional responses to BcR and/or Toll-like receptor triggering, rendering this subset a paradigmatic example for seeking evidence of antigen selection also within the T cell population. We analyzed 18 peripheral blood samples of 12 untreated subset #4 patients (samples from different time points were analyzed in 4 cases). No case had evidence of infection at sampling. PCR amplicons for TRBV-TRBD-TRBJ gene rearrangements (BIOMED2 protocol) were subcloned by transformation into E. coli/TOP10F bacteria and randomly chosen individual colonies were subjected to Sanger sequencing. Only productive rearrangements (n=320, ranging from 14–52/case) were analyzed. All cases were found to carry clusters of identical rearrangements (≥2) corresponding to distinct clonotypes; the number of expanded clonotypes/case ranged from 1–13 (median 5). The relative frequency of each clonotype/case was determined by dividing the number of the corresponding identical sequences by the total number of subcloned sequences analyzed. The frequency of the most expanded (immunodominant) clonotype/case ranged from 8.1–70.4%. Collectively, the frequency of all expanded clonotypes/case ranged from 29.7–93.3%. In 2/4 cases that were analyzed at different time points, at least one clonotype was found to persist. Importantly, cluster analysis of the TRB CDR3 sequences of all cases identified ‘public' clonotypes: 2 identical clonotypes (TRBV15*02/TRBD1*01/TRBJ2–2*01 and TRBV30*01/TRBD1*01/TRBJ2–2*01) each shared by a pairs of different patients and a highly similar clonotype shared by an additional pair of patients. In conclusion, the present study provides clear evidence of repertoire skewing among T cells in CLL patients belonging to subset #4, strongly supporting antigen selection. The finding of ‘public' clonotypes raises the possibility that shared antigenic epitopes may be relevant for clonal selection of T cells in different subset #4 cases. Whether the antigens that drive T cell repertoire restriction are identical/related to those implicated in the selection of CLL progenitors of subset #4 or even the malignant cells themselves or whether they are tumor-associated antigens remains to be clarified. Disclosures:No relevant conflicts of interest to declare.

Th17/IL-17 Might Play a Protective Role in CLL Immunity

Abstract 2843 Background:Immune system disorders play an important role in the development and progression of CLL. Many deficiencies, contributing to disease progression, were found in T cell populations. Th17 cells represent a recently-discovered distinct lineage of helper T-cells, characterized by IL-17 secretion. IL-17 play an active role in inflammation and autoimmune diseases, however the role of IL-17 and Th17 cells in CLL immunopathogenesis remains undefined. We investigated IL-17A mRNA and IL-17A protein expression in peripheral blood (PB) CD4+ T cells and the plasma levels of IL-17 in 70 untreated patients with CLL in the context of clinical and immunological parameters. Th17 cell percentage was measured also in the bone marrow of CLL patients. The control group consisted of 20 healthy age-matched subjects. Materials and methods:Quantitative ‘real-time' reverse transcription-polymerase chain reaction (qRT-PCR) was used for the analysis of IL-17A mRNA in PB CD4+ T-cells. Intracellular IL-17A expression in CD3+/CD4+cells was analyzed using a three-color flow cytometry technique. Plasma levels of IL-17A, IL-4 and IL-10 were measured by ELISA. Results:None of the CD4+ T cells in the samples obtained from healthy volunteers (HV) contained detectable amounts of IL-17 mRNA, whereas it could be found in 5 out of 70 CLL samples. All CLL patients with detectable IL-17 mRNA in T-cells were in 0 Rai stage and negative both for ZAP-70 and CD38 expression. It is possible that IL-17 mRNA in these cells is expressed only for a short time thus evading detection by PCR, or that CLL cells contain low amounts of the IL-17 transcript and the RT-PCR method was not sufficiently sensitive to detect these small amounts. Frequently, in HV as well as in CLL patients, the percentage of CD4+ T cells with intracellular IL-17 expression in non-activation assays was lower than 1%, comparable to the level of autofluorescence. Despite the fact that IL-17 mRNA was not detected in the T cells of the majority of CLL patients, IL-17 protein was present in T cells after PMA and ionomycin stimulation. We found a significantly higher median percentage of CD4+/CD3+/IL-17+ cells in CLL patients (18.2%) than in HV (2.9%) (p<0.01). Median percentage of Th17 cells was significantly higher in CLL patients in stages 0–1 according to Rai, as compared with stages 2–4 (36.2% vs 4.5%, p<0.05). There were no differences in Th17 percentages between the PB and BM of CLL patients. Accordingly, the IL-17 plasma level was significantly higher in CLL patients than in HV (101.9 pg/ml vs 75.6 pg/ml; p<0.05) and there was a significant correlation between Th17 cell percentage and IL-17 plasma level. There was also significant correlation between the percentages of Th17 and iNKT cells (R=0.56; p<0.05), and inverse correlations between the percentage of Th17 and: Treg cells (R=−0.28; p<0.05), CD4+IL-4+ (R=−0.55; p<0.05) and CD4+TNF+ T cells (R=−0.59; p<0.01). The percentage of Th17 inversely correlated with β2-microglobulin serum level (R=−0.29; p<0.05) and the expression of ZAP-70 (R=−0.244; p<0.05) and CD38 (R=−0.33; p<0.01). The plasma level of IL-17 inversely correlated with the stage of disease (R=−0.23; p<0.05), IL-10 (R=−0.56; p<0.01) and TNF (R=−0.66; p<0.01) plasma levels, CD38 antigen (R=−0.25; p<0.05) and ZAP-70 expression (R=−0.28; p<0.05). In patients requiring therapy during the observation period, the median percentage of Th17 cells and median plasma IL-17 level were significantly lower comparing to untreated ones (5.7% vs 23.1%, p<0.05; 53.8 vs 95.6 pg/ml, p<0.05; respectively). Moreover, median plasma IL-17A level correlated with the time from CLL diagnosis to the start of therapy (R=0.49; p<0.01). Conclusions: both the Th17 cell percentage, and the IL-17 plasma level are significantly higher in the PB of untreated CLL patients compared with the HV, and IL-17 mRNA expression was found only in the T cell of CLL patients, but not in the control group. Correlations found with disease activity parameters, the subsets of T cells involved in tumor surveillance, and also with the length of time from diagnosis to the start of therapy, suggest an important protective role for Th17 cells in CLL immunity. Disclosures:No relevant conflicts of interest to declare.

Juxtaposing CD20 and CD74 with Novel Bispecific Antibodies Evokes Potent Cytotoxicity in Mantle Cell Lymphoma (MCL)

Abstract 599 Introduction:Initial response rates with first-line conventional or high-dose chemotherapy, are high in MCL, but most patients relapse. With 3,500 new cases detected every year in the US, there is an unmet medical need for new therapeutic interventions in MCL. Combination of two different targeting mAbs to achieve improved efficacy without increased toxicity was demonstrated first in NHL patients given both rituximab (anti-CD20 chimeric antibody) and epratuzumab (humanized anti-CD22 IgG). Recently, the potential advantage of targeting both CD20 and CD74 was reported in a preclinical study showing that combining rituximab and milatuzumab (humanized anti-CD74 IgG) together with a crosslinking antibody resulted in anti-tumor activity in MCL lines and patient samples in vitro (Alinari L, et al. Blood 2011; 117:4530-41). Here we describe the generation of two novel bispecific hexavalent antibodies [HexAbs; IgG-(Fab)4] constructed from veltuzumab (humanized anti-CD20 IgG) and milatuzumab (anti-CD74 IgG), and show both are capable of inducing potent cytotoxicity in mantle cell lymphoma (MCL) and other lymphoma/leukemia cell lines, as well as primary MCL and CLL patient samples, without requiring a crosslinking antibody. To identify these HexAbs, we assign each of them a code of X-(Y)-(Y), where × and Y are specific numbers given to differentiate the antibodies, and a designated number enclosed in a parenthesis representing the antibody as a stabilized Fab dimmer (e.g., 20-(74)-(74) designates the bispecific HexAb comprising a divalent anti-CD20 IgG of veltuzumab and a pair of stabilized dimers of Fab derived from milatuzumab). Methods: The two bispecific HexAbs, 74-(20)-(20) and 20-(74)-(74), as well as the two monospecific HexAbs, 74-(74)-(74) and 20-(20)-(20), were generated by the Dock-and-Lock method by reacting cognate CH3-AD2-IgG and CH1-DDD2-Fab modules under mild redox conditions and purified by Protein A. The in vitro activities were determined by cell viability and Annexin V binding assays. In addition, the role of signal transduction, homotypic adhesion, actin reorganization, and lysosomal volume changes were evaluated. Results: Each HexAb was purified to >95 % homogeneity and were stable in vitro and in serum. Both 20-(74)-(74) and 74-(20)-(20) potently inhibited the growth of MCL lines, JeKo-1, Granta-519 and Mino, at 10 nM. In contrast, neither parental antibody, alone or in combination, nor the two monospecific counterparts, 74-(74)-(74) and 20-(20)-(20), inhibited the growth of JeKo-1 under the same conditions, suggesting the requirement of apposing CD74 and CD20 for the observed cytotoxicity. Treatment of primary MCL and CLL patient cells with 74-(20)-(20) and 20-(74)-(74) induced 25–30% apoptosis, compared to 10–15% apoptosis with parental IgGs, alone or in combination. The two bispecific anti-CD20/CD74 HexAbs, but not the parental mAbs, induced strong homotypic adhesion, actin reorganization to cell-cell junctions, loss of mitochondrial membrane potential, generation of ROS, deactivation of the PI3K/Akt signaling pathway, as well as rapid and sustained activation of ERK and JNK MAPKs, and enlargement of lysosomes followed by release of cathepsin B in target cells. In SCID mice bearing JeKo-1 xenografts, both 20-(74)-(74) and 74-(20)-(20) were effective at the 370-μg dose level, resulting in 60% and 30% increases in median survival time, respectively, compared to saline controls (P = 0.001). Conclusion: Juxtaposing CD20 and CD74 in close proximity by HexAbs induces potent in vitro cytotoxicity in MCL lines and primary samples. These novel bispecific mAbs warrant further evaluation in B-cell lymphomas that are refractory or poorly responsive to anti-CD20 antibodies. Disclosures:Gupta:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Rossi:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment. Furman:Centocor Ortho Biotech Research & Development: Research Funding. Chang:Immunomedics, Inc.: Employment, Equity Ownership, Patents & Royalties.

Fc-Engineered NKG2D-IgG1 Fusion Proteins Target Leukemia Cells for Antibody-Dependent Cellular Cytotoxicity (ADCC) of NK Cells

Abstract 1537The capability of anti-tumor antibodies to recruit Fc-receptor (FcR) bearing effector cells like NK cells, a feature considered critical for therapeutic success, can be markedly improved by modifications of the human IgG1 part. At present, Fc-engineered antibodies targeting leukemia cells are yet not available. The various ligands of the NK cell-activating immunoreceptor NKG2D (NKG2DL) are generally absent on healthy cells but upregulated on malignant cells of various origins including leukemia. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as target-antigens for Fc-optimized NKG2D-IgG1 fusion proteins targeting leukemia cells for antibody-dependent cellular cytotoxicity (ADCC) and IFN-g production of NK cells. NKG2D-IgG1 fusion proteins with distinct modifications in their Fc portion were generated as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT), the mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcgRIIIa receptor but comparable binding to NKG2DL-expressing target cells. Functional analyses with allogenic NK cells and leukemia cell lines as well as primary leukemic cells of AML and CLL patients revealed that NKG2D-Fc-KO significantly (p<0.05, Mann-Whitney U test) reduced NK cytotoxicity and IFN-g production (about 20% and 30% reduction, respectively), which can be attributed to blockade of NKG2DL-mediated activating signals. Treatment with NKG2D-Fc-WT significantly (p<0.05, Mann-Whitney U test) enhanced NK reactivity (about 20% and 100% increase in cytotoxicity and cytokine production, respectively). The effects observed upon treatment with NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT resulting in at least doubled NK ADCC and IFN-g production compared to NKG2D-Fc-WT. When applied in combination with Rituximab in analyses with CLL cells, a clear additive effect resulting in a more than four-fold increase of ADCC and FcgRIIIa-induced IFN-g production was observed. The NKG2D-Fc fusion proteins did not induce NK reactivity against healthy blood cells, which is in line with the tumor-restricted expression of NKG2DL. Of note, treatment with NKG2D-Fc-ADCC also significantly (p<0.05, Mann-Whitney U test) enhanced reactivity (up to 70% increase) of NK cells against NKG2DL-positive AML and CLL cells among patient PBMC in an autologous setting. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing leukemia cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to their highly increased affinity to the FcgRIIIa receptor, NKG2D-Fc-ADCC potently enhances NK anti-leukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, Fc-modified NKG2D-Ig may thus constitute an attractive means for immunotherapy of leukemia. Disclosures:No relevant conflicts of interest to declare.

Defective Triggering of NK Cells Results in Primary CLL Cells Resistance to Cytotoxicity,

Abstract 3876Chronic lymphocytic leukemia (CLL) remains an incurable disease except after allogenic transplantation. Natural killer (NK) cells are one of the main effectors of immune surveillance involved in tumor control. Alterations of NK cells functions have been well characterized in myeloid malignancies. However the role of NK cells in immune escape of CLL in less known and controversial. Here we describe extensive phenotypic and functional characterization of NK cells and primary CLL cells and their interactions in vitro and in vivo.Twenty eight untreated CLL patients, twenty four age-matched healthy donors and ten AML patients were enrolled in the study.We have previously shown that expression and function of NK cell-triggering receptors is defective in AML. We then assessed the phenotypic and functional properties of NK cells from CLL patients. Unlike the results found in AML, no significant differences were observed in term of activating receptors, NKp46, DNAM-1, NKG2D, 2B4 and CD16. Only the natural cytotoxicity receptor (NCR) NKp30 was weakly decreased compared to healthy donors (p=0.0107). There wasn't any difference in the expression of inhibitory receptors CD158a, b, e, ILT2 and NKG2A. Looking at the spontaneous NK-mediated cytotoxicity, CLL NK cells displayed a cytolytic activity similar to that of healthy donors against K562 cell line. To further evaluate the functional consequences of the decreased expression of NKp30, mAb redirected killing assays was performed against P815 cell lines. The NK cells killing was slightly lower in CLL patients compared to healthy donors when anti-NKp30 was used although no difference could be observed with anti-NKp46 and anti-CD16. All these results supported that NK cells cytotoxicity should be effective in CLL.We then studied the susceptibility of CLL B cells to allogenic NK killing both in vitro and in vivo. Unlike AML cells and K562 cells, CLL cells were resistant to NK cytotoxicity mediated by resting cells. Exogenous stimulation of allogenic NK cells with IL2 and IL15 restored partially CLL killing, which was nevertheless still lower than AML blasts and K562 cells killing (p=0.0288 and <0.0001 respectively). Murine xenotransplantation model using NOD/SCID g null (NSG) mice allowed us to study the anti-leukemic capacity of purified NK cells after activation with IL2. We didn’t observe any clearance of CLL cells after allogenic NK cell injection while CLL and NK cells were checked to be present in blood, bone marrow, spleen and liver. These experiments confirmed the CLL resistance to NK-mediated killing.To investigate the potential mechanisms of this resistance, we analyzed the surface expression of ligands for activating and inhibitory NK receptors on CLL cells. CLL cells displayed poor expression of ligands for activating NK receptors MICA/B, ULBP1-3, PVR, nectin-2 and CD54. Interestingly, this profile of surface expression was similar to that of normal B cells except a slight increase of ULBP3 expression on CLL cells. Regarding ligands for inhibitory NK receptors, HLA-class I molecules were significantly down-regulated while HLA-E tended to be up-regulated on CLL cells compared to normal B cells.Finally, we tested ADCC in order to overcome the resistance of CLL cells to NK killing: the presence of rituximab increased significantly CLL lysis. Of note, priming of NK cells with IL2+IL15 still increased CLL cytotoxicity (p<0.0001).Our findings demonstrate that primary CLL cells are resistant to NK mediated killing. This defect is mainly due to the lack of ligands for NK receptors on CLL cells surface leading to deficient triggering of NK cells. However NK cells of CLL patients are fully competent. Attempts to optimize NK cell therapy for treatment of CLL will require overcoming the low immunogenicity of B-CLL cells. Our xenograft model provides the tools for such preclinical development. Disclosures:No relevant conflicts of interest to declare.

Assessment of the pathway of apoptosis involving PAR-4, DAXX and ZIPK proteins in CLL patients and its relationship with the principal prognostic factors

Par-4 (prostate apoptosis response-4) protein was originally found upregulated in prostate tumor cells undergoing apoptosis. Then it was further identified as a proapoptotic protein upregulated both in normal and leukemic lymphocytes. The aim of our study was to assess PAR-4 protein expression in the B cells of CLL patients and to examine its relationship with the expression of other proteins involved in the apoptosis process, such as DAXX, ZIPK and BCL-2. We found a positive relationship between PAR-4 and BCL-2 protein expression. Additionally, there was a positive correlation between PAR-4 and both DAXX and ZIPK protein expression. The results of our research were also analyzed in association with the principal CLL prognostic factors. There was a positive correlation between the expression of PAR-4 protein and the lactate dehydrogenase (LDH) serum concentration (p < 0.005). The expression of PAR-4 protein in B cells correlated positively with the percentage of CD38(+) cells (p < 0.05), as well as with CD38(+)/ZAP-70(+) cells (p < 0.05). Moreover, we found a close relationship between LPL protein expression or LPL/ADAM29 MFI ratio and PAR-4 protein expression. Our results confirm the significance of apoptosis deregulation in CLL, and suggest a possible relationship between PAR-4 expression and the clinical course of the disease. This however requires further investigation.

Percentage of Smudge Cells Predicting Prognosis In Chronic Lymphocytic Leukemia

AbstractAbstract 4624 Background:Identifying prognostic markers is important for clinical and pathological course of chronic lymphocytic leukemia. Percentage of smudge cells in CLL patients has recently been reported as a novel prognostic factor. We present here the preliminary results of the retrospective cohort study of 90 patients diagnosed with CLL at a major referral center in the state of Alabama between 1997 and 2009. Methods:Smudge cell percentage (ratio of smudge cells to combined smudge cells and intact cells) was calculated by microscopic evaluation of archived Wright-Giemsa stained blood smears. A total of 200 cell differential was counted for inter-observer consistency. For the first time, the concordance between observers was assessed by quantization of morphological parameters of lymphocytes using computerized image analysis. Medical records of above mentioned patients were reviewed for molecular, genetic and clinical parameters including CD38, ZAP 70 expression, immunocytopenias and response to first treatment. Primary endpoint of this analysis was time to initiation of chemotherapy for first treatment (TIFT). It was calculated from date of first diagnosis until first dose of chemotherapy. Untreated patients in the follow up were censored. Kaplan Meier survival analysis and log rank test were used for statistical analysis. Prognostic factors for TIFT were analyzed with Cox's proportional hazards regression. Wilcoxon rank sum test and Fishers exact test were used to evaluate differences between groups for continuous and categorical variables, respectively. We had more than 90% Power to detect an association between smudge cell percentage and TIFT. Results:Of the 160 treatment naïve CLL patients enrolled for the study, 90 had archived blood smears available for evaluation. Patient characteristics include Rai stage 0 or 1 disease (87%), CD 38+ (30%), Zap 70 + (35%), 13q Del + (26%). Percentage of smudge cells ranged from 2 to 90 % (median 34%). Patients with CD38+ and ZAP 70+ on flow cytometry had lower percentage of smudge cell than those with negative markers and are trending towards significance (p=0.08). Our cohort included 16% of African American population and had no significant difference in TIFT when compared to Caucasians. In univariate analysis smudge cell percentage (stratified as less than or more than 30) was significantly associated with TIFT (p=0.04). Rai stages 0, 1 and 2 were significantly associated with longer TIFT than stage 3 and 4 (p= 0.01). Genetic parameters- deletion 13 q and 11q were associated with significantly greater TIFT (p= 0.008 and P=0.001 respectively). Patients who developed immunocytopenias during the course of study had shorter TIFT (p=0.01). We performed Step wise multivariate Cox regression analysis and identified del11q (HR=0.02, p=0.001) and Rai stage (HR=0.008, p <0.0001) as independent predictors of TIFT. Patients with longer TIFT had better response to treatment when compared to shorter TIFT (68% vs 32%, p=0.05). Contrary to our expectation, we found that, in multivariate analysis, we found smudge cell percentage is not significantly associated with TIFT (p= 0.75). Conclusion:Time for initiation of first treatment is significantly shorter in CLL patients with del11q and del13q and those who develop immunocytopenias. Smudge cell percentage did not significantly predict TIFT in this single center study. We speculate this could be due to subjective decision on initiation of chemotherapy by physicians, based on several other clinical parameters and co-morbidities. Larger studies are needed that concurrently assess any confounders between smudge cell percentage and TIFT. Alternatively, computerized image analysis is superior to observer measurements and can be considered for accuracy and reproducibility of cytological evaluation. It can ascertain the objectivity of concordance in differential cell counting in similar studies. Disclosures:No relevant conflicts of interest to declare.

Dinaciclib (SCH727965) Is a Novel Cyclin Dependent Kinase Inhibitor That Promotes Selective Apoptosis In CLL Cells and Abrogates the Protective Effects of Microenvironment Cytokines

AbstractAbstract 971The first generation cyclin dependent kinase (CDK) inhibitor flavopiridol has significant clinical activity in refractory CLL including those with del(17p13.1). These successes have prompted development of second generation molecules that lack the protein binding, off-target effects (i.e. GSK3-β inhibition), and complex enterohepatic circulation that exist with flavopiridol. Dinaciclib is a selective inhibitor of CDK 1, 2, 5 and 9 (IC50 of < 5nM) and lacks GSK3-β inhibition. Dinaciclib was selected as a lead CDK inhibitor candidate by an in vivo screen that identified it as having a favorable therapeutic index. Completed phase I studies in solid tumor cancer patients with dinaciclib have confirmed this favorable toxicity profile. We sought to explore the pre-clinical activity of dinaciclib in CLL to support its clinical development. Our studies demonstrate that dinaciclib promotes concentration-dependent apoptosis that is superior to flavopiridol following a clinically relevant 2-hour exposure (74.4% viable with 1μM flavopiridol compared to 26.7% viable with 1μM dinaciclib, p<0.0001). Additionally, dinaciclib, like flavopiridol, lacks cytotoxic effects on normal T-cells under these same conditions. Dinaciclib induced cytotoxicity was independent of IgVH mutational status and fludarabine refractoriness. Surprisingly, CLL cells from patients with del(17p13.1) were significantly more resistant than from patients with normal cytogenetics (68.2% viable versus 19.6%, p=0.0006) to dinaciclib induced cytotoxicity. This is in contrast to flavopiridol (54.1% viable versus 50.0%, p=0.2119) where this has not been seen. This finding is being investigated in the current phase 1 clinical trial of dinaciclib in CLL. Apoptosis induced by dinaciclib is caspase dependent, as co-incubation with the caspase inhibitor z-VAD-fmk rescued dinaciclib induced cytotoxicity. As seen with other CDK inhibitors, dinaciclib potently down-regulated Mcl-1 and Bcl-2 expression at a mRNA and protein level in CLL cells. Given the importance of microenvironment stimuli to CLL survival, we sought to determine how soluble and contact microenvironment effects influence dinaciclib cytotoxicity. T-cells and stromal cells promote CLL cell survival in part through the production of cytokines including CD40 ligand, BAFF, TNF-α, and IL-4. While CD40 ligand, BAFF, TNF-α, and IL-4 all protected CLL cells from spontaneous apoptosis, they did not significantly abrogate dinaciclib-mediated cytotoxicity. Consequently dinaciclib inhibited CD40 ligand induced up-regulation of Mcl-1 protein expression. The mechanism by which this protection is abrogated appears to involve down-regulation of the respective ligand receptor, as expression of each of these receptors was notably reduced at 2 hours in 1μM dinaciclib-treated CLL cells. CLL cells also derive protection from both spontaneous and drug-induced apoptosis by direct co-culture (and contact) with stromal cells. We therefore assessed the cytotoxic effect of dinaciclib on CLL patient cells when cultured in the presence of stromal cells. These studies surprisingly demonstrated loss of adhesion of stromal cells and abrogation of CLL cell protection. To avoid any direct effect of dinaciclib on the stromal cells, we then pre-treated CLL cells with 1μM dinaciclib for 2 hours, followed by washout and incubation on stromal cells. In these experiments, co-culture of CLL cells on stromal cells almost completely abrogated dinaciclib-mediated cytotoxicity. Similar findings were observed with fibronectin-coated plates. Together, these data indicate that stromal protection of CLL cells against dinaciclib is mediated either by cell-cell contact or by cytokines other than those tested. Collectively, these studies support the development of dinaciclib for the treatment of CLL, and also identify distinct microenvironment signals that may modulate response to this novel agent. Additionally, the differential response to del(17p13.1) CLL cells suggest distinct differences between dinaciclib and flavopiridol that are currently under investigation.This work was supported by the National Cancer Institute Leukemia SPORE P50-CA140158, The Leukemia and Lymphoma Society and The D. Warren Brown Foundation. AJJ is a Paul Calabresi Scholar. Disclosures:Jones:Glaxo Smith-Kline: Consultancy; Abbott: Research Funding. Bannerji:Merck & Co: Employment, Equity Ownership.

Expression of Lipoprotein Lipase In Chronic Lymphocytic Leukemia Cells: Is There a Biological Function?

AbstractAbstract 3580The expression of lipoprotein lipase (LPL) in CLL cells is an established mRNA surrogate marker for immunoglobulin heavy chain (IgVH) mutational status. High expression of LPL correlates with poor prognosis. However, the possible functional role of LPL in CLL is still unclear. LPL is normally expressed in muscle cells, adipose tissue and macrophages, transported to the luminal surface of endothelial cells where it is bound heparan sulfate-proteoglycans (HSPG). Heparin competes with HSPG for the binding sites and intravenous injection leads to elevated plasma LPL protein levels and enzymatic activity (“heparin release test”). LPL mRNA levels correlate with intracellular protein expression (Heintel et al. Leukemia. 2005; Mansouri et al. Leuk Res. 2010). Moreover cellular lysates from CLL patients contain elevated LPL enzymatic activity compared to healthy donors (Pallasch et al. Leukemia. 2008.). In this study, we investigated the basal (pre-heparin) LPL protein levels by enzyme-linked immunosorbent assay in the serum of 42 CLL patients, 14 non-CLL patients (lymphoma in remission), and 4 healthy donors (HD): Median pre-heparin LPL protein levels were 40.10 ng/ml (range: 5.66–108.44), 44.11 ng/ml (18.26-84.08), and 68.14 ng/ml (33.28-174.38), respectively. Among CLL patients there were no significant differences between those with high (N=16; median LPL protein in serum: 38.10 ng/ml (8.72-73.49)) and low (N=26; 43.12 ng/ml (5.66-108.44) (p=0.354) LPL mRNA expression. Thirteen patients with known LPL mRNA expression were investigated for LPL protein “release” after heparin injection. Ten and twenty minutes after 50 U/kg heparin injection, the elevation of both parameters, LPL protein amount in serum and enzymatic activity in plasma, was similar to those of HD normal values. In detail, medium serum protein levels in samples with high LPL mRNA (N=5) increased from 16.11 to 214.33 and 332.78 ng/ml and in the samples with low mRNA (N=8) from 13.08 to 219.68 and 386.65 ng/ml, respectively. The corresponding median values of the LPL enzymatic activities in high vs. low expressors were: 7.25/15.52/20.01 and 7.45/19.13/20.57 μ M/ml/h. In addition, release of LPL from peripheral mononuclear cells (PBMC) of CLL patients (N=3) by heparin in vitro was absent. Cell viability and LPL mRNA expression remained unaffected in both in vivo and in vitro samples after heparin addition. In order to assess the impact of LPL on cell survival, CLL cells were cultured (N=3) for up to 72 hours with different doses of purified LPL protein. There was no positive effect on cell survival irrespective of primary LPL mRNA expression or culture conditions (with or without FCS). Since these results point to an intracellular effect of LPL, we aimed to identify downstream targets by knock down with siRNA against LPL in 7 CLL samples and 5 cell lines (hepatocellular carcinoma, cervix carcinoma, colon carcinoma, multiple myeloma and acute monocytic leukemia) with high LPL mRNA expression. Gene expression changes were analyzed by microarrays (GeneChip® Human Gene 1.0 ST Array, Affymetrix). Fifteen genes were up- (N=4) or downregulated (N=11) in at least 3 of 5 cell lines by more than 1.5-fold (e.g. GSTP1, COROC1). Nine genes were at least 1.5-fold downregulated in parallel with LPL in the CLL samples only. These genes belong to various pathways (e.g. cell cycle, signaling in immune system, metabolism of carbohydrates) and seem to be specific for CLL. Cross-validation of individual genes is under way.Our data suggest that (1) neither basal serum LPL protein levels nor heparin-induced LPL release in CLL patients are suitable clinical prognostic markers; (2) Stimulation with external LPL protein does not affect CLL cell survival; (3) siRNA knock-down of LPL induces changes in various functional pathways. We conclude that the key role of LPL expression in high-risk CLL is related to its (intra)cellular expression. Disclosures:No relevant conflicts of interest to declare.

The HSP90 Inhibitor 17-DMAG Targets NF-κB to Induce Apoptosis in CLL and Prolongs Survival in a CLL Mouse Model.

Abstract 732▪▪This icon denotes an abstract that is clinically relevant.One attractive therapeutic target currently being explored in CLL is HSP90, a chaperone which stabilizes various client proteins (AKT, Raf, ZAP-70) which are important for survival of CLL cells. Interfering with HSP90 protein binding to these client proteins leads to their rapid degradation. Our group and others have demonstrated that 17-allylamino-17-demethoxy-geldanamycin (17-AAG) depletes only select chaperone proteins and promotes modest cytotoxicity in CLL patient cells. 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) is a novel HSP90 inhibitor with improved solubility, bioavailability and cytotoxicity in cancer cell lines as compared to 17-AAG. We demonstrate that 17-DMAG more potently induces caspase-dependent apoptosis of primary CLL cells compared to 17-AAG. This enhanced cytotoxicity is tumor cell selective as it spares normal T-cells and NK-cells, and only modestly affects normal B cells. In addition to the broad range of Hsp90 client proteins which are regulated by 17-AAG (AKT, CDK9, ZAP-70), we found that 17-DMAG was also able to deplete both IKKαa and IKKβ, the activating kinases of the NF-κB family of transcription factors. The impact of 17-DMAG on both IKKαa and β is very relevant, as there have been several studies investigating the effect of IKK inhibitors on in vitro apoptosis in CLL, however there is still no clear therapeutic option for targeting these kinases in the clinic. Most IKK inhibitors including 17-AAG are specific for the IKKβ subunit that mediates classical NF-κB signaling, leaving IKKαa and the alternative NF-κB pathway intact. This is particularly important given recent evidence by Lam et al. (Clin Can Res 2005 Jan 1;11(1):28-40) demonstrating that IKKαa is able to compensate for the loss of IKKβ in DLBCL cells lines. Therefore the ability of 17-DMAG to target both subunits of the IKK complex potentially makes it a very potent and effective NF-κB inhibitor in CLL. To validate the downstream significance of this NF-κB regulation, we show that 17-DMAG effectively reduces NF-κB nuclear localization DNA binding in CLL patient cells resulting in decreased transcript and protein levels of NF-κB targets MCL1 and BCL2, known to be major factors in CLL cell survival and drug resistance. The decrease in MCL1 and BCL2 following 17-DMAG treatment was not prevented by treatment with the caspase inhibitor zVAD-fmk even though viability was rescued, suggesting that the decrease in these survival proteins precedes cell death and is not simply a consequence of the apoptotic process. Finally, we determined the in vivo significance of 17-DMAG treatment using a TCL1-SCID transplant model. We found that NF-κB targets genes (A20, BCL2, cIAP, MCL1 and XIAP) were decreased in vivo following treatment with 17-DMAG. In addition to typical genes which are activated by NF-κB, we also determined the in vivo effect of 17-DMAG on NF-κB mediated transcriptional repression. Our group has recently determined that the transcription factor FOXD3 is transcriptionally silenced by an NF-κB repressor complex very early in the disease progression of the TCL1 mouse model, an event which facilitates subsequent global DNA methylation and gene silencing in CLL. We found that treatment with 17-DMAG leads to re-expression of FOXD3 suggesting that 17-DMAG is an effective therapeutic tool to target NF-κB mediated gene repression as well as NF-κB mediated expression of survival genes. Furthermore, 17-DMAG treatment significantly prolonged the survival of these mice (75 days vs. 66 days, p=0.027, n=10/group). Together, our data demonstrate that the HSP90 inhibitor 17-DMAG represents a novel multi-subunit IKK inhibitor leading to a decrease in anti-apoptosis genes relevant to CLL survival while increasing the expression of genes silenced during CLL disease progression. Given its oral formulation, which allows administration of 17-DMAG by continuous dosing and uninterrupted inhibition of HSP90, initiation of phase II clinical trials in CLL that include detailed pharmacodynamic studies monitoring NF-κB target genes are indicated. Disclosures:No relevant conflicts of interest to declare.

Therapy of B-Cell Malignancies by Anti-HLA-DR Humanized Monoclonal Antibody, IMMU-114, Is Mediated through Hyperactivation of ERK and JNK MAP Kinase Signaling Pathways.

Abstract 3738Poster Board III-674 IntroductionHLA-DR is currently under investigation as a target for antibody (mAb) therapy of B-cell malignancies. IMMU-114, a humanized IgG4 form of the murine anti-HLA-DR mAb, L243, recognizes a conformational epitope in the a-chain of HLA-DR. We have previously reported that IMMU-114 lacks effector-cell functions while displaying potent antitumor activity against B-cell lymphomas in vitro and in vivo. Here, we further investigated the cytotoxic effects of IMMU-114 on leukemia, lymphoma, and multiple myeloma (MM) cell lines, as well as clinical specimens of CLL, and explored the signaling pathways involved. MethodsBinding and cytotoxicity of IMMU-114 was examined on a panel of NHL, AML, MCL, ALL, and CLL cell lines. Induction of apoptosis (Annexin V binding), ROS generation (dihydoehidium staining), changes in mitochondrial membrane potential (ψM, TMRE staining) and effects on the ERK1/2 and JNK pathways (immunoblot analyses) were evaluated on selected cell lines. The effects of various inhibitors on IMMU-114-mediated apoptosis also were determined. Studies were performed in tumor-bearing SCID mice. ResultsHigh expression of HLA-DR was detected on the cell surface of all MCL, ALL, HC, CLL, and NHL cell lines tested, as well as 2/3 AML and 5/6 MM cell lines. IMMU-114 was cytotoxic to 2/2 MCL, 2/2 CLL, 2/4 ALL, 1/1 HC, 2/2 NHL and 2/2 MM cell lines without any added anti-Fc second crosslinking antibody. IMMU-114 was consistently more cytotoxic than anti-CD20 mAbs on these cell lines, as well as on rituximab-resistant variants of Raji and SU-DHL-4. Despite positive staining, AML cell lines, Kasumi-3 and GDM-1, were not killed by IMMU-114. Four of 6 CLL patient samples expressed HLA-DR at markedly higher levels than CD20, with the remaining 2 showing similar HLA-DR and CD20 expression. Approximately 60% cytotoxicity was obtained after incubation with IMMU-114 in CLL patient cells. Induction of apoptosis as well as changes in ψM and ROS correlated with sensitivity of cell lines to IMMU-114-mediated cytotoxicity. Time-course analyses demonstrated that IMMU-114 induced approximately 46% change of ψM and 24% enhancement of ROS in as little as 30 min in Raji cells. These changes were abrogated in the presence of ROS inhibitor, N-acetyl cysteine. Immunoblot analyses revealed that IMMU-114 induces phosphorylation of ERK and JNK mitogen-activated protein (MAP) kinases in all cells defined as IMMU-114-sensitive by the cytotoxicity assays, but not IMMU-114-resistant cell lines. The p38 pathway was found to be constitutively active in these cell lines. Inhibition of ERK, JNK, or ROS by their respective inhibitors decreased apoptosis, although the inhibition was not complete when any single inhibitor was used. This could be due to activation of multiple pathways, since inhibition of 2 or more pathways by specific inhibitors abolishes the apoptosis induced by IMMU-114. Activation (5-fold increases of phosphorylation) of ERK1/2 and JNK signaling pathways also was observed in CLL patient samples following IMMU-114 incubation. Further studies revealed caspase-independent apoptosis and activation of apoptosis-inducing factor (AIF) mediated by IMMU-114. The therapeutic activity of IMMU-114 was evaluated in vivo in 3 NHL models, WSU-FSCCL, Namalwa, and Raji. IMMU-114 was more effective than anti-B-cell mAbs such as rituximab, in all models. For example, in WSU-FSCCL, IMMU-114 treatment yielded 100% long-term survival. Although rituximab also induced significant responses compared to untreated mice (median survival time of 87.5 vs. 36 days for untreated controls, P<0.0001), no long-term survivors were seen. ConclusionIMMU-114 is cytotoxic to a variety of hematological malignancies, and its potent activity can be related to high antigen expression of target cells and hyperactivation of ERK and JNK pathways upon binding to HLA-DR. Clinical evaluation of IMMU-114 is planned. Disclosures:Gupta:Immunomedics Inc.: Employment. Stein:Garden State Cancer Center: Employment. Cardillo:Immunomedics Inc.: Employment. Chang:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

A Fc-Engineered RANK-Fc Fusion Protein Neutralizes Soluble RANK Ligand and Targets Malignant Hematopoietic Cells for NK Cell Reactivity.

Abstract 411Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NFκB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) as key regulators of bone resorption. Multiple myeloma (MM) disrupts the balance within this molecule system, and severe bone destruction due to inappropriate osteoclastogenesis is a prominent feature of this disease. Besides MM cells, other malignant hematopoietic cells have also been found to express RANKL at the cell surface and to release this molecule in soluble form (sRANKL). Neutralization of RANKL using RANK-Fc fusion protein or monoclonal antibody (Denosumab/AMG162), which mimics the RANKL-neutralizing endogenous effects of osteoprotegerin, decreases osteolysis in multiple in vivo models and is presently being evaluated as a means to treat both non-malignant and malignant osteolysis. We here confirmed and extended previously published data and report that all investigated MM cell lines (n = 5) as well as primary leukemic cells of CLL patients (n = 12) displayed substantial levels of RANKL mRNA and surface expression. Moreover, we report that substantial levels of sRANKL can be detected in culture supernatants of MM and primary CLL cells, but not in supernatants of healthy PBMC. Next we engineered RANK-Fc fusion proteins with modified affinity to FcγR by mutating amino acids in the Fc portion as previously described (Lazar et al., PNAS 2006; Armour et al., Eur J Immunol 1999). Compared to wildtype RANK-Fc (RANK-Fc-wt), our mutants (S239D/I332E and E233P/L234V/L235A/DeltaG236/A327G/A330S) displayed highly enhanced and abrogated (RANK-Fc-ADCC+ and RANK-Fc-KO, respectively) affinity to FcγRIIIa expressed on NK cells, which play an important role in anti-tumor immunity due to their ability to lyse target cells directly and to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of therapeutic antibodies. The RANK-Fc-ADCC+ displayed similar capacitiy to neutralize sRANKL compared to the RANK-Fc-KO and the RANK-Fc-wt as revealed by binding competition assays. Next we cultured NK cells with L cells or P815 cells transfected to express RANKL and the parental, RANKL-negative controls in the presence or absence of the different RANK-Fc constructs. Addition of RANK-Fc-KO or RANK-Fc-wt did not substantially alter NK cell reactivity against the target cells. However, presence of the RANK-Fc-ADCC+ dramatically enhanced NK cell cytotoxicity and cytokine production in cultures with the RANKL-expressing target cells (increase from 20% to 89%, E:T ratio 30:1 and 12 pg/ml to 290 pg/ml respectively; both p<0.01, Student's t-test). Neither of the three RANK-Fc proteins altered NK cell cytotoxicity and cytokine production in cultures with the RANKL negative controls demonstrating that the RANK-Fc-ADCC+ specifically induced NK cell reactivity against RANKL-expressing malignant cells. Moreover, treatment with RANK-Fc-ADCC+ also significantly augmented NK cell anti-tumor reactivity in cultures with RANKL-expressing primary CLL cells of patients, and this was observed both in settings using allogenic NK cells and analyzing autologous NK cells among PBMC of the leukemia patients (both p<0.01, Student's t-test). Taken together, our Fc-engineered RANK-Fc-ADCC+ fusion protein may neutralize detrimental effects of sRANKL, can target RANKL-expressing malignant cells for NK cell anti-tumor reactivity and may thus constitute an attractive immunotherapeutic means for treatment of hematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

Automated Classification of Normal and Leukemic B Cells on the ImageStream® Imaging Flow Cytometer Using Immunocytomorphological Image Characteristics

Leukemic B cells can be distinguished from normal B cells based on their surface staining patterns and their morphology, but the differences can be subtle and the abnormal events rare. In order to reliably differentiate normal from leukemic B cells, objective quantitation of large data sets is required. This is accomplished with the ImageStream cytometer, which automatically acquires multispectral images of thousands of individual cells in flow at very high speeds. The associated IDEAS data analysis software measures hundreds of photometric and morphometric features per cell, thereby enabling simultaneous immunophenotyping and morphology-based measurements. The features quantify individual image characteristics such as shape, size, signal strength and texture. We have leveraged these features by implementing an automated classification routine that identifies the feature combination that best separates different cell types of interest. Our automated classification routine utilizes a modification of Fisher's Linear Discriminant criterion to measure discrimination power between two or more populations of cells. The classifier generates a composite feature consisting of a weighted linear combination of basic image features that provides maximum discrimination. Thus, our method transforms a multi-parameter classification task that can be difficult to conceptualize into a classification based on a single composite feature that can be readily conceptualized and visualized using a simple histogram output. Since the composite feature is generated from, and tested and optimized on, large numbers of cells, we obtain results that are highly objective, repeatable, statistically significant and scalable with data size. We used this classification approach to study normal and tumor cells of the hematopoietic lineage with the goal of improving diagnosis of hematological disorders. Here we show the results of using automated classification to distinguish the subtle differences between B cells of CLL patients and those of normal patients. We also used the classifier to distinguish B-cell ALL from B-cell CLL. In both cases, we used a combination of shape, size, texture and signal strength features from the SSC, brightfield and nuclear imagery generated by the ImageStream system. The data presented demonstrate the application of a novel automated cell classification method to the discrimination of normal and tumor cells within large and heterogeneous cell samples.

The Aiolos transcription factor is up-regulated in chronic lymphocytic leukemia

AbstractThe Aiolos transcription factor, member of the Ikaros family of zinc finger proteins, plays an important role in the control of mature B lymphocyte differentiation and proliferation, and its function appears to be modulated through alternative splicing. To assess Aiolos isoform role in humans' pathologies, we studied Aiolos variant distribution and expression in mature B lymphoproliferative disorders (chronic lymphocytic leukemia [CLL] and other B-cell lymphomas). We demonstrated that more than 80% of expressed Aiolos in normal as well as in malignant B cells is of the hAio1 type, and we showed for the first time a homogeneous overexpression of the total amounts of Aiolos transcripts in the B cells of CLL patients, independently of ZAP-70 and IgVH mutational status prognosis factors. This up-regulation of Aiolos, confirmed at protein level, seems independent of Aiolos promoter H3K9 acetylation and H3K4 trimethylation.

Comparison of Gene Expression between T Cells from CLL Patients, Myeloma Patients and Healthy Individuals.

A unique feature of chronic lymphocytic leukemia is the expansion in absolute numbers of circulating T cells, accompanying the increase in the leukemic B cell clone. T cells of CLL patients may have a role in contributing to the microenvironment that favors the survival and progression of the malignant clone. We have looked at the gene profile of highly purified peripheral T cells from indolent CLL patients using Affymetrix global gene arrays and compared them with purified T cells from multiple myeloma patients as well as healthy individuals. T cells from five indolent, treatment-naïve CLL patients were examined for gene expression by Affymetrix-based microarray alongside T cells from 5 stage I, multiple myeloma patients and 5 age-matched healthy volunteers. Differential expression analysis revealed that 356 genes were differentially expressed by T cells from CLL patients. Based on a two-fold or greater difference, 107 genes were specifically upregulated and 247 genes were noted to be specifically downregulated in T cells of CLL patients. The highest level of upregulation was noted for the chemokines XCL1, XCL2, and the cytokine IFN-g. CCL4 and CCL5 were two other important chemokines that also were found to be specifically upregulated in T cells of B-CLL patients as well as KLF6 and TRAF1. The findings of the microarray analysis was further confirmed using quantitative real-time PCR and immunoblotting with specific antibodies. using T cells from an additional 14 CLL, 6 multiple myeloma and 10 healthy individuals. Our results indicate that T cells from B CLL patients may express a variety of genes that contribute to the creation of a microenvironment that sustains the survival of the leukemic clone and may produce soluble factors that inhibit apoptosis and facilitate proliferation of the leukemic B cells.