Donor Vδ1+ γδ T cells expand after allogeneic hematopoietic stem cell transplantation and show reactivity against CMV-infected cells but not against progressing B-CLL.

PubMed

Prinz, Immo; Thamm, Kristina; Port, Matthias; Weissinger, Eva M; Stadler, Michael; Gabaev, Ildar; Jacobs, Roland; Ganser, Arnold; Koenecke, Christian

2013-05-11

γδ T lymphocytes play an important role in immune reactions towards infections and malignancies. In particular, Vγ9-Vδ1+ T lymphocytes are thought to play protective antiviral roles in human CMV infection. Recently, Vδ1+ T lymphocytes were proposed to also have anti- B-CLL reactivity. Here we report a case of 48-year-old man who received allogeneic stem cell transplantation for progressive B-CLL. Within one year after transplantation, lymphoma relapsed despite a dramatic increase of Vδ1+ T cells in the patient's blood. In vitro killing assays revealed activity of patient's γδ cells against CMV target cells, but not against the relapsing lymphoma-cells. This argues for a contribution of Vδ1+ cells in the immune reaction against CMV reactivation, but does not support a strong correlation of expanded Vδ1+ T cells and favorable disease outcome in B-CLL patients.

Microenvironmental agonists generate de novo phenotypic resistance to combined ibrutinib plus venetoclax in CLL and MCL

PubMed Central

Portell, Craig A.; Gordon, Vicki L.; Capaldo, Brian J.; Bekiranov, Stefan; Axelrod, Mark J.; Brett, L. Kyle; Wulfkuhle, Julia D.; Gallagher, Rosa I.; Petricoin, Emanuel F.; Bender, Timothy P.; Williams, Michael E.

2017-01-01

De novo resistance and rapid recurrence often characterize responses of B-cell malignancies to ibrutinib (IBR), indicating a need to develop drug combinations that block compensatory survival signaling and give deeper, more durable responses. To identify such combinations, we previously performed a combinatorial drug screen and identified the Bcl-2 inhibitor venetoclax (VEN) as a promising partner for combination with IBR in mantle cell lymphoma (MCL). We have opened a multi-institutional clinical trial to test this combination. However, analysis of primary samples from patients with MCL as well as chronic lymphocytic leukemia (CLL) revealed unexpected heterogeneous de novo resistance even to the IBR+VEN combination. In the current study, we demonstrate that resistance to the combination can be generated by microenvironmental agonists: interleukin-10 (IL-10), CD40L and, most potently, cytosine guanine dinucleotide–oligodeoxynucleotides (CpG-ODNs), which is a surrogate for unmethylated DNA and a specific agonist for Toll-like receptor 9 (TLR9) signaling. Incubation with these agonists caused robust activation of NF-κB signaling, especially alternative NF-κB, which led to enhanced expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and survivin, thus decreasing dependence on Bcl-2. Inhibitors of NF-κB signaling blocked overexpression of these antiapoptotic proteins and overcame resistance. Inhibitors of Mcl-1, Bcl-xL, or survivin also overcame this resistance, and showed synergistic benefit with the IBR+VEN combination. We conclude that microenvironmental factors, particularly the TLR9 agonist, can generate de novo resistance to the IBR+VEN combination in CLL and MCL cells. This signaling pathway presents targets for overcoming drug resistance induced by extrinsic microenvironmental factors in diverse B-cell malignancies. PMID:29034364

Biological and clinical characterization of recurrent 14q deletions in CLL and other mature B-cell neoplasms.

PubMed

Reindl, Lena; Bacher, Ulrike; Dicker, Frank; Alpermann, Tamara; Kern, Wolfgang; Schnittger, Susanne; Haferlach, Torsten; Haferlach, Claudia

2010-10-01

14q-deletions have been repeatedly described in mature B-cell neoplasms, but not yet characterized in a larger cohort. Based on chromosome banding analysis, the present study identified 47 del(14q) cases in 3054 mature B-cell neoplasms (1·5%) (chronic lymphocytic leukaemia [CLL]: 1·9%; CLL/prolymphocytic leukaemia [PL]: 9·0%; others: 0·2%). Interphase fluorescence in situ hybridization was performed with probes for 14q22.1, 14q24.1, 14q32.33, and IGH@ (14q32.3). The del(14q) had heterogeneous size but showed a breakpoint cluster at the centromeric site in 14q24.1 (62% of cases). At the telomeric side, the most frequent breakpoint was within the IGH@ locus (14q32.3) between IGH@ 3'-flanking and IGHV (IgVH) probes (45%). In 16 cases (34%), breakpoints occurred within 14q24.1 and 14q32.3. Eighty-one percent of del(14q) cases showed 1-3 additional cytogenetic alterations (in 45%, +12), and 56% were IGHV-unmutated. In all cases (16/16) with breakpoints in 14q24.1 and 14q32.3, a B-CLL immunophenotype was found. Clinical follow-up in 32 del(14q) patients was compared to 383 CLL and CLL/PL patients without del(14q). While 3-year-overall survival did not differ significantly, time to treatment was significantly shorter in the del(14q) cohort (21·0 months vs. 80·1 months, P = 0·015). In conclusion, the del(14q) is a rare recurrent alteration in diverse mature B-cell neoplasms, shows variable size but distinct clustering of breakpoints, and is associated with short time to treatment. © 2010 Blackwell Publishing Ltd.

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

PubMed

Winkler, Mark T; Bushey, Ryan T; Gottlin, Elizabeth B; Campa, Michael J; Guadalupe, Eross S; Volkheimer, Alicia D; Weinberg, J Brice; Patz, Edward F

2017-01-01

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

PubMed

Bruno, Silvia; Ledda, Bernardetta; Tenca, Claudya; Ravera, Silvia; Orengo, Anna Maria; Mazzarello, Andrea Nicola; Pesenti, Elisa; Casciaro, Salvatore; Racchi, Omar; Ghiotto, Fabio; Marini, Cecilia; Sambuceti, Gianmario; DeCensi, Andrea; Fais, Franco

2015-09-08

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

CLL Cells Respond to B-Cell Receptor Stimulation with a MicroRNA/mRNA Signature Associated with MYC Activation and Cell Cycle Progression

PubMed Central

Pede, Valerie; Rombout, Ans; Vermeire, Jolien; Naessens, Evelien; Mestdagh, Pieter; Robberecht, Nore; Vanderstraeten, Hanne; Van Roy, Nadine; Vandesompele, Jo; Speleman, Frank; Philippé, Jan; Verhasselt, Bruno

2013-01-01

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation. PMID:23560086

Activation of the PI3K/AKT pathway by microRNA-22 results in CLL B-cell proliferation.

PubMed

Palacios, F; Abreu, C; Prieto, D; Morande, P; Ruiz, S; Fernández-Calero, T; Naya, H; Libisch, G; Robello, C; Landoni, A I; Gabus, R; Dighiero, G; Oppezzo, P

2015-01-01

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal B cells arrested in G0/G1 stages that coexist, in different proportions, with proliferative B cells. Understanding the crosstalk between the proliferative subsets and their milieu could provide clues on CLL biology. We previously identified one of these subpopulations in the peripheral blood from unmutated patients that appears to be a hallmark of a progressive disease. Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis comparing the global mRNA and microRNA expression of this leukemic subpopulation, and compared it with their quiescent counterparts. Our results suggest that proliferation of this fraction depend on microRNA-22 overexpression that induces phosphatase and tensin homolog downregulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. Transfection experiments demonstrated that miR-22 overexpression in CLL B cells switches on PI3K/AKT, leading to downregulation of p27(-Kip1) and overexpression of Survivin and Ki-67 proteins. We also demonstrated that this pathway could be triggered by microenvironment signals like CD40 ligand/interleukin-4 and, more importantly, that this regulatory loop is also present in lymph nodes from progressive unmutated patients. Altogether, these results underline the key role of PI3K/AKT pathway in the generation of the CLL proliferative pool and provide additional rationale for the usage of PI3K inhibitors.

Correlation of the expression of CD32 and CD180 receptors on CLL cells and MEC1 cell line.

PubMed

Tsertsvadze, T; Mitskevich, N; Ghirdaladze, D; Porakishvili, N

2015-03-01

Chronic Lymphocytic Leukemia (CLL) presents with clonal expansion and accumulation of CD5+CD19+CD23+ cells in peripheral lymphoid organs and tissues and in bone marrow. CLL is supposedly driven by exogenous and/or endogenous (auto)antigen(s) and there is increasing evidence that CLL cells receive microenvironmental signals which support their growth, survival and expansion in vivo. We have previously shown that powerful signals are received by CLL cells through CD180 orphan toll-like receptor. Additional accessory signals could be generated through FcγRII (CD32), since both are expressed on CLL cells as well as on control B cells. Here we studied correlation of the expression of CD32 and CD180 on CLL cells as well as on MEC1 cell line. Peripheral blood mononuclear cells (PBMC) from CLL patients and age-matched healthy volunteers were separated, stained with appropriate antibodies to CD19, CD32 and CD180 and analysed by flow cytometry. CD32 and CD180 expression on MEC1 cells was studied at different time-points. The data was statistically analysed using the Mann-Whitney non-parametrical test. Our data indicates that expression of CD32 is significantly increased on CLL cells compared to control B cells as well as in long-term MEC1 cell culture. In contrast, CD180 expression on MEC1 cells significantly decreased throughout 0-96h of MEC1 cell culture. We have recently shown that CD180 ligation can redirect sIgM-mediated signaling from pro-survival to pro-apoptotic. This data indicates that a drop in the expression of CD180 on cycling CLL cells might lead to a weakening of this effect and enhance further survival and expansion of CLL cells in proliferative centres of lymphoid tissues. Since MEC1 cells are derived from a CLL patient with mutated IGVH genes (M-CLL) negative correlation between CD180 and CD32 expression on cycling MEC1 cells could be limited to M-CLL.

c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches.

PubMed

Hallaert, Delfine Y H; Jaspers, Annelieke; van Noesel, Carel J; van Oers, Marinus H J; Kater, Arnon P; Eldering, Eric

2008-12-15

In lymph node (LN) proliferation centers in chronic lymphocytic leukemia (CLL), the environment protects from apoptotic and cytotoxic triggers. Here, we aimed to define the molecular basis for the increased drug resistance and searched for novel strategies to circumvent it. The situation in CLL LN could be mimicked by prolonged in vitro CD40 stimulation, which resulted in up-regulation of antiapoptotic Bcl-xL, A1/Bfl-1, and Mcl-1 proteins, and afforded resistance to various classes of drugs (fludarabine, bortezomib, roscovitine). CD40 stimulation also caused ERK-dependent reduction of Bim-EL protein, but ERK inhibition did not prevent drug resistance. Drugs combined with sublethal doses of the BH3-mimetic ABT-737 displayed partial and variable effects per individual CD40-stimulated CLL. The antiapoptotic profile of CD40-triggered CLL resembled BCR-Abl-dependent changes seen in chronic myeloid leukemia (CML), which prompted application of c-Abl inhibitors imatinib or dasatinib. Both compounds, but especially dasatinib, prevented the entire antiapoptotic CD40 program in CLL cells, and restored drug sensitivity. These effects also occurred in CLL samples with dysfunctional p53. Importantly, ex vivo CLL LN samples also displayed strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels. These data indicate that CLL cells in chemoresistant niches may be sensitive to therapeutic strategies that include c-Abl inhibitors.

Immunotherapies in CLL.

PubMed

Park, Jae H; Brentjens, Renier J

2013-01-01

Chronic lymphocytic leukemia (CLL) is the most frequently diagnosed leukemia in the Western world, yet remains essentially incurable. Although initial chemotherapy response rates are high, patients invariably relapse and subsequently develop resistance to chemotherapy. For the moment, allogeneic hematopoietic stem cell transplant (allo-HSCT) remains the only potentially curative treatment for patients with CLL, but it is associated with high rates of treatment-related mortality. Immune-based treatment strategies to augment the cytotoxic potential of T cells offer exciting new treatment options for patients with CLL, and provide a unique and powerful spectrum of tools distinct from traditional chemotherapy. Among the most novel and promising of these approaches are chimeric antigen receptor (CAR)-based cell therapies that combine advances in genetic engineering and adoptive immunotherapy.

Immunological aspects in chronic lymphocytic leukemia (CLL) development.

PubMed

García-Muñoz, Ricardo; Galiacho, Verónica Roldan; Llorente, Luis

2012-07-01

Chronic lymphocytic leukemia (CLL) is unique among B cell malignancies in that the malignant clones can be featured either somatically mutated or unmutated IGVH genes. CLL cells that express unmutated immunoglobulin variable domains likely underwent final development prior to their entry into the germinal center, whereas those that express mutated variable domains likely transited through the germinal center and then underwent final development. Regardless, the cellular origin of CLL remains unknown. The aim of this review is to summarize immunological aspects involved in this process and to provide insights about the complex biology and pathogenesis of this disease. We propose a mechanistic hypothesis to explain the origin of B-CLL clones into our current picture of normal B cell development. In particular, we suggest that unmutated CLL arises from normal B cells with self-reactivity for apoptotic bodies that have undergone receptor editing, CD5 expression, and anergic processes in the bone marrow. Similarly, mutated CLL would arise from cells that, while acquiring self-reactivity for autoantigens-including apoptotic bodies-in germinal centers, are also still subject to tolerization mechanisms, including receptor editing and anergy. We believe that CLL is a proliferation of B lymphocytes selected during clonal expansion through multiple encounters with (auto)antigens, despite the fact that they differ in their state of activation and maturation. Autoantigens and microbial pathogens activate BCR signaling and promote tolerogenic mechanisms such as receptor editing/revision, anergy, CD5+ expression, and somatic hypermutation in CLL B cells. The result of these tolerogenic mechanisms is the survival of CLL B cell clones with similar surface markers and homogeneous gene expression signatures. We suggest that both immunophenotypic surface markers and homogenous gene expression might represent the evidence of several attempts to re-educate self-reactive B cells.

Improving Therapy of Chronic Lymphocytic Leukemia (CLL) with Chimeric Antigen Receptor (CAR) T Cells

PubMed Central

Fraietta, Joseph A.; Schwab, Robert D.; Maus, Marcela V.

2016-01-01

Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically-engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and re-directing the immune system against cancer. This review will briefly summarize T cell therapies in development for CLL disease. We discuss the role of T cell function and phenotype, T cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. PMID:27040708

Diffuse large B-cell lymphoma (Richter syndrome) in patients with chronic lymphocytic leukaemia (CLL): a cohort study of newly diagnosed patients.

PubMed

Parikh, Sameer A; Rabe, Kari G; Call, Timothy G; Zent, Clive S; Habermann, Thomas M; Ding, Wei; Leis, Jose F; Schwager, Susan M; Hanson, Curtis A; Macon, William R; Kay, Neil E; Slager, Susan L; Shanafelt, Tait D

2013-09-01

Nearly all information about patients with chronic lymphocytic leukaemia (CLL) who develop diffuse large B-cell lymphoma [Richter syndrome (RS)] is derived from retrospective case series or patients treated on clinical trials. We used the Mayo Clinic CLL Database to identify patients with newly diagnosed CLL between January 2000 and July 2011. Individuals who developed biopsy-proven RS during follow-up were identified. After a median follow-up of 4 years, 37/1641 (2·3%) CLL patients developed RS. The rate of RS was approximately 0·5%/year. Risk of RS was associated with advanced Rai stage at diagnosis (P < 0·001), high-risk genetic abnormalitites on fluorescence in situ hybridization (P < 0·0001), unmutated IGHV (P = 0·003), and expression of ZAP70 (P = 0·02) and CD38 (P = 0·001). The rate of RS doubled in patients after treatment for CLL (1%/year). Stereotyped B-cell receptors (odds-ratio = 4·2; P = 0·01) but not IGHV4-39 family usage was associated with increased risk of RS. Treatment with combination of purine analogues and alkylating agents increased the risk of RS three-fold (odds-ratio = 3·26, P = 0·0003). Median survival after RS diagnosis was 2·1 years. The RS prognosis score stratified patients into three risk groups with median survivals of 0·5 years, 2·1 years and not reached. Both underlying characteristics of the CLL clone and subsequent CLL therapy influence the risk of RS. Survival after RS remains poor and new therapies are needed. © 2013 John Wiley & Sons Ltd.

Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.

PubMed

Barnidge, David R; Jelinek, Diane F; Muddiman, David C; Kay, Neil E

2005-01-01

Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.

MFI ratio estimation of ZAP-70 in B-CLL by flow cytometry can be improved by considering the isotype-matched antibody signal.

PubMed

Marquez, M-E; Deglesne, P-A; Suarez, G; Romano, E

2011-04-01

The IgV(H) mutational status of B-cell chronic lymphocytic leukemia (B-CLL) is of prognostic value. Expression of ZAP-70 in B-CLL is a surrogate marker for IgV(H) unmutated (UM). As determination of IgV(H) mutational status involves a methodology currently unavailable for most clinical laboratories, it is important to have available a reliable technique for ZAP-70 estimation in B-CLL. Flow cytometry (FC) is a convenient technique for this purpose. However, there is still no adequate way for data analysis, which would prevent the assignment of false positive or negative expression. We have modified the currently most accepted technique, which uses the ratio of the mean fluorescent index (MFI) of B-CLL to T cells. The MFI for parallel antibody isotype staining is subtracted from the ZAP-70 MFI of both B-CLL and T cells. We validated this technique comparing the results obtained for ZAP-70 expression by FC with those obtained with quantitative PCR for the same patients. We applied the technique in a series of 53 patients. With this modification, a better correlation between ZAP-70 expression and IgV(H) UM was obtained. Thus, the MFI ratio B-CLL/T cell corrected by isotype is a reliable analysis technique to estimate ZAP-70 expression in B-CLL. © 2010 Blackwell Publishing Ltd.

Stimulation of Chronic Lymphocytic Leukemia (CLL) Cells with CpG Oligodeoxynucleotide (ODN) Gives Consistent Karyotypic Results among Laboratories: a CLL Research Consortium (CRC)h Study

PubMed Central

Heerema, Nyla A.; Byrd, John C.; Cin, Paola Dal; Dell’ Aquila, Marie L.; Koduru, Prasad; Aviram, Ayala; Smoley, Stephanie; Rassenti, Laura Z.; Greaves, Andrew W.; Brown, Jennifer R.; Rai, Kanti R.; Kipps, Thomas J.; Kay, Neil E.; van Dyke, Daniel

2010-01-01

Cytogenetic abnormalities in CLL are important prognostic indicators. Historically, only interphase cytogenetics was clinically useful in CLL because traditional mitogens are not effective mitotic stimulants. Recently, CpG-oligodeoxynucleotide (ODN) stimulation has shown effectiveness in CLL. The CLL Research Consortium (CRC) tested the effectiveness and reproducibility of CpG-ODN stimulation to detect chromosomally abnormal clones by five laboratories. More clonal abnormalities were observed after culture of CLL cells with CpG-ODN than with pokeweed mitogen (PWM)+12-O-tetradecanoyl-phorobol-13-acetate (TPA). All clonal abnormalities in PWM+TPA cultures were observed in CpG-ODN cultures, whereas CpG-ODN identified some clones not found by PWM+TPA. CpG-ODN stimulation of one normal control and 12 CLL samples showed that excepting clones of del(13q) in low frequencies and one translocation, results in all five laboratories were consistent, and all abnormalities were concordant with FISH. Thus, abnormal clones in CLL are more readily detected with CpG-ODN stimulation than with traditional B-cell mitogens. After CpG-ODN stimulation, abnormalities were reproducible among cytogenetic laboratories. CpG-ODN did not appear to induce aberrations in cell culture and enhanced detection of abnormalities and complexity in CLL. Since karyotypic complexity is prognostic and is not detectable by standard FISH analyses, stimulation with CpG-ODN is useful to identify this additional prognostic factor in CLL. PMID:21156225

The survival decrease in gastric cancer is associated with the methylation of B-cell CLL/lymphoma 6 member B promoter.

PubMed

Deng, Jingyu; Liang, Han; Dong, Qiuping; Hou, Yachao; Xie, Xingming; Yu, Jun; Fan, Daiming; Hao, Xishan

2014-07-01

The methylation of B-cell CLL/lymphoma 6 member B (BCL6B) DNA promoter was detected in several malignancies. Here, we quantitatively detect the methylated status of CpG sites of BCL6B DNA promoter of 459 patients with gastric cancer (GC) by using bisulfite gene sequencing. We show that patients with three or more methylated CpG sites in the BCL6B promoter were significantly associated with poor survival. Furthermore, by using the Akaike information criterion value calculation, we show that the methylated count of BCL6B promoter was identified to be the optimal prognostic predictor of GC patients.

Isolation of a novel chronic lymphocytic leukemic (CLL) cell line and development of an in vivo mouse model of CLL.

PubMed

Kellner, Joshua; Wierda, William; Shpall, Elizabeth; Keating, Michael; McNiece, Ian

2016-01-01

Leukemic cell lines have become important tools for studies of disease providing a monoclonal cell population that can be extensively expanded in vitro while preserving leukemic cellular characteristics. However, studies of chronic lymphocytic leukemia (CLL) have been impeded in part by the lack of continuous human cell lines. CLL cells have a high spontaneous apoptosis rate in vitro and exhibit minimal proliferation in xenograft models. Therefore, there is a need for development of primary CLL cell lines and we describe the isolation of such a line from the bone marrow of a CLL patient (17p deletion and TP53 mutation) which has been in long term culture for more than 12 months with continuous proliferation. The CLL cell line (termed MDA-BM5) which was generated in vitro with continuous co-culture on autologous stromal cells is CD19+CD5+ and shows an identical pattern of somatic hypermutation as determined in the patient's bone marrow (BM), confirming the origin of the cells from the original CLL clone. MDA-BM5 cells were readily transplantable in NOD/SCID gamma null mice (NSG) with disease developing in the BM, liver and spleen. BM cells from quaternary serial transplantation in NSG mice demonstrated the presence of CD19+CD5+ cells with Ig restricted to lambda which is consistent with the original patient cells. These studies describe a new CLL cell line from a patient with del(17p) that provides a unique model for in vitro and in vivo studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of interleukins on the proliferation and survival of B cell chronic lymphocytic leukaemia cells.

PubMed Central

Mainou-Fowler, T; Copplestone, J A; Prentice, A G

1995-01-01

AIMS--To investigate the effects of interleukin (IL) 1, 2, 4, and 5 on the proliferation and survival of peripheral blood B cells from patients with B chronic lymphocytic leukaemia (B-CLL) and compare them with the effects on normal peripheral blood B cells. METHODS--The proliferation and survival of pokeweed mitogen (PWM) activated B cells from B-CLL (n = 12) and normal peripheral blood (n = 5) were studied in vitro in response to IL-1, IL-2 IL-4, and IL-5. Survival of cells in cultures with or without added interleukins was studied by microscopic examination of cells and DNA agarose gel electrophoresis. RESULTS--Proliferation was observed in both B-CLL and normal peripheral blood cells on culture with IL-2 alone and also in some, but not all, B-CLL and normal peripheral blood cells with IL-1 and IL-4. However, there was greater variability in B-CLL cell responses than in normal peripheral blood cells. Il-5 did not affect normal peripheral blood cell proliferation but it increased proliferation in two B-CLL cases. Synergistic effects of these cytokines were not detected. IL-4 inhibited normal peripheral blood and B-CLL cell proliferation after the addition of IL-2. Inhibition of B-CLL cell responses to IL-2 was also observed with IL-5 and Il-1. Survival of B-CLL cells in cultures was enhanced with IL-4 not by an increase in proliferation but by reduced apoptosis. No such effect was seen in normal peripheral blood cells. IL-2 had a less noticeable antiapoptotic effect; IL-5 enhanced apoptosis in B-CLL cells. CONCLUSIONS--B-CLL and normal peripheral blood cells proliferated equally well in response to IL-2. IL-4 had a much lower effect on B-CLL cell proliferation, but had noticeable antiapoptotic activity. IL-5 enhanced cell death by apoptosis. Images PMID:7629299

A pilot trial of the mTOR (mammalian target of rapamycin) inhibitor RAD001 in patients with advanced B-CLL.

PubMed

Decker, Thomas; Sandherr, Michael; Goetze, Katharina; Oelsner, Madlen; Ringshausen, Ingo; Peschel, Christian

2009-03-01

Although B-cell chronic lymphocytic leukemia (CLL) is treatable, it remains an incurable disease and most patients inevitably suffer relapse. Many therapeutic options exist for those requiring therapy, including monoclonal antibodies and stem cell transplantation, but remissions tend to last shorter in the course of the disease. Targeting the cell cycle has recently been realized to be an attractive therapeutic approach in solid and hematological malignancies, and the proliferative nature of B-CLL is increasingly accepted. Here, we report data on a phase II pilot trial with the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 5 mg/daily in patients with advanced B-CLL who had progressive disease after at least two lines of treatment. After treatment of seven patients, this trial was stopped because of toxicity concerns, although some degree of activity was observed (one partial remission, three patients with stable disease). Interestingly, cyclin E expression decreased in responding patients. Further strategies of mTOR inhibition by RAD001 in B-CLL should focus on different treatment schedules, adequate anti-infectious prophylaxis, or combinations with cytotoxic drugs.

STAT3-Activated GM-CSFRα Translocates to the Nucleus and Protects CLL Cells from Apoptosis

PubMed Central

Li, Ping; Harris, David; Liu, Zhiming; Rozovski, Uri; Ferrajoli, Alessandra; Wang, Yongtao; Bueso-Ramos, Carlos; Hazan-Halevy, Inbal; Grgurevic, Srdana; Wierda, William; Burger, Jan; O'Brien, Susan; Faderl, Stefan; Keating, Michael; Estrov, Zeev

2014-01-01

Here it was determined that Chronic Lymphocytic Leukemia (CLL) cells express the α-subunit but not the β-subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF3R). GM-CSFRα was detected on the surface, in the cytosol, and the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL-6 to activate STAT3 to identify STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL-6 stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3 siRNA or CLL cells with STAT3 shRNA significantly down-regulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell-survival pathways, and the proteins KAP1 (TRIM28) and ISG15 co-immunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. PMID:24836891

Acute myeloid leukemia stem cell markers in prognosis and targeted therapy: potential impact of BMI-1, TIM-3 and CLL-1.

PubMed

Darwish, Noureldien H E; Sudha, Thangirala; Godugu, Kavitha; Elbaz, Osama; Abdelghaffar, Hasan A; Hassan, Emad E A; Mousa, Shaker A

2016-09-06

Acute myeloid leukemia (AML) patients show high relapse rates and some develop conventional chemotherapy resistance. Leukemia Stem Cells (LSCs) are the main player for AML relapses and drug resistance. LSCs might rely on the B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) in promoting cellular proliferation and survival. Growth of LSCs in microenvironments that are deprived of nutrients leads to up-regulation of the signaling pathways during the progression of the disease, which may illustrate the sensitivity of LSCs to inhibitors of those signaling pathways as compared to normal cells. We analyzed the expression of LSC markers (CD34, CLL-1, TIM-3 and BMI-1) using quantitative RT-PCR in bone marrow samples of 40 AML patients of different FAB types (M1, M2, M3, M4, M5, and M7). We also studied the expression of these markers in 2 AML cell lines (Kasumi-1 and KG-1a) using flow cytometry and quantitative RT-PCR. The overexpression of TIM-3, CLL-1, and BMI-1 was markedly correlated with poor prognosis in these patients. Our in vitro findings demonstrate that targeting BMI-1, which markedly increased in the leukemic cells, was associated with marked decrease in leukemic burden. This study also presents results for blocking LSCs' surface markers CD44, CLL-1, and TIM-3. These markers may play an important role in elimination of AML. Our study indicates a correlation between the expression of markers TIM-3, CLL-1, and especially of BMI-1 and the aggressiveness of AML and thus the potential impact of prognosis and therapies that target LSCs on improving the cure rates.

Ph I/II Study of Subcutaneously Administered Veltuzumab (hA20) in NHL and CLL

ClinicalTrials.gov

2013-03-25

NHL; Lymphoma, Non-Hodgkin; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Intermediate-Grade; Lymphoma, Large-Cell; Lymphoma, Low-Grade; Lymphoma, Mixed-Cell; Lymphoma, Small-Cell; Leukemia, Lymphocytic, Chronic; Leukemia, B-Cell, Chronic; Leukemia, Prolymphocytic; Leukemia, Small Lymphocytic; Lymphoma, Small Lymphocytic; Lymphoma, Lymphoplasmacytoid, CLL; Lymphoplasmacytoid Lymphoma, CLL; CLL; SLL

S-allyl derivatives of 6-mercaptopurine are highly potent drugs against human B-CLL through synergism between 6-mercaptopurine and allicin.

PubMed

Miron, Talia; Wilchek, Meir; Shvidel, Lev; Berrebi, Alain; Arditti, Fabian D

2012-12-01

S-allylthio-6-mercaptopurine and its ribose derivative were tested for anti-leukemic activity, using a human- mouse B-CLL model. The novel prodrugs contain two components, a purine analog, which interferes with DNA synthesis, and an S-allylthio, readily engaging in thiol-disulfide exchange reactions. The latter component targets the redox homeostasis which is more sensitive in leukemic cells, than in normal B-cells. Upon administration, the prodrug permeates cells, instantly reacts with free thiol, forming S-allyl mixed disulfides and releasing purine. Several cycles of thiol-disulfide exchange reactions occur, thus extending the duration of the prodrug effects. The concerted action of 2 components, as compared with purine alone, boosted in vitro apoptotis in B-CLL cells from 10% to 38%, and decreased in vivo engraftment of B-CLL from 30% to 0.7%. Copyright © 2012 Elsevier Ltd. All rights reserved.

The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.

PubMed

van Rhenen, Anna; van Dongen, Guus A M S; Kelder, Angèle; Rombouts, Elwin J; Feller, Nicole; Moshaver, Bijan; Stigter-van Walsum, Marijke; Zweegman, Sonja; Ossenkoppele, Gert J; Jan Schuurhuis, Gerrit

2007-10-01

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.

Notch signaling sustains the expression of Mcl-1 and the activity of eIF4E to promote cell survival in CLL

PubMed Central

De Falco, Filomena; Sabatini, Rita; Del Papa, Beatrice; Falzetti, Franca; Di Ianni, Mauro; Sportoletti, Paolo; Baldoni, Stefano; Screpanti, Isabella; Marconi, Pierfrancesco; Rosati, Emanuela

2015-01-01

In chronic lymphocytic leukemia (CLL), Notch1 and Notch2 signaling is constitutively activated and contributes to apoptosis resistance. We show that genetic inhibition of either Notch1 or Notch2, through small-interfering RNA, increases apoptosis of CLL cells and is associated with decreased levels of the anti-apoptotic protein Mcl-1. Thus, Notch signaling promotes CLL cell survival at least in part by sustaining Mcl-1 expression. In CLL cells, an enhanced Notch activation also contributes to the increase in Mcl-1 expression and cell survival induced by IL-4. Mcl-1 downregulation by Notch targeting is not due to reduced transcription or degradation by caspases, but in part, to increased degradation by the proteasome. Mcl-1 downregulation by Notch targeting is also accompanied by reduced phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), suggesting that this protein is another target of Notch signaling in CLL cells. Overall, we show that Notch signaling sustains CLL cell survival by promoting Mcl-1 expression and eIF4E activity, and given the oncogenic role of these factors, we underscore the therapeutic potential of Notch inhibition in CLL. PMID:26041884

CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia.

PubMed

Wang, Jinghua; Chen, Siyu; Xiao, Wei; Li, Wende; Wang, Liang; Yang, Shuo; Wang, Weida; Xu, Liping; Liao, Shuangye; Liu, Wenjian; Wang, Yang; Liu, Nawei; Zhang, Jianeng; Xia, Xiaojun; Kang, Tiebang; Chen, Gong; Cai, Xiuyu; Yang, Han; Zhang, Xing; Lu, Yue; Zhou, Penghui

2018-01-10

Acute myeloid leukemia (AML) is one of the most common types of adult acute leukemia. Standard chemotherapies can induce complete remission in selected patients; however, a majority of patients eventually relapse and succumb to the disease. Thus, the development of novel therapeutics for AML is urgently needed. Human C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells and the majority of AML blasts. Moreover, CLL-1 is expressed in leukemia stem cells (LSCs), but absent in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment. We tested the expression of CLL-1 antigen on peripheral blood cells and bone marrow cells in healthy donor and AML patients. Then, we developed a chimeric antigen receptor (CAR) containing a CLL1-specific single-chain variable fragment, in combination with CD28, 4-1BB costimulatory domains, and CD3-ζ signaling domain. We further investigate the function of CLL-1 CAR-T cells. The CLL-1 CAR-T cells specifically lysed CLL-1 + cell lines as well as primary AML patient samples in vitro. Strong anti-leukemic activity was observed in vivo by using a xenograft model of disseminated AML. Importantly, CLL-1 + myeloid progenitor cells and mature myeloid cells were specifically eliminated by CLL-1 CAR-T cells, while normal HSCs were not targeted due to the lack of CLL-1 expression. CLL-1 CAR-T represents a promising immunotherapy for the treatment of AML.

EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

PubMed Central

Trinidad, Eva M.; García, Dolores; Soler, Gloria; Ortuño, Francisco J.; Zapata, Agustín G.; Alonso, Luis M.

2016-01-01

A role of endothelial cells in the survival of CLL cells during extravasation is presently unknown. Herein we show that CLL cells but not normal B cells can receive apoptotic signals through physical contact with TNF-α activated endothelium impairing survival in transendothelial migration (TEM) assays. In addition, the CLL cells of patients having lymphadenopathy (LApos) show a survival advantage during TEM that can be linked to increased expression of α4 and αL integrin chains. Within this context, ephrinA4 expressed on the surface of CLL cells sequestrates integrins and inactivates them resulting in reduced adhesion and inhibition of apoptotic/survival signals through them. In agreement, ephrinA4 silencing resulted in increased survival of CLL cells of LApos patients but not LA neg patients. Similarly was observed when a soluble ephrinA4 isoform was added to TEM assays strongly suggesting that accumulation of this isoform in the serum of LApos patients could contribute to CLL cells dissemination and survival in vivo. In supporting, CLL lymphadenopathies showed a preferential accumulation of apoptotic CLL cells around high endothelial venules lacking ephrinA4. Moreover, soluble ephrinA4 isolated from sera of patients increased the number and viability of CLL cells recovered from the lymph nodes of adoptively transferred mice. Finally, we present evidence suggesting that soluble ephrinA4 mediated survival during TEM could enhance a transcellular TEM route of the CLL cells. Together these findings point to an important role of ephrinA4 in the nodal dissemination of CLL cells governing extravasation and survival. PMID:27374180

Direct in vivo Evidence for Increased Proliferation of CLL Cells in Lymph Nodes Compared to Bone Marrow and Peripheral Blood

PubMed Central

Saba, Nakhle S.; Valdez, Janet; Emson, Claire; Gatmaitan, Michelle; Tian, Xin; Hughes, Thomas E.; Sun, Clare; Arthur, Diane C.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Niemann, Carsten U.; Marti, Gerald E.; Aue, Georg; Soto, Susan; Farooqui, Mohammed Z.H.; Herman, Sarah E.M.; Chiorazzi, Nicholas; Wiestner, Adrian

2016-01-01

Chronic Lymphocytic Leukemia (CLL) is a progressive malignancy of mature B-cells that involves the peripheral blood (PB), lymph nodes (LNs) and bone marrow (BM). While the majority of CLL cells are in a resting state, small populations of proliferating cells exist; however, the anatomical site of active cell proliferation remains to be definitively determined. Based on findings that CLL cells in LNs have increased expression of B-cell activation genes, we tested the hypothesis that the fraction of “newly born” cells would be highest in the LNs. Using a deuterium oxide (2H) in vivo labeling method in which patients consumed deuterated (heavy) water (2H2O), we determined CLL cell kinetics in concurrently obtained samples from LN, PB, and BM. The LN was identified as the anatomical site harboring the largest fraction of newly born cells, compared to PB and BM. In fact, the calculated birth rate in the LN reached as high a 3.3% of the clone per day. Subdivision of the bulk CLL population by flow cytometry identified the subpopulation with the CXCR4dimCD5bright phenotype as containing the highest proportion of newly born cells within each compartment, including the LN, identifying this subclonal population as an important target for novel treatment approaches. PMID:28074063

MALT1 Inhibition Is Efficacious in Both Naïve and Ibrutinib-Resistant Chronic Lymphocytic Leukemia.

PubMed

Saba, Nakhle S; Wong, Deanna H; Tanios, Georges; Iyer, Jessica R; Lobelle-Rich, Patricia; Dadashian, Eman L; Liu, Delong; Fontan, Lorena; Flemington, Erik K; Nichols, Cydney M; Underbayev, Chingiz; Safah, Hana; Melnick, Ari; Wiestner, Adrian; Herman, Sarah E M

2017-12-15

The clinical efficacy displayed by ibrutinib in chronic lymphocytic leukemia (CLL) has been challenged by the frequent emergence of resistant clones. The ibrutinib target, Bruton's tyrosine kinase (BTK), is essential for B-cell receptor signaling, and most resistant cases carry mutations in BTK or PLCG2 , a downstream effector target of BTK. Recent findings show that MI-2, a small molecule inhibitor of the para-caspase MALT1, is effective in preclinical models of another type of BCR pathway-dependent lymphoma. We therefore studied the activity of MI-2 against CLL and ibrutinib-resistant CLL. Treatment of CLL cells in vitro with MI-2 inhibited MALT1 proteolytic activity reduced BCR and NF-κB signaling, inhibited nuclear translocation of RelB and p50, and decreased Bcl-xL levels. MI-2 selectively induced dose and time-dependent apoptosis in CLL cells, sparing normal B lymphocytes. Furthermore, MI-2 abrogated survival signals provided by stromal cells and BCR cross-linking and was effective against CLL cells harboring features associated with poor outcomes, including 17p deletion and unmutated IGHV Notably, MI-2 was effective against CLL cells collected from patients harboring mutations conferring resistance to ibrutinib. Overall, our findings provide a preclinical rationale for the clinical development of MALT1 inhibitors in CLL, in particular for ibrutinib-resistant forms of this disease. Cancer Res; 77(24); 7038-48. ©2017 AACR . ©2017 American Association for Cancer Research.

Chronic Lymphocytic Leukemia B-Cell Normal Cellular Counterpart: Clues From a Functional Perspective

PubMed Central

Darwiche, Walaa; Gubler, Brigitte; Marolleau, Jean-Pierre; Ghamlouch, Hussein

2018-01-01

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of small mature-looking CD19+ CD23+ CD5+ B-cells that accumulate in the blood, bone marrow, and lymphoid organs. To date, no consensus has been reached concerning the normal cellular counterpart of CLL B-cells and several B-cell types have been proposed. CLL B-cells have remarkable phenotypic and gene expression profile homogeneity. In recent years, the molecular and cellular biology of CLL has been enriched by seminal insights that are leading to a better understanding of the natural history of the disease. Immunophenotypic and molecular approaches (including immunoglobulin heavy-chain variable gene mutational status, transcriptional and epigenetic profiling) comparing the normal B-cell subset and CLL B-cells provide some new insights into the normal cellular counterpart. Functional characteristics (including activation requirements and propensity for plasma cell differentiation) of CLL B-cells have now been investigated for 50 years. B-cell subsets differ substantially in terms of their functional features. Analysis of shared functional characteristics may reveal similarities between normal B-cell subsets and CLL B-cells, allowing speculative assignment of a normal cellular counterpart for CLL B-cells. In this review, we summarize current data regarding peripheral B-cell differentiation and human B-cell subsets and suggest possibilities for a normal cellular counterpart based on the functional characteristics of CLL B-cells. However, a definitive normal cellular counterpart cannot be attributed on the basis of the available data. We discuss the functional characteristics required for a cell to be logically considered to be the normal counterpart of CLL B-cells. PMID:29670635

A novel CD44-binding peptide from the pro-matrix metalloproteinase-9 hemopexin domain impairs adhesion and migration of chronic lymphocytic leukemia (CLL) cells.

PubMed

Ugarte-Berzal, Estefanía; Bailón, Elvira; Amigo-Jiménez, Irene; Albar, Juan Pablo; García-Marco, José A; García-Pardo, Angeles

2014-05-30

(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4β1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC50 value of 90 μM. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4β1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Ibrutinib treatment improves T cell number and function in CLL patients

PubMed Central

Long, Meixiao; Do, Priscilla; Mundy, Bethany L.; Gordon, Amber; Lehman, Amy M.; Maddocks, Kami J.; Cheney, Carolyn; Jones, Jeffrey A.; Flynn, Joseph M.; Andritsos, Leslie A.; Fraietta, Joseph A.; June, Carl H.; Maus, Marcela V.; Woyach, Jennifer A.; Caligiuri, Michael A.; Johnson, Amy J.

2017-01-01

BACKGROUND. Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton’s tyrosine kinase (BTK) and IL-2–inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. METHODS. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. RESULTS. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. CONCLUSIONS. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. TRIAL REGISTRATION. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. FUNDING. The National Cancer Institute. PMID:28714866

Ibrutinib treatment improves T cell number and function in CLL patients.

PubMed

Long, Meixiao; Beckwith, Kyle; Do, Priscilla; Mundy, Bethany L; Gordon, Amber; Lehman, Amy M; Maddocks, Kami J; Cheney, Carolyn; Jones, Jeffrey A; Flynn, Joseph M; Andritsos, Leslie A; Awan, Farrukh; Fraietta, Joseph A; June, Carl H; Maus, Marcela V; Woyach, Jennifer A; Caligiuri, Michael A; Johnson, Amy J; Muthusamy, Natarajan; Byrd, John C

2017-08-01

Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton's tyrosine kinase (BTK) and IL-2-inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. The National Cancer Institute.

Gliotoxin is a potent NOTCH2 transactivation inhibitor and efficiently induces apoptosis in chronic lymphocytic leukaemia (CLL) cells.

PubMed

Hubmann, Rainer; Hilgarth, Martin; Schnabl, Susanne; Ponath, Elena; Reiter, Marlies; Demirtas, Dita; Sieghart, Wolfgang; Valent, Peter; Zielinski, Christoph; Jäger, Ulrich; Shehata, Medhat

2013-03-01

Chronic lymphocytic leukaemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC)- dependent manner. The transcriptional activity of NOTCH2 correlates not only with the expression of its target gene FCER2 (CD23) but is also functionally linked with CLL cell viability. In the majority of CLL cases, DNA-bound NOTCH2 complexes are less sensitive to the γ-secretase inhibitor (GSI) DAPT. Therefore, we searched for compounds that interfere with NOTCH2 signalling at the transcription factor level. Using electrophoretic mobility shift assays (EMSA), we identified the Aspergillum-derived secondary metabolite gliotoxin as a potent NOTCH2 transactivation inhibitor. Gliotoxin completely blocked the formation of DNA-bound NOTCH2 complexes in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signalling by gliotoxin was associated with down regulation of CD23 (FCER) expression and induction of apoptosis. Short time exposure of CLL cells indicated that the early apoptotic effect of gliotoxin is independent of proteasome regulated nuclear factor κB activity, and is associated with up regulation of NOTCH3 and NR4A1 expression. Gliotoxin could overcome the supportive effect of primary bone marrow stromal cells in an ex vivo CLL microenvironment model. In conclusion, we identified gliotoxin as a potent NOTCH2 inhibitor with a promising therapeutic potential in CLL. © 2012 Blackwell Publishing Ltd.

Sensitization of B-cell chronic lymphocytic leukemia cells to recombinant immunotoxin by immunostimulatory phosphorothioate oligodeoxynucleotides.

PubMed

Decker, Thomas; Hipp, Susanne; Kreitman, Robert J; Pastan, Ira; Peschel, Christian; Licht, Thomas

2002-02-15

A recombinant anti-CD25 immunotoxin, LMB-2, has shown clinical efficacy in hairy cell leukemia and T-cell neoplasms. Its activity in B-cell chronic lymphocytic leukemia (B-CLL) is inferior but might be improved if B-CLL cells expressed higher numbers of CD25 binding sites. It was recently reported that DSP30, a phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN) induces immunogenicity of B-CLL cells by up-regulation of CD25 and other antigens. The present study investigated the antitumor activity of LMB-2 in the presence of DSP30. To this end, B-CLL cells from peripheral blood of patients were isolated immunomagnetically to more than 98% purity. Incubation with DSP30 for 48 hours augmented CD25 expression in 14 of 15 B-CLL samples, as assessed by flow cytometry. DSP30 increased LMB-2 cytotoxicity dose dependently whereas a control ODN with no CpG motif did not. LMB-2 displayed no antitumor cell activity in the absence of CpG-ODN as determined colorimetrically with an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. In contrast, B-CLL growth was inhibited in 12 of 13 samples with 50% inhibition concentrations (IC(50)) in the range of LMB-2 plasma levels achieved in clinical studies. Two samples were not evaluable because of spontaneous B-CLL cell death in the presence of DSP30. Control experiments with an immunotoxin that does not recognize hematopoietic cells, and an anti-CD22 immunotoxin, confirmed that sensitization to LMB-2 was specifically due to up-regulation of CD25. LMB-2 was much less toxic to normal B and T lymphocytes compared with B-CLL cells. In summary, immunostimulatory CpG-ODNs efficiently sensitize B-CLL cells to a recombinant immunotoxin by modulation of its target. This new treatment strategy deserves further attention.

Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL.

PubMed

Sharma, Shruti; Galanina, Natalie; Guo, Ailin; Lee, Jimmy; Kadri, Sabah; Van Slambrouck, Charles; Long, Bradley; Wang, Weige; Ming, Mei; Furtado, Larissa V; Segal, Jeremy P; Stock, Wendy; Venkataraman, Girish; Tang, Wei-Jen; Lu, Pin; Wang, Yue Lynn

2016-10-18

Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL). However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months and many patients discontinue the treatment due to leukemia progression or Richter transformation. Mutations affecting the C481 residue of BTK disrupt ibrutinib binding and have been characterized by us and others as the most common mechanism of ibrutinib resistance. Thus far, all described BTK mutations are located in its kinase domain and mutations outside this domain have never been described. Herein, we report a patient whose CLL progressed, was salvaged with ibrutinib and then relapsed. Serial analysis of samples throughout patient's clinical course identified a structurally novel mutation (BTKT316A) in the SH2 domain, but not kinase domain, of Bruton tyrosine kinase which was associated with disease relapse. Functionally, cells carrying BTKT316A show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTKC481S. Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.

JAK2 mutation in a patient with CLL with coexistent myeloproliferative neoplasm (MPN).

PubMed

Kodali, Srinivas; Chen, Chi; Rathnasabapathy, Chenthilmurugan; Wang, Jen Chin

2009-12-01

JAK2 mutation has not been described in patients with chronic lymphocytic leukemia (CLL). We found JAK2 mutation in a patient with CLL and coexisting myeloproliferative neoplasm (MPN). In this patient, we demonstrated the presence of the JAK2 mutation in CD34(+) progenitor cells, myeloid lineage cells, megakaryocytes, B lymphocytes but not in T lymphocytes. This case represents the first case report of JAK2 mutation in CLL and may also suggest that, JAK2 mutation most likely represents a secondary event from primary gene mutations involving the primitive stem cells which give rise to MPN and CLL. Furthermore, in this case, we believe that we are the first to demonstrate that JAK2 mutation in myeloid and B lymphoid cells but not T lymphocytes in a case of coexisting CLL and MPN.

CD47 Agonist Peptides Induce Programmed Cell Death in Refractory Chronic Lymphocytic Leukemia B Cells via PLCγ1 Activation: Evidence from Mice and Humans

PubMed Central

Attout, Tarik; Boullet, Heloïse; Herbi, Linda; Vela, Laura; Barbier, Sandrine; Chateau, Danielle; Chapiro, Elise; Nguyen-Khac, Florence; Davi, Frédéric; Le Garff-Tavernier, Magali; Moumné, Roba; Sarfati, Marika; Karoyan, Philippe; Merle-Béral, Hélène; Launay, Pierre; Susin, Santos A.

2015-01-01

Background Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides. Methods and Findings In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides

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.

PubMed

Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

2009-11-12

Allogeneic 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.

Chemokines and chemokine receptors in chronic lymphocytic leukemia (CLL): from understanding the basics towards therapeutic targeting.

PubMed

Burger, Jan A

2010-12-01

Chemokines and their receptors organize the recruitment and positioning of cells at each stage of the immune response, a system critically dependent upon coordination to get the right cells to the right place at the right time. Chemokine receptors expressed on CLL B cells are thought to function in a similar fashion, regulating the trafficking of the leukemia cells between blood, lymphoid organs, and the bone marrow, and within sub compartments within these tissues, in concert with adhesion molecules and other guidance cues. CLL cells not only respond to chemokines secreted in the microenvironment, the leukemia cells also secrete chemokines in response to external signals, such as B cell receptor engagement. These CLL cell-derived chemokines facilitate interactions between CLL cells, T cells, and other immune cells that shape the CLL microenvironment. CXCR4, the most prominent chemokine receptor in CLL, is now targeted in a first clinical trial, emphasizing that chemokines and their receptors have become a highly dynamic translational research field. Copyright © 2010 Elsevier Ltd. All rights reserved.

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease.

PubMed

Palacios, Florencia; Moreno, Pilar; Morande, Pablo; Abreu, Cecilia; Correa, Agustín; Porro, Valentina; Landoni, Ana Ines; Gabus, Raul; Giordano, Mirta; Dighiero, Guillermo; Pritsch, Otto; Oppezzo, Pablo

2010-06-03

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

Genetic and epigenetic profiling of CLL disease progression reveals limited somatic evolution and suggests a relationship to memory-cell development.

PubMed

Smith, E N; Ghia, E M; DeBoever, C M; Rassenti, L Z; Jepsen, K; Yoon, K-A; Matsui, H; Rozenzhak, S; Alakus, H; Shepard, P J; Dai, Y; Khosroheidari, M; Bina, M; Gunderson, K L; Messer, K; Muthuswamy, L; Hudson, T J; Harismendy, O; Barrett, C L; Jamieson, C H M; Carson, D A; Kipps, T J; Frazer, K A

2015-04-10

We examined genetic and epigenetic changes that occur during disease progression from indolent to aggressive forms of chronic lymphocytic leukemia (CLL) using serial samples from 27 patients. Analysis of DNA mutations grouped the leukemia cases into three categories: evolving (26%), expanding (26%) and static (47%). Thus, approximately three-quarters of the CLL cases had little to no genetic subclonal evolution. However, we identified significant recurrent DNA methylation changes during progression at 4752 CpGs enriched for regions near Polycomb 2 repressive complex (PRC2) targets. Progression-associated CpGs near the PRC2 targets undergo methylation changes in the same direction during disease progression as during normal development from naive to memory B cells. Our study shows that CLL progression does not typically occur via subclonal evolution, but that certain CpG sites undergo recurrent methylation changes. Our results suggest CLL progression may involve developmental processes shared in common with the generation of normal memory B cells.

Kinetics of CLL cells in tissues and blood during therapy with the BTK inhibitor ibrutinib.

PubMed

Wodarz, Dominik; Garg, Naveen; Komarova, Natalia L; Benjamini, Ohad; Keating, Michael J; Wierda, William G; Kantarjian, Hagop; James, Danelle; O'Brien, Susan; Burger, Jan A

2014-06-26

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has excellent clinical activity in patients with chronic lymphocytic leukemia (CLL). Characteristically, ibrutinib causes CLL cell redistribution from tissue sites into the peripheral blood during the initial weeks of therapy. To better characterize the dynamics of this redistribution phenomenon, we correlated serial lymphocyte counts with volumetric changes in lymph node and spleen sizes during ibrutinib therapy. Kinetic parameters were estimated by applying a mathematical model to the data. We found that during ibrutinib therapy, 1.7% ± 1.1% of blood CLL cells and 2.7% ± 0.99% of tissue CLL cells die per day. The fraction of the tissue CLL cells that was redistributed into the blood during therapy was estimated to be 23.3% ± 17% of the total tissue disease burden. These data indicate that the reduction of tissue disease burden by ibrutinib is due more to CLL cell death and less to egress from nodal compartments. © 2014 by The American Society of Hematology.

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.

PubMed

Stempin, Cinthia C; Chi, Liying; Giraldo-Vela, Juan P; High, Anthony A; Häcker, Hans; Redecke, Vanessa

2011-10-28

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.

Role of BAFF and APRIL in human B-cell chronic lymphocytic leukaemia

PubMed Central

Haiat, Stéphanie; Billard, Christian; Quiney, Claire; Ajchenbaum-Cymbalista, Florence; Kolb, Jean-Pierre

2006-01-01

B-cell chronic lymphocytic leukaemia (B-CLL) is the most prevalent leukaemia in Western countries and is characterized by the gradual accumulation in patients of small mature B cells. Since the vast majority of tumoral cells are quiescent, the accumulation mostly results from deficient apoptosis rather than from acute proliferation. Although the phenomenon is relevant in vivo, B-CLL cells die rapidly in vitro as a consequence of apoptosis, suggesting a lack of essential growth factors in the culture medium. Indeed, the rate of B-CLL cell death in vitro is modulated by different cytokines, some favouring the apoptotic process, others counteracting it. Two related members of the tumour necrosis factor family, BAFF (B-cell activating factor of the TNF family) and APRIL (a proliferation-inducing ligand), already known for their crucial role in normal B-cell survival, differentiation and apoptosis, were recently shown to be expressed by B-CLL cells. These molecules are able to protect the leukaemic cells against spontaneous and drug-induced apoptosis via autocrine and/or paracrine pathways. This review will focus on the role of BAFF and APRIL in the survival of tumoral cells. It will discuss the expression of these molecules by B-CLL cells, their regulation, transduction pathways and their effects on leukaemic cells. The design of reagents able to counteract the effects of these molecules seems to be a new promising therapeutic approach for B-CLL and is already currently developed in the treatment of autoimmune diseases. PMID:16827889

The top ten clues to understand the origin of chronic lymphocytic leukemia (CLL).

PubMed

García-Muñoz, Ricardo; Feliu, Jesús; Llorente, Luis

2015-01-01

The fundamental task of the immune system is to protect the individual from infectious organisms without serious injury to self. The essence of acquired immunity is molecular self/non self discrimination. Chronic lymphocytic leukemia is characterized by a global failure of immune system that begins with the failure of immunological tolerance mechanisms (autoimmunity) and finish with the incapacity to response to non-self antigens (immunodeficiency). Immunological tolerance mechanisms are involved in chronic lymphocytic leukemia (CLL) development. During B cell development some self-reactive B cells acquire a special BCR that recognize their own BCR. This self-autoantibody-self BCR interaction promotes survival, differentiation and proliferation of self-reactive B cells. Continuous self-autoantibody-self BCR interaction cross-linking induces an increased rate of surface BCR elimination, CD5+ expression, receptor editing and anergy. Unfortunately, some times this mechanisms increase genomic instability and promote additional genetic damage that immortalize self-reactive B cells and convert them into CLL like clones with the capability of clonal evolution and transformed CLL B cells. This review summarizes the immunological effects of continuous self-autoantibody-self BCR interaction cross-linking in the surface of self-reactive B cells and their role in CLL development. Copyright © 2014 Elsevier Ltd. All rights reserved.

Angiogenic factors in chronic lymphocytic leukaemia (CLL): Where do we stand?

PubMed

Aguirre Palma, Luis Mario; Gehrke, Iris; Kreuzer, Karl-Anton

2015-03-01

The role of angiogenesis in haematological malignancies such as chronic lymphocytic leukaemia (CLL) is difficult to envision, because leukaemia cells are not dependent on a network of blood vessels to support basic physiological requirements. Regardless, CLL cells secrete high levels of major angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet derived growth factor (PDGF). Nonetheless, it remains unclear how most angiogenic factors regulate accumulation and delayed apoptosis of CLL cells. Angiogenic factors such as leptin, granulocyte colony-stimulating factor (G-CSF), follistatin, angiopoietin-1 (Ang1), angiogenin (ANG), midkine (MK), pleiotrophin (PTN), progranulin (PGRN), proliferin (PLF), placental growth factor (PIGF), and endothelial locus-1 (Del-1), represent novel therapeutic targets of future CLL research but have remained widely overlooked. This review aims to outline our current understanding of angiogenic growth factors and their relationship with CLL, a still uncured haematopoietic malignancy. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Targeted Axl Inhibition Primes Chronic Lymphocytic Leukemia B Cells to Apoptosis and Shows Synergistic/Additive Effects in Combination with BTK Inhibitors.

PubMed

Sinha, Sutapa; Boysen, Justin; Nelson, Michael; Secreto, Charla; Warner, Steven L; Bearss, David J; Lesnick, Connie; Shanafelt, Tait D; Kay, Neil E; Ghosh, Asish K

2015-05-01

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite aggressive therapeutic approaches. We previously found that Axl receptor tyrosine kinase (RTK) plays a critical role in CLL B-cell survival. Here, we explored the possibility of using a high-affinity Axl inhibitor as a single agent or in combination with Bruton's tyrosine kinase (BTK) inhibitors for future clinical trial to treat patients with CLL. Expression/activation status of other members of the TAM (e.g., Tyro3, Axl, and MER) family of RTKs in CLL B cells was evaluated. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 with or without the presence of CLL bone marrow stromal cells (BMSCs). Inhibitory effects of TP-0903 on the Axl signaling pathway were also evaluated in CLL B cells. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination. CLL B cells overexpress Tyro3, but not MER. Of interest, Tyro3 remains as constitutively phosphorylated and forms a complex with Axl in CLL B cells. TP-0903 induces massive apoptosis in CLL B cells with LD50 values of nanomolar ranges. Importantly, CLL BMSCs could not protect the leukemic B cells from TP-0903-induced apoptosis. A marked reduction of the antiapoptotic proteins Mcl-1, Bcl-2, and XIAP and upregulation of the proapoptotic protein BIM in CLL B cells was detected as a result of Axl inhibition. Finally, combination of TP-0903 with BTK inhibitors augments CLL B-cell apoptosis. Administration of TP-0903 either as a single agent or in combination with BTK inhibitors may be effective in treating patients with CLL. ©2015 American Association for Cancer Research.

Recognition of Antigen-Specific B Cell Receptors From Chronic Lymphocytic Leukemia Patients By Synthetic Antigen Surrogates

PubMed Central

Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas

2014-01-01

In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe discovery of non-peptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used for discovery of other classes of antigen surrogates. PMID:25467125

Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

PubMed

Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

2014-12-18

In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia.

PubMed

Lanemo Myhrinder, Anna; Hellqvist, Eva; Bergh, Ann-Charlotte; Jansson, Mattias; Nilsson, Kenneth; Hultman, Per; Jonasson, Jon; Buhl, Anne Mette; Bredo Pedersen, Lone; Jurlander, Jesper; Klein, Eva; Weit, Nicole; Herling, Marco; Rosenquist, Richard; Rosén, Anders

2013-08-01

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface membrane receptors determines whether the cells will be proliferating, anergic or apoptotic. To better understand the role of antigen in leukemogenesis, CLL cell lines producing monoclonal antibodies (mAbs) will facilitate structural analysis of antigens and supply DNA for genetic studies. We present here a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA/short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA.

Understanding Drug Resistance to Targeted Therapeutics in Malignant B-Cell Lymphoproliferative Disorders (B-LPDs)

DTIC Science & Technology

2014-10-01

18. Munari F et al. Tumor-associated macrophages as major source of APRIL in gastric MALT lymphoma . Blood 2011,117(24)6612-6616. 19. Mackay F et al... Lymphoma (CLL/SLL)  microenvironment  B-cell receptor (BCR)  C-X-C chemokine receptor type 4 (CXCR-4/CD184)  lenalidomide  plerixafor...AND LICENSES: Nothing to report VIII. REPORTABLE OUTCOMES: Nothing to report IX. OTHER ACHIEVEMENTS: A. Funding Applications 1. Lymphoma

A drive through cellular therapy for CLL in 2015: allogeneic cell transplantation and CARs.

PubMed

Mato, Anthony; Porter, David L

2015-07-23

Over the past decade the development of safer reduced-intensity conditioning regimens, expanded donor pools, advances in supportive care, and prevention/management of graft-versus-host disease have expanded stem cell transplantation (SCT) availability for chronic lymphocytic leukemia (CLL) patients. However, there are now increasingly active treatment options available for CLL patients with favorable toxicity profiles and convenient administration schedules. This raises the critical issue of whether or not attainment of cure remains a necessary goal. It is now less clear that treatment with curative intention and with significant toxicity is required for long-term survival in CLL. In addition, the demonstrated safety and activity of genetically modified chimeric antigen receptor (CAR) T cells present the opportunity of harnessing the power of the immune system to kill CLL cells without the need for SCT. We attempt to define the role of SCT in the era of targeted therapies and discuss questions that remain to be answered. Furthermore, we highlight the potential for exciting new cellular therapy using genetically modified anti-CD19 CAR T cells and discuss its potential to alter treatment paradigms for CLL. © 2015 by The American Society of Hematology.

Ibrutinib modulates the immunosuppressive CLL microenvironment through STAT3-mediated suppression of regulatory B-cell function and inhibition of the PD-1/PD-L1 pathway.

PubMed

Kondo, K; Shaim, H; Thompson, P A; Burger, J A; Keating, M; Estrov, Z; Harris, D; Kim, E; Ferrajoli, A; Daher, M; Basar, R; Muftuoglu, M; Imahashi, N; Alsuliman, A; Sobieski, C; Gokdemir, E; Wierda, W; Jain, N; Liu, E; Shpall, E J; Rezvani, K

2018-04-01

Ibrutinib, a covalent inhibitor of Bruton Tyrosine Kinase (BTK), is approved for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). Besides directly inhibiting BTK, ibrutinib possesses immunomodulatory properties through targeting multiple signaling pathways. Understanding how this ancillary property of ibrutinib modifies the CLL microenvironment is crucial for further exploration of immune responses in this disease and devising future combination therapies. Here, we investigated the mechanisms underlying the immunomodulatory properties of ibrutinib. In peripheral blood samples collected prospectively from CLL patients treated with ibrutinib monotherapy, we observed selective and durable downregulation of PD-L1 on CLL cells by 3 months post-treatment. Further analysis showed that this effect was mediated through inhibition of the constitutively active signal transducer and activator of transcription 3 (STAT3) in CLL cells. Similar downregulation of PD-1 was observed in CD4+ and CD8+ T cells. We also demonstrated reduced interleukin (IL)-10 production by CLL cells in patients receiving ibrutinib, which was also linked to suppression of STAT3 phosphorylation. Taken together, these findings provide a mechanistic basis for immunomodulation by ibrutinib through inhibition of the STAT3 pathway, critical in inducing and sustaining tumor immune tolerance. The data also merit testing of combination treatments combining ibrutinib with agents capable of augmenting its immunomodulatory effects.

Autologous CLL cell vaccination early after transplant induces leukemia-specific T cells.

PubMed

Burkhardt, Ute E; Hainz, Ursula; Stevenson, Kristen; Goldstein, Natalie R; Pasek, Mildred; Naito, Masayasu; Wu, Di; Ho, Vincent T; Alonso, Anselmo; Hammond, Naa Norkor; Wong, Jessica; Sievers, Quinlan L; Brusic, Ana; McDonough, Sean M; Zeng, Wanyong; Perrin, Ann; Brown, Jennifer R; Canning, Christine M; Koreth, John; Cutler, Corey; Armand, Philippe; Neuberg, Donna; Lee, Jeng-Shin; Antin, Joseph H; Mulligan, Richard C; Sasada, Tetsuro; Ritz, Jerome; Soiffer, Robert J; Dranoff, Glenn; Alyea, Edwin P; Wu, Catherine J

2013-09-01

Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8+ T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. Clinicaltrials.gov NCT00442130. NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Translational Research Program.

The HDAC inhibitor valproate induces a bivalent status of the CD20 promoter in CLL patients suggesting distinct epigenetic regulation of CD20 expression in CLL in vivo

PubMed Central

Scialdone, Annarita; Hasni, Muhammad Sharif; Damm, Jesper Kofoed; Lennartsson, Andreas; Gullberg, Urban; Drott, Kristina

2017-01-01

Treatment with anti-CD20 antibodies is only moderately efficient in chronic lymphocytic leukemia (CLL), a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown that CD20 is epigenetically regulated and that histone deacetylase inhibitors (HDACis) can increase CD20 expression in vitro in CLL. To assess whether HDACis can upregulate CD20 also in vivo in CLL, the HDACi valproate was given to three del13q/NOTCH1wt CLL patients and CD20 levels were analysed (the PREVAIL study). Valproate treatment resulted in expected global activating histone modifications suggesting HDAC inhibitory effects. However, although valproate induced expression of CD20 mRNA and protein in the del13q/NOTCH1wt I83-E95 CLL cell line, no such effects were observed in the patients studied. In contrast to the cell line, in patients valproate treatment resulted in transient recruitment of the transcriptional repressor EZH2 to the CD20 promoter, correlating to an increase of the repressive histone mark H3K27me3. This suggests that valproate-mediated induction of CD20 may be hampered by EZH2 mediated H3K27me3 in vivo in CLL. Moreover, valproate treatment resulted in induction of EZH2 and global H3K27me3 in patient cells, suggesting transcriptionally repressive effects of valproate in CLL. Our results suggest new in vivo mechanisms of HDACis which may have implications on the design of future clinical trials in B-cell malignancies. PMID:28445158

Early autologous stem cell transplantation for chronic lymphocytic leukemia: long-term follow-up of the German CLL Study Group CLL3 trial.

PubMed

Dreger, Peter; Döhner, Hartmut; McClanahan, Fabienne; Busch, Raymonde; Ritgen, Matthias; Greinix, Hildegard; Fink, Anna-Maria; Knauf, Wolfgang; Stadler, Michael; Pfreundschuh, Michael; Dührsen, Ulrich; Brittinger, Günter; Hensel, Manfred; Schetelig, Johannes; Winkler, Dirk; Bühler, Andreas; Kneba, Michael; Schmitz, Norbert; Hallek, Michael; Stilgenbauer, Stephan

2012-05-24

The CLL3 trial was designed to study intensive treatment including autologous stem cell transplantation (autoSCT) as part of first-line therapy in patients with chronic lymphocytic leukemia (CLL). Here, we present the long-term outcome of the trial with particular focus on the impact of genomic risk factors, and we provide a retrospective comparison with patients from the fludarabine-cyclophosphamide-rituximab (FCR) arm of the German CLL Study Group (GCLLSG) CLL8 trial. After a median observation time of 8.7 years (0.3-12.3 years), median progression-free survival (PFS), time to retreatment, and overall survival (OS) of 169 evaluable patients, including 38 patients who did not proceed to autoSCT, was 5.7, 7.3, and 11.3 years, respectively. PFS and OS were significantly reduced in the presence of 17p- and of an unfavorable immunoglobulin heavy variable chain mutational status, but not of 11q-. Five-year nonrelapse mortality was 6.5%. When 110 CLL3 patients were compared with 126 matched patients from the FCR arm of the CLL8 trial, 4-year time to retreatment (75% vs 77%) and OS (86% vs 90%) was similar despite a significant benefit for autoSCT in terms of PFS. In summary, early treatment intensification including autoSCT can provide very effective disease control in poor-risk CLL, although its clinical benefit in the FCR era remains uncertain. The trial has been registered with www.clinicaltrials.gov as NCT00275015.

CD38 is a signaling molecule in B-cell chronic lymphocytic leukemia cells.

PubMed

Deaglio, Silvia; Capobianco, Andrea; Bergui, Luciana; Dürig, Jan; Morabito, Fortunato; Dührsen, Ulrich; Malavasi, Fabio

2003-09-15

The prognosis for patients with B-cell chronic lymphocytic leukemia (B-CLL) is generally less favorable for those expressing CD38. Our working hypothesis is that CD38 is not merely a marker in B-CLL, but that it plays a receptor role with pathogenetic potential ruling the proliferation of the malignant clone. CD38 levels were generally low in the patients examined and monoclonal antibody (mAb) ligation was inefficient in signaling. Other cellular models indicated that molecular density and surface organization are critical for CD38 functionality. Interleukin 2 (IL-2) induced a marked up-modulation and surface rearrangement of CD38 in all the patients studied. On reaching a specific expression threshold, CD38 becomes an efficient receptor in purified B-CLL cells. Indeed, mAb ligation is followed by Ca2+ fluxes and by a markedly increased proliferation. The unsuitability of CD38 to perform as a receptor is obviated through close interaction with the B-cell-receptor (BCR) complex and CD19. On mAb binding, CD38 translocates to the membrane lipid microdomains, as shown by a colocalization with the GM1 ganglioside and with CD81, a raft-resident protein. Finally, CD38 signaling in IL-2-treated B-CLL cells prolonged survival and induced the appearance of plasmablasts, providing a pathogenetic hypothesis for the occurrence of Richter syndrome.

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia.

PubMed

Hing, Zachary A; Mantel, Rose; Beckwith, Kyle A; Guinn, Daphne; Williams, Erich; Smith, Lisa L; Williams, Katie; Johnson, Amy J; Lehman, Amy M; Byrd, John C; Woyach, Jennifer A; Lapalombella, Rosa

2015-05-14

Despite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance. © 2015 by The American Society of Hematology.

Interphase cytogenetics of B-cell chronic lymphocytic leukemia by FISH-technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peddanna, N.; Gogineni, S.K.; Rosenthal, C.J.

Chronic lymphocytic leukemia [CLL] accounts for about 30% of all lymphoproliferative disorders. In over 95% of these cases, the leukemia is caused by B-cells, rarely T-cells. Fifty percent of B-CLL have chromosomal aberrations and of such cases, one-third have trisomy 12. Malignant B-cells have a very low mitotic index and those metaphases that can be analyzed usually represent the normal T-cell population. Retrospectively, we decided to identify the additional chromosome 12 (trisomy 12) directly at interphase by the FISH-technique using centrometric 12 specific alphoid probe (Oncor, Gaithersburg, MD). Preparations were made from 9 patients with B-CLL. All cultures except onemore » failed to produce metaphases for conventional karyotyping. Eighty percent of the cells have two dots (normal cells) over the interphase nuclei while the remaining 20% have three dots (trisomy 12). The clinical implication of trisomy 12 in the pathogenesis of CLL including age, staging and duration of disease, differentials and immunological markers are correlated with interphase cytogenetic data. The loss and/or gain of specific chromosomes in human neoplasia is common and rapid evaluation of such cases should be considered as a routine approach.« less

Identification of three subgroups of B cell chronic lymphocytic leukemia based upon mutations of BCL-6 and IgV genes.

PubMed

Capello, D; Fais, F; Vivenza, D; Migliaretti, G; Chiorazzi, N; Gaidano, G; Ferrarini, M

2000-05-01

Although B cell chronic lymphocytic leukemia (B-CLL) has been traditionally viewed as a tumor of virgin B cells, this notion has been recently questioned by data suggesting that a fraction of B-CLL derives from antigen experienced B cells. In order to further clarify the histogenetic derivation of this lymphoproliferation, we have analyzed the DNA sequences of the 5' non-coding region of BCL-6 proto-oncogene in 28 cases of B-CLL. Mutations of BCL-6 proto-oncogene, a zinc finger transcription factor implicated in lymphoma development, represent a histogenetic marker of B cell transit through the germinal center (GC) and occur frequently in B cell malignancies derived from GC or post-GC B cells. For comparison, the same tumor panel was analyzed for somatic mutations of the rearranged immunoglobulin variable (IgV) genes, which are known to be acquired at the time of B cell transit through the GC. Sequence analyses of BCL-6 and IgV genes allowed the definition of three groups of B-CLL. Group I B-CLL displayed mutations of both BCL-6 and IgV genes (10/28; 36%). Group II B-CLL displayed mutated IgV genes, but a germline BCL-6 gene (5/28; 18%). Finally, group III B-CLL included the remaining cases (13/28; 46%) that were characterized by the absence of somatic mutations of both BCL-6 and IgV genes. Overall, the distribution of BCL-6 and IgV mutations in B-CLL reinforce the notion that this leukemia is histogenetically heterogeneous and that a substantial subgroup of these lymphoproliferations derives from post-germinal center B cells.

CLL Exosomes Modulate the Transcriptome and Behaviour of Recipient Stromal Cells and Are Selectively Enriched in miR-202-3p

PubMed Central

Farahani, Mosavar; Rubbi, Carlos; Liu, Luning; Slupsky, Joseph R.; Kalakonda, Nagesh

2015-01-01

Bi-directional communication with the microenvironment is essential for homing and survival of cancer cells with implications for disease biology and behaviour. In chronic lymphocytic leukemia (CLL), the role of the microenvironment on malignant cell behaviour is well described. However, how CLL cells engage and recruit nurturing cells is poorly characterised. Here we demonstrate that CLL cells secrete exosomes that are nanovesicles originating from the fusion of multivesicular bodies with the plasma membrane, to shuttle proteins, lipids, microRNAs (miR) and mRNAs to recipient cells. We characterise and confirm the size (50–100 nm) and identity of the CLL-derived exosomes by Electron microscopy (EM), Atomic force microscopy (AFM), flow cytometry and western blotting using both exosome- and CLL-specific markers. Incubation of CLL-exosomes, derived either from cell culture supernatants or from patient plasma, with human stromal cells shows that they are readily taken up into endosomes, and induce expression of genes such as c-fos and ATM as well as enhance proliferation of recipient HS-5 cells. Furthermore, we show that CLL exosomes encapsulate abundant small RNAs and are enriched in certain miRs and specifically hsa-miR-202-3p. We suggest that such specific packaging of miR-202-3p into exosomes results in enhanced expression of ‘suppressor of fused’ (Sufu), a Hedgehog (Hh) signalling intermediate, in the parental CLL cells. Thus, our data show that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology. PMID:26509439

Multidimensional Single-Cell Analysis of BCR Signaling Reveals Proximal Activation Defect As a Hallmark of Chronic Lymphocytic Leukemia B Cells

PubMed Central

Palomba, M. Lia; Piersanti, Kelly; Ziegler, Carly G. K.; Decker, Hugo; Cotari, Jesse W.; Bantilan, Kurt; Rijo, Ivelise; Gardner, Jeff R.; Heaney, Mark; Bemis, Debra; Balderas, Robert; Malek, Sami N.; Seymour, Erlene; Zelenetz, Andrew D.

2014-01-01

Purpose Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation. Experimental Design We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled BCR signaling in 67 CLL patients using Partial Least Squares Regression (PLSR). Results from multidimensional modeling were validated using an independent test cohort of 38 patients. Results We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as a hallmark of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness. Conclusion Single-cell multidimensional analysis of BCR signaling permitted focused analysis of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Disruption of the pSYK/pPLCγ2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior. Together, these observations implicate novel elements of the BCR signal transduction as potential therapeutic targets. PMID:24489640

Promoter methylation patterns in Richter syndrome affect stem-cell maintenance and cell cycle regulation and differ from de novo diffuse large B-cell lymphoma.

PubMed

Rinaldi, Andrea; Mensah, Afua Adjeiwaa; Kwee, Ivo; Forconi, Francesco; Orlandi, Ester M; Lucioni, Marco; Gattei, Valter; Marasca, Roberto; Berger, Françoise; Cogliatti, Sergio; Cavalli, Franco; Zucca, Emanuele; Gaidano, Gianluca; Rossi, Davide; Bertoni, Francesco

2013-10-01

In a fraction of patients, chronic lymphocytic leukaemia (CLL) can transform to Richter syndrome (RS), usually a diffuse large B-cell lymphoma (DLBCL). We studied genome-wide promoter DNA methylation in RS and clonally related CLL-phases of transformed patients, alongside de novo DLBCL (of non-germinal centre B type), untransformed-CLL and normal B-cells. The greatest differences in global DNA methylation levels were observed between RS and DLBCL, indicating that these two diseases, although histologically similar, are epigenetically distinct. RS was more highly methylated for genes involved in cell cycle regulation. When RS was compared to the preceding CLL-phase and with untransformed-CLL, RS presented a higher degree of methylation for genes possessing the H3K27me3 mark and PRC2 targets, as well as for gene targets of TP53 and RB1. Comparison of the methylation levels of individual genes revealed that OSM, a stem cell regulatory gene, exhibited significantly higher methylation levels in RS compared to CLL-phases. Its transcriptional repression by DNA methylation was confirmed by 5-aza-2'deoxycytidine treatment of DLBCL cells, determining an increased OSM expression. Our results showed that methylation patterns in RS are largely different from de novo DLBCL. Stem cell-related genes and cell cycle regulation genes are targets of DNA methylation in RS. © 2013 John Wiley & Sons Ltd.

Managing high-risk CLL during transition to a new treatment era: stem cell transplantation or novel agents?

PubMed

Dreger, Peter; Schetelig, Johannes; Andersen, Niels; Corradini, Paolo; van Gelder, Michel; Gribben, John; Kimby, Eva; Michallet, Mauricette; Moreno, Carol; Stilgenbauer, Stephan; Montserrat, Emili

2014-12-18

Allogeneic hematopoietic stem cell transplantation (HSCT) has been considered as the treatment of choice for patients with high-risk chronic lymphocytic leukemia (HR-CLL; ie, refractory to purine analogs, short response [<24 months] to chemoimmunotherapy, and/or presence of del[17p]/TP53 mutations). Currently, treatment algorithms for HR-CLL are being challenged by the introduction of novel classes of drugs. Among them, BCR signal inhibitors (BCRi) and B-cell lymphoma 2 antagonists (BCL2a) appear particularly promising. As a result of the growing body of favorable outcome data reported for BCRi/BCL2a, uncertainty is emerging on how to advise patients with HR-CLL about indication for and timing of HSCT. This article provides an overview of currently available evidence and theoretical considerations to guide this difficult decision process. Until the risks and benefits of different treatment strategies are settled, all patients with HR-CLL should be considered for treatment with BCRi/BCL2a. For patients who respond to these agents, there are 2 treatment possibilities: (1) performing an HSCT or (2) continuing treatment with the novel drug. Individual disease-specific and transplant-related risk factors, along with patient's preferences, should be taken into account when recommending one of these treatments over the other. © 2014 by The American Society of Hematology.

[Ibrutinib: A new drug of B-cell malignancies].

PubMed

Thieblemont, Catherine

2015-06-01

Ibrutinib (Imbruvica®) is a first-in-class, orally administered once-daily, that inhibits B-cell antigen receptor signaling downstream of Bruton's tyrosine kinase (BTK). Ibrutinib has been approved in USA in February 2014 and in France in October 2014 for the treatment of patients with relapsed/refractory mantle cell lymphoma (MCL) or chronic lymphocytic leukaemia (CLL) and for the treatment of patients with CLL and a chromosome 17 deletion (del 17p) or TP53 mutation. In clinical studies, ibrutinib induced an impressive overall response rate (68%) in patients with relapsed/refractory MCL (phase II study). In CLL, ibrutinib has shown to significantly improve progression-free survival, response rate and overall survival in patients with relapsed/refractory CLL, including in those with del 17p. Ibrutinib had an acceptable tolerability profile. Less than 10% of patients discontinued their treatment because of adverse events. Results are pending in other B-cell lymphomas subtypes such as in diffuse large B-cell lymphoma and in follicular lymphoma. An approval extension has already been enregistered for Waldenström disease in USA in January 2015. Given its efficacy and tolerability, ibrutinib is an emerging treatment option for patients with B-cell malignancies. Copyright © 2015 Société Françise du Cancer. Publié par Elsevier Masson SAS. Tous droits réservés. Published by Elsevier Masson SAS. All rights reserved.

Combined BTK and PI3Kδ Inhibition with Acalabrutinib and ACP-319 Improves Survival and Tumor Control in CLL Mouse Model.

PubMed

Niemann, Carsten U; Mora-Jensen, Helena I; Dadashian, Eman L; Krantz, Fanny; Covey, Todd; Chen, Shih-Shih; Chiorazzi, Nicholas; Izumi, Raquel; Ulrich, Roger; Lannutti, Brian J; Wiestner, Adrian; Herman, Sarah E M

2017-10-01

Purpose: Targeting the B-cell receptor (BCR) pathway with inhibitors of Bruton tyrosine kinase (BTK) and PI3Kδ is highly effective for the treatment of chronic lymphocytic leukemia (CLL). However, deep remissions are uncommon, and drug resistance with single-agent therapy can occur. In vitro studies support the effectiveness of combing PI3Kδ and BTK inhibitors. Experimental Design: As CLL proliferation and survival depends on the microenvironment, we used murine models to assess the efficacy of the BTK inhibitor acalabrutinib combined with the PI3Kδ inhibitor ACP-319 in vivo We compared single-agent with combination therapy in TCL1-192 cell-injected mice, a model of aggressive CLL. Results: We found significantly larger reductions in tumor burden in the peripheral blood and spleen of combination-treated mice. Although single-agent therapy improved survival compared with control mice by a few days, combination therapy extended survival by over 2 weeks compared with either single agent. The combination reduced tumor proliferation, NF-κB signaling, and expression of BCL-xL and MCL-1 more potently than single-agent therapy. Conclusions: The combination of acalabrutinib and ACP-319 was superior to single-agent treatment in a murine CLL model, warranting further investigation of this combination in clinical studies. Clin Cancer Res; 23(19); 5814-23. ©2017 AACR . ©2017 American Association for Cancer Research.

Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL).

PubMed

Woyach, Jennifer A; Bojnik, Engin; Ruppert, Amy S; Stefanovski, Matthew R; Goettl, Virginia M; Smucker, Kelly A; Smith, Lisa L; Dubovsky, Jason A; Towns, William H; MacMurray, Jessica; Harrington, Bonnie K; Davis, Melanie E; Gobessi, Stefania; Laurenti, Luca; Chang, Betty Y; Buggy, Joseph J; Efremov, Dimitar G; Byrd, John C; Johnson, Amy J

2014-02-20

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

Genetic and cytokine changes associated with symptomatic stages of CLL.

PubMed

Agarwal, Amit; Cooke, Lawrence; Riley, Christopher; Qi, Wenqing; Mount, David; Mahadevan, Daruka

2014-09-01

The pathogenesis and drug resistance of symptomatic CLL patients involves genetic changes associated with the CLL clone as well as changes within the microenvironment. To further understand these processes, we compared early stage CLL to symptomatic late stage using gene expression and serum cytokine profiling to gain insight of the genetic and microenvironment changes associated with the most severe form of the disease. Patients were classified into low stage (Rai stage 0/I/II) and high stage (Rai stage III/IV). Gene expression profiles were obtained on pretreatment samples using the HG-U133A 2.0 Affymetrix platform. A comparison of low versus high stage CLL revealed a set of 21 genes differentially expressed genes. 15 genes were up regulated in the high stage compared to low stage while 6 genes were down regulated. Analysis of GO molecular function revealed 9 of 21 genes were involved in transcription factor activity. Serum cytokine profiles showed six cytokines to be significantly different in high stage patients. Two chemokines, SDF-1/CXCL12 and uPAR known to be involved in stem cell mobilization and homing were increased in serum of high stage patients. This study has identified therapeutic targets for symptomatic CLL patients. Copyright © 2014 Elsevier Ltd. All rights reserved.

Modulation of the human equilibrative nucleoside transporter1 (hENT1) activity by IL-4 and PMA in B cells from chronic lymphocytic leukemia.

PubMed

Fernández Calotti, Paula; Galmarini, Carlos María; Cañones, Cristian; Gamberale, Romina; Saénz, Daniel; Avalos, Julio Sánchez; Chianelli, Mónica; Rosenstein, Ruth; Giordano, Mirta

2008-02-15

Nucleoside transporters (NTs) are essential for the uptake of therapeutic nucleoside analogs, broadly used in cancer treatment. The mechanisms responsible for NT regulation are largely unknown. IL-4 is a pro-survival signal for chronic lymphocytic leukemia (CLL) cells and has been shown to confer resistance to nucleoside analogs. The aim of this study was to investigate whether IL-4 is able to modulate the expression and function of the human equilibrative NT1 (hENT1) in primary cultures of CLL cells and, consequently, to affect cytotoxicity induced by therapeutic nucleosides analogs. We found that treatment with IL-4 (20 ng/ml for 24 h) increased mRNA hENT1 expression in CLL cells without affecting that of normal B cells. Given that the enhanced mRNA levels of hENT1 in CLL cells did not result in increased transport activity, we examined the possibility that hENT1 induced by IL-4 may require post-translational modifications to become active. We found that the acute stimulation of PKC in IL-4-treated CLL cells by short-term incubation with PMA significantly increased hENT1 transport activity and favoured fludarabine-induced apoptosis. By contrast, and in line with previous reports, IL-4 plus PMA protected CLL cells from a variety of cytotoxic agents. Our findings indicate that the combined treatment with IL-4 and PMA enhances hENT1 activity and specifically sensitizes CLL cells to undergo apoptosis induced by fludarabine.

Study of the quantitative, functional, cytogenetic, and immunoregulatory properties of bone marrow mesenchymal stem cells in patients with B-cell chronic lymphocytic leukemia.

PubMed

Pontikoglou, Charalampos; Kastrinaki, Maria-Christina; Klaus, Mirjam; Kalpadakis, Christina; Katonis, Pavlos; Alpantaki, Kalliopi; Pangalis, Gerassimos A; Papadaki, Helen A

2013-05-01

The bone marrow (BM) microenvironment has clearly been implicated in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL). However, the potential involvement of BM stromal progenitors, the mesenchymal stem cells (MSCs), in the pathophysiology of the disease has not been extensively investigated. We expanded in vitro BM-MSCs from B-CLL patients (n=11) and healthy individuals (n=16) and comparatively assessed their reserves, proliferative potential, differentiation capacity, and immunoregulatory effects on T- and B-cells. We also evaluated the anti-apoptotic effect of patient-derived MSCs on leukemic cells and studied their cytogenetic characteristics in comparison to BM hematopoietic cells. B-CLL-derived BM MSCs exhibit a similar phenotype, differentiation potential, and ability to suppress T-cell proliferative responses as compared with MSCs from normal controls. Furthermore, they do not carry the cytogenetic abnormalities of the leukemic clone, and they exert a similar anti-apoptotic effect on leukemic cells and healthy donor-derived B-cells, as their normal counterparts. On the other hand, MSCs from B-CLL patients significantly promote normal B-cell proliferation and IgG production, in contrast to healthy-donor-derived MSCs. Furthermore, they have impaired reserves, defective cellular growth due to increased apoptotic cell death and exhibit aberrant production of stromal cell-derived factor 1, B-cell activating factor, a proliferation inducing ligand, and transforming growth factor β1, cytokines that are crucial for the survival/nourishing of the leukemic cells. We conclude that ex vivo expanded B-CLL-derived MSCs harbor intrinsic qualitative and quantitative abnormalities that may be implicated in disease development and/or progression.

Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

PubMed

Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

2015-07-09

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents

PubMed Central

Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

2015-01-01

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

Salvage therapy for CLL and the role of stem cell transplantation.

PubMed

Gribben, John G

2005-01-01

Chronic lymphocytic leukaemia (CLL) remains an incurable disease and, notwithstanding the excellent remission rates now achieved with purine analogs and monoclonal antibodies, the vast majority of patients with CLL are destined to relapse after primary treatment. The management of relapsed CLL patients is then dependent upon a number of factors, most importantly age, performance status, previous therapy administered, the response and duration of response to such therapy, and time from last therapy. Although prior therapy and response to such therapy are important factors in determining next therapy, it is often difficult to determine their importance from published studies. Furthermore, the goal of therapy, whether palliative or aggressive, must also be weighed into the decision when deciding on the next line of treatment. With many potential treatments available, the sequence of treatments and the timing of procedures such as stem cell transplantation remain controversial and are the focus of ongoing clinical trials.

Novel role of prostate apoptosis response-4 tumor suppressor in B-cell chronic lymphocytic leukemia.

PubMed

McKenna, Mary K; Noothi, Sunil K; Alhakeem, Sara S; Oben, Karine Z; Greene, Joseph T; Mani, Rajeswaran; Perry, Kathryn L; Collard, James P; Rivas, Jacqueline R; Hildebrandt, Gerhard; Fleischman, Roger; Durbin, Eric B; Byrd, John C; Wang, Chi; Muthusamy, Natarajan; Rangnekar, Vivek M; Bondada, Subbarao

2018-04-25

Prostate apoptosis response-4 (Par-4), a pro-apoptotic tumor suppressor protein, is down regulated in many cancers including renal cell carcinoma, glioblastoma, endometrial and breast cancer. Par-4 induces apoptosis selectively in various types of cancer cells but not normal cells. We found that chronic lymphocytic leukemia (CLL) cells from human patients and from the Eµ-Tcl1 mice constitutively express Par-4 in greater amounts than normal B-1 or B-2 cells. Interestingly, knockdown of Par-4 in human CLL derived Mec-1 cells results in a robust increase in p21/WAF1 expression and decreased growth due to delayed G1 to S cell cycle transition. Lack of Par-4 also increased the expression of p21 and delayed CLL growth in Eμ-Tcl1 mice. Par-4 expression in CLL cells required constitutively active B-cell receptor (BCR) signaling, as inhibition of BCR signaling with FDA approved drugs caused a decrease in Par-4 mRNA and protein, and an increase in apoptosis. In particular, activities of Lyn, a Src family kinase, spleen tyrosine kinase and Bruton's tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling. Together, these results suggest that Par-4 may play a novel pro-growth rather than pro-apoptotic role in CLL and could be targeted to enhance the therapeutic effects of BCR signaling inhibitors. Copyright © 2018 American Society of Hematology.

CMC-544 (inotuzumab ozogamicin) shows less effect on multidrug resistant cells: analyses in cell lines and cells from patients with B-cell chronic lymphocytic leukaemia and lymphoma.

PubMed

Takeshita, Akihiro; Shinjo, Kaori; Yamakage, Nozomi; Ono, Takaaki; Hirano, Isao; Matsui, Hirotaka; Shigeno, Kazuyuki; Nakamura, Satoki; Tobita, Tadasu; Maekawa, Masato; Ohnishi, Kazunori; Sugimoto, Yoshikazu; Kiyoi, Hitoshi; Naoe, Tomoki; Ohno, Ryuzo

2009-06-01

The effect of CMC-544, a calicheamicin-conjugated anti-CD22 monoclonal antibody, was analysed in relation to CD22 and P-glycoprotein (P-gp) in B-cell chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) in vitro. The cell lines used were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR. Cells obtained from 19 patients with B-cell CLL or NHL were also used. The effect of CMC-544 was analysed by viable cell count, morphology, annexin-V staining, and cell cycle distribution. A dose-dependent, selective cytotoxic effect of CMC-544 was observed in cell lines that expressed CD22. CMC-544 was not effective on Daudi/MDR and Raji/MDR cells compared with their parental cells. The MDR modifiers, PSC833 and MS209, restored the cytotoxic effect of CMC-544 in P-gp-expressing sublines. In clinical samples, the cytotoxic effect of CMC-544 was inversely related to the amount of P-gp (P = 0.003), and to intracellular rhodamine-123 accumulation (P < 0.001). On the other hand, the effect positively correlated with the amount of CD22 (P = 0.010). The effect of CMC-544 depends on the levels of CD22 and P-gp. Our findings will help to predict the clinical effectiveness of this drug on these B-cell malignancies, suggesting a beneficial effect with combined use of CMC-544 and MDR modifiers.

Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia

PubMed Central

Minici, Claudia; Gounari, Maria; Übelhart, Rudolf; Scarfò, Lydia; Dühren-von Minden, Marcus; Schneider, Dunja; Tasdogan, Alpaslan; Alkhatib, Alabbas; Agathangelidis, Andreas; Ntoufa, Stavroula; Chiorazzi, Nicholas; Jumaa, Hassan; Stamatopoulos, Kostas; Ghia, Paolo; Degano, Massimo

2017-01-01

Cell-autonomous B-cell receptor (BcR)-mediated signalling is a hallmark feature of the neoplastic B lymphocytes in chronic lymphocytic leukaemia (CLL). Here we elucidate the structural basis of autonomous activation of CLL B cells, showing that BcR immunoglobulins initiate intracellular signalling through homotypic interactions between epitopes that are specific for each subgroup of patients with homogeneous clinicobiological profiles. The molecular details of the BcR–BcR interactions apparently dictate the clinical course of disease, with stronger affinities and longer half-lives in indolent cases, and weaker, short-lived contacts mediating the aggressive ones. The diversity of homotypic BcR contacts leading to cell-autonomous signalling reconciles the existence of a shared pathogenic mechanism with the biological and clinical heterogeneity of CLL and offers opportunities for innovative treatment strategies. PMID:28598442

Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling.

PubMed

Yeh, Yuh-Ying; Ozer, Hatice Gulcin; Lehman, Amy M; Maddocks, Kami; Yu, Lianbo; Johnson, Amy J; Byrd, John C

2015-05-21

Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile. © 2015 by The American Society of Hematology.

Durable responses to ibrutinib in patients with relapsed CLL after allogeneic stem cell transplantation.

PubMed

Link, C S; Teipel, R; Heidenreich, F; Rücker-Braun, E; Schmiedgen, M; Reinhardt, J; Oelschlägel, U; von Bonin, M; Middeke, J M; Muetherig, A; Trautmann-Grill, K; Platzbecker, U; Bornhäuser, M; Schetelig, J

2016-06-01

Ibrutinib, a recently approved inhibitor of Bruton's tyrosine kinase (BTK), has shown great efficacy in patients with high-risk CLL. Nevertheless, there are few data regarding its use in patients who relapsed after allogeneic stem cell transplantation (alloSCT). We report clinical data from five CLL patients treated with ibrutinib for relapse after first or even second allogeneic transplantation. Additionally, we performed analyses on cytokine levels and direct measuring of CD4 Th1 and CD4 Th2 cells to evaluate possible clinically relevant immunomodulatory effects of ibrutinib. All patients achieved partial responses including one minimal residual disease (MRD)-negative remission. Within 1 year of follow-up, no relapse was observed. One patient died of severe pneumonia while on ibrutinib treatment. Beside this, no unexpected adverse events were observed. Flow cytometry and analyses of T cell-mediated cytokine levels (IL10 and TNFα) did not reveal substantial changes in T-cell distribution in favor of a CD4 Th1 T-cell shift in our patients. No acute exacerbation of GvHD was reported. In conclusion, these results support further evaluation of ibrutinib in CLL patients relapsing after alloSCT.

Different sensitivity of germinal center B cell-like diffuse large B cell lymphoma cells towards ibrutinib treatment

PubMed Central

2014-01-01

Background Although rituximab in the combination of CHOP chemotherapy has been widely used as the standard treatment for several kinds of B-cell non-Hodgkin lymphoma (B-NHL), a great number of B-NHL patients treated with this immunotherapy still develop primary and secondary resistance. Recently Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib showed promising therapeutic effect in relapsed/refractory CLL and B-cell NHL, which provided essential alternatives for these patients. Methods The proliferation and apoptosis induction of tumor cells were measured by cell viability assay and Annexin-V staining. Western Blotting analysis and real-time PCR were used to detect the expression level of target proteins and chemokines production. Results We demonstrated that ibrutinib inhibited the proliferation and induced apoptosis of GCB-DLBCL cell lines through suppression of BCR signaling pathway and activation of caspase-3. Furthermore, the chemokines CCL3 and CCL4 production from tumor cells were also found to be attenuated by ibrutinib treatment. But different cell lines exhibited distinct sensitivity after ibrutinib treatment. Interestingly, the decreasing level of p-ERK after ibrutinib treatment, but not the basal expression level of Btk, correlated with different drug sensitivity. Conclusions Ibrutinib could be a potentially useful therapy for GCB-DLBCL and the decreasing level of p-ERK could become a useful biomarker to predict related therapeutic response. PMID:24693884

Ongoing In Vivo Immunoglobulin Class Switch DNA Recombination in Chronic Lymphocytic Leukemia B Cells1

PubMed Central

Cerutti, Andrea; Zan, Hong; Kim, Edmund C.; Shah, Shefali; Schattner, Elaine J.; Schaffer, András; Casali, Paolo

2015-01-01

Chronic lymphocytic leukemia (CLL) results from the expansion of malignant CD5+ B cells that usually express IgD and IgM. These leukemic cells can give rise in vivo to clonally related IgG+ or IgA+ elements. The requirements and modalities of this process remain elusive. Here we show that leukemic B cells from 14 of 20 CLLs contain the hallmarks of ongoing Ig class switch DNA recombination (CSR), including extrachromosomal switch circular DNAs and circle transcripts generated by direct Sμ→Sγ, Sμ→Sα, and Sμ→Sε as well as sequential Sγ→Sα and Sγ→Sε CSR. Similar CLL B cells express transcripts for activation-induced cytidine deaminase, a critical component of the CSR machinery, and contain germline IH-CH and mature VHDJH-CH transcripts encoded by multiple Cγ, Cα, and Cε genes. Ongoing CSR occurs in only a fraction of the CLL clone, as only small proportions of CD5+CD19+ cells express surface IgG or IgA and lack IgM and IgD. In vivo class-switching CLL B cells down-regulate switch circles and circle transcripts in vitro unless exposed to exogenous CD40 ligand and IL-4. In addition, CLL B cells that do not class switch in vivo activate the CSR machinery and secrete IgG, IgA, or IgE upon in vitro exposure to CD40 ligand and IL-4. These findings indicate that in CLL at least some members of the malignant clone actively differentiate in vivo along a pathway that induces CSR. They also suggest that this process is elicited by external stimuli, including CD40 ligand and IL-4, provided by bystander immune cells. PMID:12444172

Automated quantification of apoptosis in B-cell chronic lymphoproliferative disorders: a prognostic variable obtained with the Cell-Dyn Sapphire (Abbott) automated hematology analyzer.

PubMed

Fumi, M; Martins, D; Pancione, Y; Sale, S; Rocco, V

2014-12-01

B-chronic lymphocytic leukemia CLL, a neoplastic clonal disorder with monomorphous small B lymphocytes with scanty cytoplasm and clumped chromatin, can be morphologically differentiated in typical and atypical forms with different prognosis: Smudge cells (Gumprecht's shadows) are one of the well-known features of the typical CLL and are much less inconsistent in other different types CLPD. Abbott Cell-Dyn Sapphire uses the fluorescence after staining with the DNA fluorochrome propidium iodide for the measurement of nucleated red blood cells (NRBCs) and nonviable cells (FL3+ cell fraction): We have studied the possible correlation between presence and number of morphologically identifiable smudge cells on smears and the percentage of nonviable cells produced by Cell-Dyn Sapphire. 305 blood samples from 224 patients with B-cell lymphoproliferative disorders and 40 healthy blood donors were analyzed by CBC performed by Cell-Dyn Sapphire, peripheral blood smear, and immunophenotype characterization. FL3+ fraction in CLPD directly correlated with the percentage of smudge cells and is significantly increased in patients with typical B-CLL. This phenomenon is much less evident in patients with atypical/mixed B-CLL and B-NHL. In small laboratories without FCM and cytogenetic, smudge cells%, can be utilized as a preliminary diagnostic and prognostic tool in differential diagnosis of CLPD. © 2014 John Wiley & Sons Ltd.

The dual Syk/JAK inhibitor cerdulatinib antagonises B-cell receptor and microenvironmental signaling in chronic lymphocytic leukemia

PubMed Central

Blunt, Matthew D; Koehrer, Stefan; Dobson, Rachel; Larrayoz, Marta; Wilmore, Sarah; Hayman, Alice; Parnell, Jack; Smith, Lindsay; Davies, Andrew; Johnson, Peter W; Conley, Pamela B; Pandey, Anjali; Strefford, Jon C; Stevenson, Freda K; Packham, Graham; Forconi, Francesco; Coffey, Greg; Burger, Jan A; Steele, Andrew J

2017-01-01

Purpose B-cell receptor (BCR)-associated kinase inhibitors such as ibrutinib have revolutionised the treatment of chronic lymphocytic leukemia (CLL). However, these agents are not curative and resistance is already emerging in a proportion of patients. Interleukin-4 (IL-4), expressed in CLL lymph nodes, can augment BCR-signalling and reduce the effectiveness of BCR-kinase inhibitors. Therefore simultaneous targeting of the IL-4- and BCR-signalling pathways by cerdulatinib, a novel dual Syk/JAK inhibitor currently in clinical trials (NCT01994382), may improve treatment responses in patients. Experimental Design PBMCs from CLL patients were treated with cerdulatinib alone or in combination with venetoclax. Cell death, chemokine and cell signalling assay were performed and analysed by flow cytometry, immunoblotting, Q-PCR and ELISA as indicated. Results At concentrations achievable in patients, cerdulatinib inhibited BCR- and IL-4-induced downstream signalling in CLL cells using multiple read-outs and prevented anti-IgM- and nurse-like cell (NLC)-mediated CCL3/CCL4 production. Cerdulatinib induced apoptosis of CLL cells, in a time- and concentration-dependent manner, and particularly in IGHV unmutated samples with greater BCR-signalling capacity and response to IL-4, or samples expressing higher levels of sIgM, CD49d+ or ZAP70+. Cerdulatinib overcame anti-IgM, IL-4/CD40L or NLC-mediated protection by preventing upregulation of MCL-1- and BCL-XL, however BCL-2 expression was unaffected. Furthermore in samples treated with IL-4/CD40L, cerdulatinib synergised with venetoclax in vitro to induce greater apoptosis than either drug alone. Conclusion Cerdulatinib is a promising therapeutic for the treatment of CLL either alone or in combination with venetoclax, with the potential to target critical survival pathways in this currently incurable disease. PMID:27697994

Therapeutic Implications of Activation of the Host Gene (Dleu2) Promoter for miR-15a/16-1 in Chronic Lymphocytic Leukemia (CLL)

PubMed Central

Kasar, S; Underbayev, C; Yuan, Y; Hanlon, M; Aly, S; Chang, V; Batish, M; Gavrilova, T; Badiane, F; Degheidy, H; Marti, G; Raveche, E

2014-01-01

Genetic lesions and other regulatory events lead to silencing of the 13q14 locus in a majority of chronic lymphocytic leukemia (CLL) patients. This locus encodes a pair of critical pro-apoptotic microRNAs, miR-15a/16-1. Decreased levels of miR-15a/16-1 are critical for the increased survival exhibited by CLL cells. Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1. In this paper we have identified additional molecular mechanisms regulating miR-15a/16-1 levels and shown that the transcription factor BSAP (B cell Specific Activator Protein) directly interacts with Dleu2, the host gene containing the mir-15a/16-1 loci and via negative regulation of the Dleu2 promoter results in repression of mir-15a/16 expression. CLL patient B cell expression levels of BSAP were increased compared to control sources of B cells. With the use of siRNA mediated repression, the levels of BSAP were decreased in vitro in the NZB derived malignant B1 cell line, LNC, and in ex vivo CLL patient PBMC. BSAP knockdown led to an increase in the expression of miR-15a/16-1 and an increase in apoptosis and a cell cycle arrest in both the cell line and patient PBMC. Moreover, using Dleu2 promoter analysis by chromatin immunoprecipitation (ChIP) assay we have shown that BSAP directly interacts with the Dleu2 promoter. Derepression of the Dleu2 promoter via inhibition of histone deacetylation combined with BSAP knockdown increased miR-15a/16 expression and increased malignant B cell death. In summary, therapy targeting enhanced host gene Dleu2 transcription may augment CLL therapy. PMID:23995789

Kolmogorov-Smirnov statistical test for analysis of ZAP-70 expression in B-CLL, compared with quantitative PCR and IgV(H) mutation status.

PubMed

Van Bockstaele, Femke; Janssens, Ann; Piette, Anne; Callewaert, Filip; Pede, Valerie; Offner, Fritz; Verhasselt, Bruno; Philippé, Jan

2006-07-15

ZAP-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgV(H)) mutation status, which is known as a prognostic marker in B-cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP-70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov-Smirnov (KS) statistical test to make analysis more straightforward. We examined ZAP-70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764-1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver-operating-characteristics (ROC)-curve analyses were performed to determine the optimal cut-off values for ZAP-70 measured by the two approaches. ZAP-70 protein expression was compared with ZAP-70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgV(H) mutation status. Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgV(H) mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo-method. The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP-70 expression in CLL. (c) 2006 International Society for Analytical Cytology.

The BCL2 selective inhibitor venetoclax induces rapid onset apoptosis of CLL cells in patients via a TP53-independent mechanism

PubMed Central

Anderson, Mary Ann; Deng, Jing; Seymour, John F.; Tam, Constantine; Kim, Su Young; Fein, Joshua; Yu, Lijian; Brown, Jennifer R.; Westerman, David; Si, Eric G.; Majewski, Ian J.; Segal, David; Heitner Enschede, Sari L.; Huang, David C. S.; Davids, Matthew S.; Letai, Anthony

2016-01-01

BCL2 blunts activation of the mitochondrial pathway to apoptosis, and high-level expression is required for chronic lymphocytic leukemia (CLL) survival. Venetoclax (ABT-199) is a small-molecule selective inhibitor of BCL2 currently in clinical trials for CLL and other malignancies. In conjunction with the phase 1 first-in-human clinical trial of venetoclax in patients with relapsed or refractory CLL (M12-175), we investigated the mechanism of action of venetoclax in vivo, explored whether in vitro sensitivity assays or BH3 profiling correlated with in vivo responses in patients, and determined whether loss of TP53 function affected responses in vitro and in vivo. In all samples tested, venetoclax induced death of CLL cells in vitro at concentrations achievable in vivo, with cell death evident within 4 hours. Apoptotic CLL cells were detected in vivo 6 or 24 hours after a single 20-mg or 50-mg dose in some patients. The extent of mitochondrial depolarization by a BIM BH3 peptide in vitro was correlated with percentage reduction of CLL in the blood and bone marrow in vivo, whereas the half lethal concentration derived from standard cytotoxicity assays was not. CLL cell death in vitro and the depth of clinical responses were independent of deletion of chromosome 17p, TP53 mutation, and TP53 function. These data provide direct evidence that venetoclax kills CLL cells in a TP53-independent fashion by inhibition of BCL2 in patients and support further assessment of BH3 profiling as a predictive biomarker for this drug. PMID:27069256

New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies.

PubMed

Gamonet, Clémentine; Bole-Richard, Elodie; Delherme, Aurélia; Aubin, François; Toussirot, Eric; Garnache-Ottou, Francine; Godet, Yann; Ysebaert, Loïc; Tournilhac, Olivier; Caroline, Dartigeas; Larosa, Fabrice; Deconinck, Eric; Saas, Philippe; Borg, Christophe; Deschamps, Marina; Ferrand, Christophe

2015-01-01

CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors. Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL. The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.

Metformin combined with sodium dichloroacetate promotes B leukemic cell death by suppressing anti-apoptotic protein Mcl-1

PubMed Central

Melloni, Elisabetta; Gilli, Paola; Bertolasi, Valerio; Casciano, Fabio; Rigolin, Gian Matteo; Zauli, Giorgio; Secchiero, Paola

2016-01-01

Metformin and the mitochondrial targeting dichloroacetate (DCA) have recently received attention due to their ability to inhibit anaerobic glycolysis, which renders most cancer cells resistant to apoptosis induction. We observed that Metformin alone exhibited a dose-dependent anti-leukemic activity in both B leukemic cell lines and primary B-chronic lymphocytic leukemia (B-CLL) patients' cells and its anti-leukemic activity was enhanced when used in combination with DCA. In order to overcome the problems of poor bioavailability and cellular uptake, which limit DCA efficacy, we have designed and synthetized cocrystals consisting of Metformin and DCA (Met-DCA) at different stoichiometric ratios. Of note, the MetH2++•2DCA− cocrystal exhibited enhanced in vitro anti-leukemic activity, with respect to the treatment with the mix consisting of Metformin plus DCA. In particular, the treatment with the cocrystal MetH2++•2DCA− induced a synergistic apoptotic cell death coupled to a marked down-modulation of the anti-apoptotic Mcl-1 protein. Taken together, our data emphasize that innovative compounds based on Metformin-DCA combination merit to be further evaluated as chemotherapeutic agents for the treatment of B-CLL. PMID:26959881

Intraclonal Cell Expansion and Selection Driven by B Cell Receptor in Chronic Lymphocytic Leukemia

PubMed Central

Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Fabris, Sonia; Neri, Antonino; Fabbi, Marina; Quintana, Giovanni; Quarta, Giovanni; Ghiotto, Fabio; Fais, Franco; Ferrarini, Manlio

2011-01-01

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These “stereotyped” BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone. PMID:21541442

How and when I do allogeneic transplant in CLL.

PubMed

Gribben, John G

2018-05-11

Allogenic stem cell transplantation (allo-SCT) has been considered the treatment of choice for high-risk patients with chronic lymphocytic leukemia (CLL) and the only approach offered with curative intent in this disease. The availability novel agents including the B cell receptor inhibitors (BCRi) ibrutinib, acalabrutinib and idelalisib, as well as venetoclax which targets the BCL2 pathway and the success of these agents in treating high-risk disease patients has made it more difficult to assess who and when in their treatment course allo-SCT should be considered. In this review, I will discuss the different treatment options available for the treatment of high-risk CLL and how allo-SCT fits into the treatment algorithm in the era of novel agents. Copyright © 2018 American Society of Hematology.

Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates.

PubMed

Sarkar, Mohosin; Liu, Yun; Qi, Junpeng; Peng, Haiyong; Morimoto, Jumpei; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

2016-04-01

Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

CpG Oligonucleotide and Interleukin 2 stimulation enables higher cytogenetic abnormality detection rates than 12-o-tetradecanolyphorbol-13-acetate in Asian patients with B-cell chronic lymphocytic leukemia (B-CLL).

PubMed

Liaw, Fiona Pui San; Lau, Lai Ching; Lim, Alvin Soon Tiong; Lim, Tse Hui; Lee, Geok Yee; Tien, Sim Leng

2014-12-01

The present study was designed to compare abnormality detection rates using DSP30 + IL2 and 12-O-Tetradecanoylphorbol-13-acetate (TPA) in Asian patients with B-CLL. Hematological specimens from 47 patients (29 newly diagnosed, 18 relapsed) were established as 72 h-DSP30 + IL2 and TPA cultures. Standard methods were employed to identify clonal aberrations by conventional cytogenetics (CC). The B-CLL fluorescence in situ hybridization (FISH) panel comprised ATM, CEP12, D13S25, and TP53 probes. DSP30 + IL2 cultures had a higher chromosomal abnormality detection rate (67 %) compared to TPA (44 %, p < 0.001). The mean number of analyzable metaphases and abnormal metaphases per slide was also higher (p < 0.005, p < 0.001, respectively). Culture success rate, percentage of complex karyotype, and percentage of non-clonal abnormal cell were not significantly different (p > 0.05). Thirteen cases with abnormalities were found exclusively in DSP30 + IL2 cultures compared to one found solely in TPA cultures. DSP30 + IL2 cultures were comparable to the FISH panel in detecting 11q-, +12 and 17p- but not 13q-. It also has a predilection for 11q- bearing leukemic cells compared to TPA. FISH had a higher abnormality detection rate (84.1 %) compared to CC (66.0 %) with borderline significance (p = 0.051), albeit limited by its coverage. In conclusion, DSP30 + IL2 showed a higher abnormality detection rate. However, FISH is indispensable to circumvent low mitotic indices and detect subtle abnormalities.

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells.

PubMed

Lilienthal, Nils; Lohmann, Gregor; Crispatzu, Giuliano; Vasyutina, Elena; Zittrich, Stefan; Mayer, Petra; Herling, Carmen Diana; Tur, Mehmet Kemal; Hallek, Michael; Pfitzer, Gabriele; Barth, Stefan; Herling, Marco

2016-05-01

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR. ©2016 American Association for Cancer Research.

High Mitochondrial DNA Stability in B-Cell Chronic Lymphocytic Leukemia

PubMed Central

Cerezo, María; Bandelt, Hans-Jürgen; Martín-Guerrero, Idoia; Ardanaz, Maite; Vega, Ana; Carracedo, Ángel; García-Orad, África; Salas, Antonio

2009-01-01

Background Chronic Lymphocytic Leukemia (CLL) leads to progressive accumulation of lymphocytes in the blood, bone marrow, and lymphatic tissues. Previous findings have suggested that the mtDNA could play an important role in CLL. Methodology/Principal Findings The mitochondrial DNA (mtDNA) control-region was analyzed in lymphocyte cell DNA extracts and compared with their granulocyte counterpart extract of 146 patients suffering from B-Cell CLL; B-CLL (all recruited from the Basque country). Major efforts were undertaken to rule out methodological artefacts that would render a high false positive rate for mtDNA instabilities and thus lead to erroneous interpretation of sequence instabilities. Only twenty instabilities were finally confirmed, most of them affecting the homopolymeric stretch located in the second hypervariable segment (HVS-II) around position 310, which is well known to constitute an extreme mutational hotspot of length polymorphism, as these mutations are frequently observed in the general human population. A critical revision of the findings in previous studies indicates a lack of proper methodological standards, which eventually led to an overinterpretation of the role of the mtDNA in CLL tumorigenesis. Conclusions/Significance Our results suggest that mtDNA instability is not the primary causal factor in B-CLL. A secondary role of mtDNA mutations cannot be fully ruled out under the hypothesis that the progressive accumulation of mtDNA instabilities could finally contribute to the tumoral process. Recommendations are given that would help to minimize erroneous interpretation of sequencing results in mtDNA studies in tumorigenesis. PMID:19924307

The BCL2 selective inhibitor venetoclax induces rapid onset apoptosis of CLL cells in patients via a TP53-independent mechanism.

PubMed

Anderson, Mary Ann; Deng, Jing; Seymour, John F; Tam, Constantine; Kim, Su Young; Fein, Joshua; Yu, Lijian; Brown, Jennifer R; Westerman, David; Si, Eric G; Majewski, Ian J; Segal, David; Heitner Enschede, Sari L; Huang, David C S; Davids, Matthew S; Letai, Anthony; Roberts, Andrew W

2016-06-23

BCL2 blunts activation of the mitochondrial pathway to apoptosis, and high-level expression is required for chronic lymphocytic leukemia (CLL) survival. Venetoclax (ABT-199) is a small-molecule selective inhibitor of BCL2 currently in clinical trials for CLL and other malignancies. In conjunction with the phase 1 first-in-human clinical trial of venetoclax in patients with relapsed or refractory CLL (M12-175), we investigated the mechanism of action of venetoclax in vivo, explored whether in vitro sensitivity assays or BH3 profiling correlated with in vivo responses in patients, and determined whether loss of TP53 function affected responses in vitro and in vivo. In all samples tested, venetoclax induced death of CLL cells in vitro at concentrations achievable in vivo, with cell death evident within 4 hours. Apoptotic CLL cells were detected in vivo 6 or 24 hours after a single 20-mg or 50-mg dose in some patients. The extent of mitochondrial depolarization by a BIM BH3 peptide in vitro was correlated with percentage reduction of CLL in the blood and bone marrow in vivo, whereas the half lethal concentration derived from standard cytotoxicity assays was not. CLL cell death in vitro and the depth of clinical responses were independent of deletion of chromosome 17p, TP53 mutation, and TP53 function. These data provide direct evidence that venetoclax kills CLL cells in a TP53-independent fashion by inhibition of BCL2 in patients and support further assessment of BH3 profiling as a predictive biomarker for this drug. © 2016 by The American Society of Hematology.

Targeting of colony-stimulating factor 1 receptor (CSF1R) in the CLL microenvironment yields antineoplastic activity in primary patient samples.

PubMed

Edwards V, David K; Sweeney, David Tyler; Ho, Hibery; Eide, Christopher A; Rofelty, Angela; Agarwal, Anupriya; Liu, Selina Qiuying; Danilov, Alexey V; Lee, Patrice; Chantry, David; McWeeney, Shannon K; Druker, Brian J; Tyner, Jeffrey W; Spurgeon, Stephen E; Loriaux, Marc M

2018-05-15

In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.

[Expression of Notch Gene and Its Role of Anti-apoptosis and Drug-resistance of Cells in Chronic Lymphocytic Leukemia].

PubMed

Zhang, Jin-Yan; Zhang, Ju-Shun; Xu, Zhen-Shu

2015-08-01

To investigate the expression of Notch gene in chronic lymphocytic leukemia cells and to explore the change of Notch protein after the therapy with cytosine arabinoside or dexmethasone, and the mechanism of Notch mediated anti-apoptosis and drug-resistance in chronic lymphocytic leukemia cells. The mononuclear cells from bone marrow or peripheral blood of chronic lymphocytic leukemia patients (24 cases) and healthy donors (14 cases) were collected, then the expression of Notch gene, BCL-2, as well as NF-κB gene were detected by real-time fluorescent quantitative PCR (qRT-PCR) at the level of transcription. The change of Notch protein in L1210 cell lines after therapy with cytosine arabinoside and dexmethasone was determined by Western blot. mRNA expression levels of Notch1, Notch2, BCL-2 and NF-κB gene in CLL group were significantly higher than those in healthy control group (0.8556 ± 0.8726 vs 0.6731 ± 0.5334, P = 0.0182; 1.2273 ± 0.8207 vs 0.6577 ± 0.6424, P < 0.0001; 8.0960 ± 7.5661 vs 0.5969 ± 0.4976, P < 0.0001; 1.0966 ± 0.6925 vs 0.5373 ± 0.7180, P < 0.0001, respectively), but no significant difference was found between Notch3 and Notch4 gene (1.1914 ± 2.4219 vs 0.8713 ± 0.7937, P = 0.3427; 0.8174 ± 1.0869 vs 0.9752 ± 1.3446, P = 0.2402, respectively). Notch1 protein expression in L1210 cells were significantly decreased after treating with cytosine arabinoside of low and middle concentrations, but increased after treating with cytosine arabinoside of high concentration or prolonging time of cytosine arabinoside of middle con-centration. Notch1 protein expression in L1210 cells dereased after treating with dexamethasone, but did not be changed with the different concentrations and different times of dexmethason. The transcription level of Notch gene in CLL patients significantly higher than that in normal controls. The Notch1 protein expression is down-regulated in process of inhibiting L1210 cell proliferation by Ara-C and dexmethason. Notch

Loss of Dnmt3a induces CLL and PTCL with distinct methylomes and transcriptomes in mice.

PubMed

Haney, Staci L; Upchurch, Garland M; Opavska, Jana; Klinkebiel, David; Appiah, Adams Kusi; Smith, Lynette M; Heavican, Tayla B; Iqbal, Javeed; Joshi, Shantaram; Opavsky, Rene

2016-09-28

Cytosine methylation of DNA is an epigenetic modification involved in the repression of genes that affect biological processes including hematopoiesis. It is catalyzed by DNA methyltransferases, one of which -DNMT3A- is frequently mutated in human hematologic malignancies. We have previously reported that Dnmt3a inactivation in hematopoietic stem cells results in chronic lymphocytic leukemia (CLL) and CD8-positive peripheral T cell lymphomas (PTCL) in EμSRα-tTA;Teto-Cre;Dnmt3a fl/fl ; Rosa26LOXP EGFP/EGFP (Dnmt3a Δ/Δ ) mice. The extent to which molecular changes overlap between these diseases is not clear. Using high resolution global methylation and expression analysis we show that whereas patterns of methylation and transcription in normal B-1a cells and CD8-positive T cells are similar, methylomes and transcriptomes in malignant B-1a and CD8+ T cells are remarkably distinct, suggesting a cell-type specific function for Dnmt3a in cellular transformation. Promoter hypomethylation in tumors was 10 times more frequent than hypermethylation, three times more frequent in CLL than PTCL and correlated better with gene expression than hypermethylation. Cross-species molecular comparison of mouse and human CLL and PTCL reveals significant overlaps and identifies putative oncogenic drivers of disease. Thus, Dnmt3a Δ/Δ mice can serve as a new mouse model to study CLL and PTCL in relevant physiological settings.

Clinical roundtable monograph. New alternatives in CLL therapy: managing adverse events.

PubMed

Chanan-Khan, Asher; Kipps, Thomas; Stilgenbauer, Stephan

2010-08-01

Chronic lymphocytic leukemia (CLL) is a B-cell leukemia mainly affecting older adults. Historically, CLL has been regarded as an incurable disease, and treatment has been confined to cytotoxic chemotherapy regimens. However, prognosis for patients treated with these agents remained poor, prompting the development of new, targeted agents. The introduction of rituximab, a CD20-targeted monoclonal antibody, revolutionized the treatment for this disease. Rituximab in combination with fludarabine improved response rates and length of progression-free survival. The success of rituximab in this setting has prompted the development of many more investigational agents for CLL, including other antibody agents. However, as with any medication, the potential benefit achieved with CLL therapies is mitigated by the safety risk for the patient. These agents have been associated with adverse events such as immunosuppression, reactivation of cytomegalovirus, and infusion-related reactions that can occur with antibody administration. Adverse events can greatly affect the patient’s quality of life and ability to tolerate therapy. Management of adverse events is a critical component of the overall treatment strategy for CLL, particularly in elderly patients. In this clinical roundtable monograph, 3 expert physicians discuss the latest clinical studies evaluating the treatment of CLL, focusing on the adverse events associated with each agent and the potential interventions that can be used to manage their occurrence.

CD5-expressing B-cell lymphomas/leukemias: relatively high frequency of CD5+ B-cell lymphomas with an overall poor prognosis in Nagasaki Japan.

PubMed

Kamihira, S; Hirakata, Y; Atogami, S; Sohda, H; Tsuruda, K; Yamada, Y; Tomonaga, M

1996-06-01

To characterize CD5+ B-cell neoplasms in Japan, where chronic lymphocytic leukemia (CLL) is rare and of different subtypes in comparison with Western countries, we collected 58 cases of CD5+ B-cell lymphomas/leukemias and analyzed their clinicopathologic features. According to the French-American-British (FAB) and standard histologic classification, the cases corresponded to small lymphocytic lymphoma (SLL, group I; n = 22, consisting of CLL, n = 10, CLL/PL, n = 3, and CLLmixed, n = 7); intermediate differentiated lymphoma/mantle cell lymphoma (IDL/MCL, group II, n = 18); and others with CD5-positive lymphomas (group III, n = 18). The CD5+ B-cell lymphomas showed morphologic and prognostic variability among the three groups. The clinical and immunophenotypic features were remarkably consistent in leukemic disease being seen in 73% of all cases, splenomegaly in 63%, and intense CD19, CD20, surface membrane immunogobulin M (SmIgM) or SmIgM and SmIgD, light-chain expression, and no CD10 expression. The median survival time of groups I, II, and III was 7.8, 3.3, and 0.8 years, respectively. These findings suggest that CD5 antigens may serve as valid markers for the prognosis and clinical features of B-cell lymphomas and that CD5+ B-cell lymphomas with an overall poor prognosis occurs at a relatively high frequency in Japan. This also suggests that a combination of immunophenotypic and morphologic features is of value for characterizing CD5+ B-cell neoplasms.

Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl2 mRNA

PubMed Central

Otake, Yoko; Soundararajan, Sridharan; Sengupta, Tapas K.; Kio, Ebenezer A.; Smith, James C.; Pineda-Roman, Mauricio; Stuart, Robert K.; Spicer, Eleanor K.

2007-01-01

B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells that are resistant to apoptosis as a result of bcl2 oncogene overexpression. Studies were done to determine the mechanism for the up-regulation of bcl-2 protein observed in CD19+ CLL cells compared with CD19+ B cells from healthy volunteers. The 11-fold higher level of bcl-2 protein in CLL cells was positively correlated with a 26-fold elevation in the cytosolic level of nucleolin, a bcl2 mRNA–stabilizing protein. Measurements of the bcl2 heterogeneous nuclear/bcl2 mRNA (hnRNA)/mRNA ratios and the rates of bcl2 mRNA decay in cell extracts indicated that the 3-fold higher steady-state level of bcl2 mRNA in CLL cells was the result of increased bcl2 mRNA stability. Nucleolin was present throughout the nucleus and cytoplasm of CLL cells, whereas in normal B cells nucleolin was only detected in the nucleus. The addition of recombinant human nucleolin to extracts of normal B cells markedly slowed the rate of bcl2 mRNA decay. SiRNA knockdown of nucleolin in MCF-7 cells resulted in decreased levels of bcl2 mRNA and protein but no change in β-actin. These results indicate that bcl-2 overexpression in CLL cells is related to stabilization of bcl2 mRNA by nucleolin. PMID:17179226

Microenvironment interactions and B-cell receptor signaling in Chronic Lymphocytic Leukemia: implications for disease pathogenesis and treatment

PubMed Central

ten Hacken, Elisa; Burger, Jan A.

2015-01-01

Chronic Lymphocytic Leukemia (CLL) is a malignancy of mature B lymphocytes which are highly dependent on interactions with the tissue microenvironment for their survival and proliferation. Critical components of the microenvironment are monocyte-derived nurselike cells (NLCs), mesenchymal stromal cells, T cells and NK cells, which communicate with CLL cells through a complex network of adhesion molecules, chemokine receptors, tumor necrosis factor (TNF) family members, and soluble factors. (Auto-) antigens and/or autonomous mechanisms activate the B-cell receptor (BCR) and its downstream signaling cascade in secondary lymphatic tissues, playing a central pathogenetic role in CLL. Novel small molecule inhibitors, including the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib and the phosphoinositide-3-kinase delta (PI3Kδ) inhibitor idelalisib, target BCR signaling and have become the most successful new therapeutics in this disease. We here review the cellular and molecular characteristics of CLL cells, and discuss the cellular components and key pathways involved in the cross-talk with their microenvironment. We also highlight the relevant novel treatment strategies, focusing on immunomodulatory agents and BCR signaling inhibitors and how these treatments disrupt CLL-microenvironment interactions. PMID:26193078

Matrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interaction via extracellular signal-regulated kinase-1/2 signaling and is involved in CCL21-driven B-cell chronic lymphocytic leukemia cell invasion and migration.

PubMed

Redondo-Muñoz, Javier; José Terol, María; García-Marco, José A; García-Pardo, Angeles

2008-01-01

B-cell chronic lymphocytic leukemia (B-CLL) progression is frequently accompanied by clinical lymphadenopathy, and the CCL21 chemokine may play an important role in this process. Indeed, CCR7 (the CCL21 receptor), as well as matrix metalloproteinase-9 (MMP-9), are overexpressed in infiltrating B-CLL cells. We have studied whether MMP-9 is regulated by CCL21 and participates in CCL21-dependent migration. CCL21 significantly increased B-CLL MMP-9 production, measured by gelatin zymography. This was inhibited by blocking extracellular signal-regulated kinase-1/2 (ERK1/2) activity or by cell transfection with CCR7-siRNA. Accordingly, CCL21/CCR7 interaction activated the ERK1/2/c-Fos pathway and increased MMP-9 mRNA. CCL21-driven B-CLL cell migration through Matrigel or human umbilical vein endothelial cells (HUVEC) was blocked by anti-CCR7 antibodies, CCR7-siRNA transfection, or the ERK1/2 inhibitor U0126, as well as by anti-MMP-9 antibodies or tissue inhibitor of metalloproteinase 1 (TIMP-1). These results strongly suggest that MMP-9 is involved in B-CLL nodal infiltration and expand the roles of MMP-9 and CCR7 in B-CLL progression. Both molecules could thus constitute therapeutic targets for this disease.

Results of a randomized trial comparing high-dose chemotherapy plus Auto-SCT and R-FC in CLL at diagnosis.

PubMed

Magni, M; Di Nicola, M; Patti, C; Scimè, R; Mulè, A; Rambaldi, A; Intermesoli, T; Viero, P; Tarella, C; Gueli, A; Bergui, L; Trentin, L; Barzan, A; Benedetti, F; Ambrosetti, A; Di Raimondo, F; Chiarenza, A; Parvis, G; Billio, A; Attolico, I; Olivieri, A; Montanari, M; Carlo-Stella, C; Matteucci, P; Devizzi, L; Guidetti, A; Viviani, S; Valagussa, P; Gianni, A M

2014-04-01

The importance of early therapy intensification in B-cell CLL (B-CLL) patients remains to be defined. Even though several studies have been published, no randomized trials comparing directly autologous stem cell transplant (ASCT) and the accepted conventional therapy (that is, rituximab, fludarabine and CY; R-FC) have been reported so far. To assess the benefit of a first-line aggressive therapy, we designed a multicenter, randomized, phase 3 trial comparing R-FC and high-dose chemotherapy supported by ASCT in patients under 65 years of age, with stage B(II) or C B-CLL. Primary end point was CR: 96 patients were enrolled (48 in each arm). On an intent-to-treat basis, the CR rates in the ASCT and R-FC arms were 62.5% and 58%, respectively. After 5 years of follow-up, PFS was 60.4% in the ASCT arm and 65.1% in the R-FC arm, time to progression 65.8 and 70.5%, and overall survival 88% vs 88.1%, respectively. Our trial demonstrates, for the first time in a randomized manner, that frontline ASCT does not translate into a survival advantage when compared with benchmark chemoimmunotherapy in B-CLL patients; the possibility of its clinical benefit in certain subgroups remains uncertain.

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells.

PubMed

Li, L H; Biagi, E; Allen, C; Shivakumar, R; Weiss, J M; Feller, S; Yvon, E; Fratantoni, J C; Liu, L N

2006-02-01

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

Immunotherapy in Chronic Lymphocytic Leukaemia (CLL).

PubMed

Freeman, Ciara L; Gribben, John G

2016-02-01

Chronic lymphocytic leukaemia (CLL) is well known to generate impaired immune responses in the host, with the malignant clone residing in well-vascularized tissues and circulating in peripheral blood but also in close proximity to effector cells that are capable, if activated appropriately, of eliciting a cytotoxic response. These, combined with the fact that this is frequently a condition affecting older patients with co-morbidities often unfit for many "traditional" cytotoxic agents with their significant associated toxicities, make CLL an ideal candidate for the development of immunotherapy. The impressive results seen with the addition of a monoclonal antibody, rituximab, to a chemotherapy backbone, for example, is testament to how effective harnessing an immune-mediated response in CLL can be. This review serves to outline the available arsenal of immunotherapies-past and present-demonstrated to have potential in CLL with some perspectives on how the landscape in this disease may evolve in the future.

A 17-residue Sequence from the Matrix Metalloproteinase-9 (MMP-9) Hemopexin Domain Binds α4β1 Integrin and Inhibits MMP-9-induced Functions in Chronic Lymphocytic Leukemia B Cells*

PubMed Central

Ugarte-Berzal, Estefanía; Bailón, Elvira; Amigo-Jiménez, Irene; Vituri, Cidonia L.; del Cerro, Mercedes Hernández; Terol, María José; Albar, Juan P.; Rivas, Germán; García-Marco, José A.; García-Pardo, Angeles

2012-01-01

We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4β1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4β1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC50 values of 138 and 279 μm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesis. PMID:22730324

PD-1 expression and clinical PD-1 blockade in B-cell lymphomas.

PubMed

Xu-Monette, Zijun Y; Zhou, Jianfeng; Young, Ken H

2018-01-04

Programmed cell death protein 1 (PD-1) blockade targeting the PD-1 immune checkpoint has demonstrated unprecedented clinical efficacy in the treatment of advanced cancers including hematologic malignancies. This article reviews the landscape of PD-1/programmed death-ligand 1 (PD-L1) expression and current PD-1 blockade immunotherapy trials in B-cell lymphomas. Most notably, in relapsed/refractory classical Hodgkin lymphoma, which frequently has increased PD-1 + tumor-infiltrating T cells, 9p24.1 genetic alteration, and high PD-L1 expression, anti-PD-1 monotherapy has demonstrated remarkable objective response rates (ORRs) of 65% to 87% and durable disease control in phase 1/2 clinical trials. The median duration of response was 16 months in a phase 2 trial. PD-1 blockade has also shown promise in a phase 1 trial of nivolumab in relapsed/refractory B-cell non-Hodgkin lymphomas, including follicular lymphoma, which often displays abundant PD-1 expression on intratumoral T cells, and diffuse large B-cell lymphoma, which variably expresses PD-1 and PD-L1. In primary mediastinal large B-cell lymphoma, which frequently has 9p24.1 alterations, the ORR was 35% in a phase 2 trial of pembrolizumab. In contrast, the ORR with pembrolizumab was 0% in relapsed chronic lymphocytic leukemia (CLL) and 44% in CLL with Richter transformation in a phase 2 trial. T cells from CLL patients have elevated PD-1 expression; CLL PD-1 + T cells can exhibit a pseudo-exhaustion or a replicative senescence phenotype. PD-1 expression was also found in marginal zone lymphoma but not in mantle cell lymphoma, although currently anti-PD-1 clinical trial data are not available. Mechanisms and predictive biomarkers for PD-1 blockade immunotherapy, treatment-related adverse events, hyperprogression, and combination therapies are discussed in the context of B-cell lymphomas. © 2018 by The American Society of Hematology.

Cell-Intrinsic Determinants of Ibrutinib-Induced Apoptosis in Chronic Lymphocytic Leukemia.

PubMed

Amin, Nisar A; Balasubramanian, Sriram; Saiya-Cork, Kamlai; Shedden, Kerby; Hu, Nan; Malek, Sami N

2017-02-15

Purpose: Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed chronic lymphocytic leukemia (CLL) and CLL with del17p. Mechanistically, ibrutinib interferes with B-cell receptor (BCR) signaling as well as multiple CLL cell-to-microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted. Experimental Design: We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole-exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 to 5 μmol/L of ibrutinib ex vivo and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted TP53 in HG3, PGA1, and PG-EBV cell lines and measured BCR signaling and ibrutinib responses. Results: CLL samples demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib, with IC 50 values ranging from 0.4 to 9.7 μmol/L. Unmutated IGVH status, elevated ZAP70 expression, and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/ TP53 -mutated status. A validation sample of 15 CLL carrying TP53 mutations, of which 13 carried both del17p and a TP53 mutation, confirmed substantially less sensitivity to ibrutinib-induced apoptosis. Conclusions: This study identifies that CLL harboring del17p/ TP53 -mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/ TP53 wild-type cells. Clin Cancer Res; 23(4); 1049-59. ©2016 AACR . ©2016 American Association for Cancer Research.

Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

PubMed Central

Ghiotto, Fabio; Marcatili, Paolo; Tenca, Claudya; Calevo, Maria Grazia; Yan, Xiao-Jie; Albesiano, Emilia; Bagnara, Davide; Colombo, Monica; Cutrona, Giovanna; Chu, Charles C; Morabito, Fortunato; Bruno, Silvia; Ferrarini, Manlio; Tramontano, Anna; Fais, Franco; Chiorazzi, Nicholas

2011-01-01

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV–diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation. PMID:21785810

Reduced intensity versus full myeloablative stem cell transplant for advanced CLL.

PubMed

Peres, E; Braun, T; Krijanovski, O; Khaled, Y; Levine, J E; Yanik, G; Kato, K; Mineishi, S

2009-11-01

CLL remains incurable with the standard therapy. Allogeneic hematopoietic stem cell transplant may be curative. We examined 50 patients with advanced CLL who underwent allogeneic HCT at the University of Michigan between 1996 and 2006. Twenty-one patients received reduced-intensity conditioning (RIC) and twenty-nine patients received full-intensity conditioning (FIC) consisting of CY, etoposide and BCNU (n=20) or BU and CY (n=9). RIC recipients were older than FIC recipients (median age 54 vs 51, P=0.009). There were no statistically significant differences between groups in terms of the number of earlier therapies or patients with adverse cytogenetics. There were more unrelated donors in the RIC group 62% than in the FIC group 31% (P=0.030). Despite their older age and greater use of URD, the 5-year overall survival (OS) rate was 63% in the RIC group as compared with 18% in the FIC group (P=0.006). The primary cause of inferior survival in the FIC recipients was TRM, which was twice as high at day 100 for the FIC group 27% compared with the RIC group 14% (P=0.005). The relapse rate was 15% regardless with the majority of relapses occurring after day 100. These results suggest a favorable outcome for advanced CLL who undergo a RIC regimen compared with FIC.

The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia.

PubMed

de Rooij, Martin F M; Kuil, Annemieke; Geest, Christian R; Eldering, Eric; Chang, Betty Y; Buggy, Joseph J; Pals, Steven T; Spaargaren, Marcel

2012-03-15

Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α(4)β(1)-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.

Mechanisms of PD-L1/PD-1-mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model.

PubMed

McClanahan, Fabienne; Riches, John C; Miller, Shaun; Day, William P; Kotsiou, Eleni; Neuberg, Donna; Croce, Carlo M; Capasso, Melania; Gribben, John G

2015-07-09

T-cell defects, immune suppression, and poor antitumor immune responses are hallmarks of chronic lymphocytic leukemia (CLL), and PD-1/PD-L1 inhibitory signaling has emerged as a major immunosuppressive mechanism. However, the effect of different microenvironments and the confounding influence of aging are poorly understood. The current study uses the Eμ-TCL1 mouse model, which replicates human T-cell defects, as a preclinical platform to longitudinally examine patterns of T-cell dysfunction alongside developing CLL and in different microenvironments, with a focus on PD-1/PD-L1 interactions. The development of CLL was significantly associated with changes in T-cell phenotype across all organs and function. Although partly mirrored in aging wild-type mice, CLL-specific T-cell changes were identified. Murine CLL cells highly expressed PD-L1 and PD-L2 in all organs, with high PD-L1 expression in the spleen. CD3(+)CD8(+) T cells from leukemic and aging healthy mice highly expressed PD-1, identifying aging as a confounder, but adoptive transfer experiments demonstrated CLL-specific PD-1 induction. Direct comparisons of PD-1 expression and function between aging CLL mice and controls identified PD-1(+) T cells in CLL as a heterogeneous population with variable effector function. This is highly relevant for therapeutic targeting of CD8(+) T cells, showing the potential of reprogramming and selective subset expansion to restore antitumor immunity. © 2015 by The American Society of Hematology.

COUP-TFII inhibits NFkappaB activation in endocrine-resistant breast cancer cells

PubMed Central

Litchfield, Lacey M.; Appana, Savitri N.; Datta, Susmita; Klinge, Carolyn M.

2016-01-01

Reduced COUP-TFII expression contributes to endocrine resistance in breast cancer cells. Endocrine-resistant breast cancer cells have higher NFkappa B (NFκB) activity and target gene expression. The goal of this study was to determine if COUP-TFII modulates NFκB activity. Endocrine-resistant LCC9 cells with low endogenous COUP-TFII displayed ~5-fold higher basal NFκB activity than parental endocrine-sensitive MCF-7 breast cancer cells. Transient transfection of LCC9 cells with COUP-TFII inhibited NFκB activation and reduced NFκB target gene expression. COUP-TFII and NFκB were inversely correlated in breast cancer patient samples. Endogenous COUP-TFII coimmunoprecipitated with NFκB subunits RelB and NFκB1 in MCF-7 cells. COUP-TFII inhibited NFκB-DNA binding in vitro and impaired coactivator induced NFκB transactivation. LCC9 cells were growth-inhibited by an NFκB inhibitor and 4-hydroxytamoxifen compared to MCF-7 cells. Together these data indicate a novel role for COUP-TFII in suppression of NFκB activity and explain, in part, why decreased COUP-TFII expression results in an endocrine-resistant phenotype. PMID:24141032

The ibrutinib B-cell proliferation inhibition is potentiated in vitro by dexamethasone: Application to chronic lymphocytic leukemia.

PubMed

Manzoni, Delphine; Catallo, Régine; Chebel, Amel; Baseggio, Lucile; Michallet, Anne-Sophie; Roualdes, Olivier; Magaud, Jean-Pierre; Salles, Gilles; Ffrench, Martine

2016-08-01

New B-cell receptor-targeted therapies such as ibrutinib, a Bruton tyrosine kinase inhibitor, are now proposed for lymphoid pathologies. The putative benefits of its combination with glucocorticoids were evaluated here. We compared the effects of dexamethasone (DXM), ibrutinib and their in vitro combination on proliferation and metabolic stress markers in stimulated normal B-lymphocytes and in malignant lymphocytes from chronic lymphocytic leukemia (CLL) patients. In both cellular models, cell cycle progression was globally inhibited by DXM and/or ibrutinib. This inhibition was significantly amplified by DXM addition to ibrutinib and was related to a significant decrease in the expression of the cell cycle regulatory proteins CDK4 and cyclin E. Apoptosis increased especially with DXM/ibrutinib combination and was associated with a significant decrease in Mcl-1 expression. Treatment effects on metabolic stress were evaluated by DNA damage recognition after 53BP1 foci labeling. The percentage of cells with more than five 53BP1 foci decreased significantly with ibrutinib in normal and CLL lymphocytes. This decrease was strongly reinforced, in CLL, by DXM addition. Our data indicated that, in vitro, DXM potentiated antiproliferative effects of ibrutinib and decreased DNA damage in lymphoid B-cells. Thus their combination may be proposed for CLL treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

How I treat CLL up front.

PubMed

Gribben, John G

2010-01-14

Although chronic lymphocytic leukemia (CLL) remains incurable, over the past decade there have been major advances in understanding the pathophysiology of CLL and in the treatment of this disease. This has led to greatly increased response rates and durations of response but not yet improved survival. Advances in the use of prognostic factors that identify patients at high risk for progression have led us to the question whether there is still a role for a "watch and wait" approach in asymptomatic high-risk patients or whether they should be treated earlier in their disease course. Questions remain, including, what is the optimal first-line treatment and its timing and is there any role of maintenance therapy or stem cell transplantation in this disease? CLL is a disease of the elderly and not all patients are eligible for aggressive up-front chemoimmunotherapy regimens, so what is the optimal treatment approach for more frail elderly patients? It is highly likely that our treatment approaches will continue to evolve as the results of ongoing clinical trials are released and that further improvements in the outcome of this disease will result from identification of therapies that target the underlying pathophysiology of CLL.

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions.

PubMed

Tiscornia, A C; Cayota, A; Landoni, A I; Brito, C; Oppezzo, P; Vuillier, F; Robello, C; Dighiero, G; Gabús, R; Pritsch, O

2004-01-01

Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro- and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16(14del)), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16(neg)) was correlated to a decreased B-CLL cell viability. The 9-beta-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16(neg) transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16(pos)), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide (.- NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.

Symptomatic Hypercalcemia in a Patient with B-cell Chronic Lymphocytic Leukemia - A Case Report and Review of the Literature.

PubMed

Koutroumpakis, Efstratios; Lobe, Montgomery; McCarthy, Lezah; Mehdi, Syed

Hypercalcemia due to malignancy is well described in the literature and a common paraneoplastic finding in certain solid tumors. Hematologic malignancies, however, are less frequently associated with hypercalcemia with the exception of myelomas and T-cell lymphomas. This case report describes a patient with B-cell chronic lymphocytic leukemia (B-CLL) who developed symptomatic hypercalcemia. None of the pathogenetic mechanisms of malignancy-associated hypercalcemia already described in the literature could explain the pathogenesis of hypercalcemia in our patient. Calcium levels were normalized after initial treatment and remained within normal limits following treatment of the underlying B-CLL. The follow-up period was 26 months. The normalization of calcium levels was closely associated with the drop in the absolute lymphocyte count. Symptomatic hypercalcemia in B-CLL is exceedingly rare and only documented a few times in the literature. Hypercalcemia, in the present case, was not caused by any of the mechanisms already described in the literature and responded well to treatment of the underlying B-CLL. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

miR in CLL: more than mere markers of prognosis?

PubMed

Kater, Arnon P; Eldering, Eric

2014-07-03

In this issue of Blood, Mraz et al show that microRNA-150 (miR-150) is the most abundantly expressed miR in chronic lymphocytic leukemia (CLL) and affects the threshold for B-cell receptor (BCR) signaling by repressing expression levels of GAB1 and FOXP1. This functional link might explain the described association between expression levels of miR-150 and prognosis.

Clinical effect of stereotyped B-cell receptor immunoglobulins in chronic lymphocytic leukaemia: a retrospective multicentre study.

PubMed

Baliakas, Panagiotis; Hadzidimitriou, Anastasia; Sutton, Lesley-Ann; Minga, Eva; Agathangelidis, Andreas; Nichelatti, Michele; Tsanousa, Athina; Scarfò, Lydia; Davis, Zadie; Yan, Xiao-Jie; Shanafelt, Tait; Plevova, Karla; Sandberg, Yorick; Vojdeman, Fie Juhl; Boudjogra, Myriam; Tzenou, Tatiana; Chatzouli, Maria; Chu, Charles C; Veronese, Silvio; Gardiner, Anne; Mansouri, Larry; Smedby, Karin E; Pedersen, Lone Bredo; van Lom, Kirsten; Giudicelli, Véronique; Francova, Hana Skuhrova; Nguyen-Khac, Florence; Panagiotidis, Panagiotis; Juliusson, Gunnar; Angelis, Lefteris; Anagnostopoulos, Achilles; Lefranc, Marie-Paule; Facco, Monica; Trentin, Livio; Catherwood, Mark; Montillo, Marco; Geisler, Christian H; Langerak, Anton W; Pospisilova, Sarka; Chiorazzi, Nicholas; Oscier, David; Jelinek, Diane F; Darzentas, Nikos; Belessi, Chrysoula; Davi, Frederic; Rosenquist, Richard; Ghia, Paolo; Stamatopoulos, Kostas

2014-11-01

About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptor immunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggests that B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never been explored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn. For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogenetic data were available. These patients were followed up in 15 academic institutions throughout Europe (in Czech Republic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collected between June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptor immunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressing unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome of our analysis was time to first treatment, defined as the time between diagnosis and date of first treatment. 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 major subsets. Stereotyped subsets showed significant differences in terms of age, sex, disease burden at diagnosis, CD38 expression, and cytogenetic aberrations of prognostic significance. Patients within a specific subset generally followed the same clinical course, whereas patients in different stereotyped subsets-despite having the same immunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status-showed substantially different times to first treatment. By integrating B-cell receptor immunoglobulin stereotypy (for subsets 1, 2, and 4) into the well established Döhner cytogenetic prognostic model, we showed these, which collectively account for around 7% of all

Mechanisms of PD-L1/PD-1–mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model

PubMed Central

McClanahan, Fabienne; Riches, John C.; Miller, Shaun; Day, William P.; Kotsiou, Eleni; Neuberg, Donna; Croce, Carlo M.; Capasso, Melania

2015-01-01

T-cell defects, immune suppression, and poor antitumor immune responses are hallmarks of chronic lymphocytic leukemia (CLL), and PD-1/PD-L1 inhibitory signaling has emerged as a major immunosuppressive mechanism. However, the effect of different microenvironments and the confounding influence of aging are poorly understood. The current study uses the Eμ-TCL1 mouse model, which replicates human T-cell defects, as a preclinical platform to longitudinally examine patterns of T-cell dysfunction alongside developing CLL and in different microenvironments, with a focus on PD-1/PD-L1 interactions. The development of CLL was significantly associated with changes in T-cell phenotype across all organs and function. Although partly mirrored in aging wild-type mice, CLL-specific T-cell changes were identified. Murine CLL cells highly expressed PD-L1 and PD-L2 in all organs, with high PD-L1 expression in the spleen. CD3+CD8+ T cells from leukemic and aging healthy mice highly expressed PD-1, identifying aging as a confounder, but adoptive transfer experiments demonstrated CLL-specific PD-1 induction. Direct comparisons of PD-1 expression and function between aging CLL mice and controls identified PD-1+ T cells in CLL as a heterogeneous population with variable effector function. This is highly relevant for therapeutic targeting of CD8+ T cells, showing the potential of reprogramming and selective subset expansion to restore antitumor immunity. PMID:25979947

A Case of Early Ocular Manifestation of Maculopathy in a 37 year Old Male Patient of CLL.

PubMed

Mehdi, Syed Riaz; Tandon, Nishi; Khan, Sufia Ahmad; Ahmad, Sharique

2014-09-01

B cell chronic lymphocytic leukemia (CLL) also called chronic lymphocytic leukemia is a disease of elderly, but in rare cases it can occur in young adults. Majority of patients present with no specific symptoms, and CLL is suspected on the basis of high total and relative lymphocyte counts, discovered on routine blood examination. This patient was referred from medicine outpatient in our institute, Era's Lucknow Medical College & Hospital for routine complete blood count. His total leukocyte count came out to be alarmingly high. The general blood picture and bone marrow examinations were consistent with CLL. On immunophenotyping CD5 and CD23 were positive. His cervical lymph nodes biopsy was reported as a case of CLL/small lymphocytic lymphoma. On his complaint of hazy vision his funduscopy revealed maculopathy. This makes our case a unique and rare one, as maculopathy in a young patient of CLL has not yet been reported from India.

Hodgkin/Reed-Sternberg cells and Hodgkin's disease in patients with B-cell chronic lymphocytic leukaemia: an immunohistological, molecular and clinical study of four cases suggesting a heterogeneous pathogenetic background.

PubMed

Pescarmona, E; Pignoloni, P; Mauro, F R; Cerretti, R; Anselmo, A P; Mandelli, F; Baroni, C D

2000-08-01

We report the immunohistological, molecular and clinical findings in four patients affected by B-cell chronic lymphocytic leukaemia (CLL) who developed "Richter's syndrome with Hodgkin's disease (HD) features" or "CLL with Hodgkin's transformation", all characterised by the presence of typical Hodgkin/Reed-Sternberg (H/RS) cells in lymph node biopsies. In three cases the nodal involvement by CLL was demonstrated both by the presence of a predominant background of CD5/CD19/CD23+ small lymphocytes and an IgH monoclonal rearrangement revealed by PCR analysis. Conversely, in the remaining case there was neither immunohistological nor molecular evidence of lymph node involvement by CLL. In all four cases H/RS cells were Epstein-Barr virus (EBV) latent membrane protein (LMP-1) positive. These findings suggest that the presence of H/RS cells in the first three patients, who had CLL/HD nodal involvement, might be related to transformation or clonal evolution of CLL cells in H/RS cells, which is in keeping with use of the term "CLL with Hodgkin's transformation". In the fourth case a de novo HD may be postulated, representing a second malignancy presumably not clonally related to CLL. In all cases a key pathogenetic role of EBV is suggested by the expression of LMP-1 in H/RS cells. Our findings indicate that the presence of typical H/RS cells in lymph node biopsies in CLL patients may reflect a heterogeneous pathogenetic background. The different clinico-pathologic settings should be taken into consideration because of their possible implications for patients' treatment and prognosis.

Pro-Apoptotic Activity of New Honokiol/Triphenylmethane Analogues in B-Cell Lymphoid Malignancies.

PubMed

Mędra, Aleksandra; Witkowska, Magdalena; Majchrzak, Agata; Cebula-Obrzut, Barbara; Bonner, Michael Y; Robak, Tadeusz; Arbiser, Jack L; Smolewski, Piotr

2016-07-30

Honokiol and triphenylmethanes are small molecules with anti-tumor properties. Recently, we synthesized new honokiol analogues (HAs) that possess common features of both groups. We assessed the anti-tumor effectiveness of HAs in B-cell leukemia/lymphoma cells, namely in chronic lymphocytic leukemia (CLL) cells ex vivo and in pre-B-cell acute lymphoblastic leukemia (Nalm-6), Burkitt lymphoma (BL; Raji), diffuse large B-cell lymphoma (DLBCL; Toledo) and multiple myeloma (MM; RPMI 8226) cell lines. Four of these compounds appeared to be significantly active against the majority of cells examined, with no significant impact on healthy lymphocytes. These active HAs induced caspase-dependent apoptosis, causing significant deregulation of several apoptosis-regulating proteins. Overall, these compounds downregulated Bcl-2 and XIAP and upregulated Bax, Bak and survivin proteins. In conclusion, some of the HAs are potent tumor-selective inducers of apoptosis in ex vivo CLL and in BL, DLBCL and MM cells in vitro. Further preclinical studies of these agents are recommended.

The Antineoplastic Effect of Nitric Oxide-Donating Acetylsalicylic Acid (NO-ASA) in Chronic Lymphocytic Leukemia (CLL) Cells is Highly Dependent on its Positional Isomerism.

PubMed

Gehrke, Iris; Razavi, Regina; Poll-Wolbeck, Simon Jonas; Berkessel, Albrecht; Hallek, Michael; Kreuzer, Karl-Anton

2011-10-01

Chronic Lymphocytic Leukemia (CLL) is not curable in patients that are not eligible for allogeneic stem cell transplantation. Therefore, new treatment options are highly desirable. Chemically modified nonsteroidal anti-inflammatory drugs (NSAIDs), such as nitric-oxide-donating acetylsalicylic acid (NO-ASA), have been described to possess antineoplastic capacity. Recently, we could demonstrate a potent apoptosis induction in primary CLL cells in vitro and tumor growth inhibition by para-NO-ASA in a xenograft mouse model. However, little is known about the impact of positional isomerism of NO-ASA on its antineoplastic capacity in CLL. Primary CLL cells were treated with the meta-or para-isomer of NO-ASA at varying concentrations and durations. Viability was assessed flow cytometrically by annexin V-FITC/PI staining and by CellTiter-Glo luminescence cell viability assay. Caspase and PARP cleavage as well as involvement of β-catenin/Lef-1 signaling was determined by immunoblotting. For caspase inhibition, BD™ ApoBlock was used. Nude mice were xenografted with JVM3 cells and treated with meta-NO-ASA, para-NO-ASA or vehicle control. The meta-isomer was entirely ineffective in inducing CLL cell apoptosis in concentrations up to 100 μM, while para-NO-ASA acted in the low micromolar range. meta-NO-ASA, in contrast to para-NO-ASA, did not alter caspase activity. While para-NO-ASA action involved inhibition of β-catenin/Lef-1 signaling, meta-NO-ASA did not show any impact on this signaling pathway. Further, meta-NO-ASA did not significantly reduce tumor growth in a CLL xenograft mouse model, while para-NO-ASA was highly potent. We conclude that positional isomerism is crucial for the antineoplastic effect of NO-ASA in CLL. It can be suggested that the para-isomer, but not the meta-isomer, generates a chemical structure which is essential for the neoplastic effect of NO-ASA.

The Antineoplastic Effect of Nitric Oxide-Donating Acetylsalicylic Acid (NO-ASA) in Chronic Lymphocytic Leukemia (CLL) Cells is Highly Dependent on its Positional Isomerism

PubMed Central

Gehrke, Iris; Razavi, Regina; Poll-Wolbeck, Simon Jonas; Berkessel, Albrecht; Hallek, Michael; Kreuzer, Karl-Anton

2011-01-01

Background: Chronic Lymphocytic Leukemia (CLL) is not curable in patients that are not eligible for allogeneic stem cell transplantation. Therefore, new treatment options are highly desirable. Chemically modified nonsteroidal anti-inflammatory drugs (NSAIDs), such as nitric-oxide-donating acetylsalicylic acid (NO-ASA), have been described to possess antineoplastic capacity. Recently, we could demonstrate a potent apoptosis induction in primary CLL cells in vitro and tumor growth inhibition by para-NO-ASA in a xenograft mouse model. However, little is known about the impact of positional isomerism of NO-ASA on its antineoplastic capacity in CLL. Methods: Primary CLL cells were treated with the meta-or para-isomer of NO-ASA at varying concentrations and durations. Viability was assessed flow cytometrically by annexin V-FITC/PI staining and by CellTiter-Glo luminescence cell viability assay. Caspase and PARP cleavage as well as involvement of β-catenin/Lef-1 signaling was determined by immunoblotting. For caspase inhibition, BD™ ApoBlock was used. Nude mice were xenografted with JVM3 cells and treated with meta-NO-ASA, para-NO-ASA or vehicle control. Results: The meta-isomer was entirely ineffective in inducing CLL cell apoptosis in concentrations up to 100 μM, while para-NO-ASA acted in the low micromolar range. meta-NO-ASA, in contrast to para-NO-ASA, did not alter caspase activity. While para-NO-ASA action involved inhibition of β-catenin/Lef-1 signaling, meta-NO-ASA did not show any impact on this signaling pathway. Further, meta-NO-ASA did not significantly reduce tumor growth in a CLL xenograft mouse model, while para-NO-ASA was highly potent. Conclusion: We conclude that positional isomerism is crucial for the antineoplastic effect of NO-ASA in CLL. It can be suggested that the para-isomer, but not the meta-isomer, generates a chemical structure which is essential for the neoplastic effect of NO-ASA. PMID:23556096

Current strategies for the diagnosis and management of chronic lymphocytic leukemia (CLL), with a focus on poor-risk CLL: A review.

PubMed

Clanahan, Fabienne Mc; Dreger, Peter

2011-06-05

Despite substantial advancement in the understanding and treatment of chronic lymphocytic leukemia (CLL), a standard curative approach does not exist. The choice of treatment is generally based on the existence of biological and genetic factors associated with the prediction of prognosis, individual response to therapy, and duration of remission. About 20% of patients that require treatment have an aggressive disease course and die within a few years, despite early initiation of intensive therapy (poor-risk CLL). Poor-risk CLL can be predicted by the presence of genomic markers, and the quality and duration of response to purine-analogue-based treatment. Within this patient subgroup alternative treatment approaches such as alemtuzumab or new substances such as flavopiridol or IMiDs® should be considered. To date, the only treatment bearing curative potential is allogeneic stem cell transplantation; in contrast to conventional immunochemotherapy, it can provide long-term disease control, even in patients with del 17p or other unfavorable biological and clinical risk factors. The aim of this review was to outline the current strategies for the diagnosis and management of CLL, with a focus on high-risk CLL.

Autologous transplantation in CLL patients with B and C Binet stages: final results of the prospective randomized GOELAMS LLC 98 trial.

PubMed

Brion, A; Mahé, B; Kolb, B; Audhuy, B; Colombat, P; Maisonneuve, H; Foussard, C; Bureau, A; Ferrand, C; Lesesve, J F; Béné, M C; Feugier, P

2012-04-01

The relevance of high-dose chemotherapy followed by auto-SCT in CLL remains to be defined. The aim of the prospective, randomized, GOELAMS LLC 98 trial was to compare two strategies in previously untreated CLL patients aged <60 years. Conventional chemotherapy (Arm A) consisted of six monthly courses of CHOP followed by six CHOP courses in every 3 months in those achieving a complete or PR. Arm A was compared with high-dose therapy with auto-SCT (Arm B), used as consolidation after three CHOP courses in case of CR or very good PR. A total of 86 patients were enrolled, of which 39 and 43 patients were evaluable in arm A and arm B, respectively. The primary endpoint was PFS. On an intent-to-treat basis and with a median follow-up time of 77.1 (range 1-135.5) months, the median PFS was 22 months in Arm A and 53 months in Arm B (P<0.0001). Median survival time was 104.7 months in arm A and 107.4 months in arm B. This trial demonstrates that frontline high-dose therapy with auto-SCT prolongs PFS but does not translate into a survival advantage in advanced CLL patients in the pre-rituximab era.

S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression.

PubMed

Prieto, Daniel; Sotelo, Natalia; Seija, Noé; Sernbo, Sandra; Abreu, Cecilia; Durán, Rosario; Gil, Magdalena; Sicco, Estefanía; Irigoin, Victoria; Oliver, Carolina; Landoni, Ana Inés; Gabus, Raúl; Dighiero, Guillermo; Oppezzo, Pablo

2017-08-10

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL. © 2017 by The American Society of Hematology.

Richter's transformation to diffuse large B-cell lymphoma: a retrospective study reporting clinical data, outcome, and the benefit of adding rituximab to chemotherapy, from the Israeli CLL Study Group.

PubMed

Tadmor, Tamar; Shvidel, Lev; Bairey, Osnat; Goldschmidt, Neta; Ruchlemer, Rosa; Fineman, Riva; Rahimi-Levene, Naomi; Herishanu, Yair; Yuklea, Mona; Arad, Ariela; Aviv, Ariel; Polliack, Aaron

2014-11-01

Richter's syndrome (RS) is the rare development of an aggressive lymphoid malignancy in a patient with pre-existing chronic lymphocytic leukemia (CLL). Data on RS is sparse and mostly derived from case reports or small series of patients and only a few larger cohorts have been published. The purpose of this large retrospective study was to summarize our national experience with RS in CLL, examine possible risk factors, and analyze relevant demographic, laboratory and clinical parameters, including results of therapy and outcome. We first evaluated data obtained from 119 patients with RS diagnosed during 1971-2010 from 12 medical centers in Israel. The final cohort summarized consisted of 81 patients with RS who developed only diffuse large B-cell lymphoma (DLBCL) after exclusion all cases with insufficient data and those who were not DLBCL. Median overall survival from time of diagnosis of RS was 8 months; after applying the Richter score, patients could be stratified into three prognostic groups, while all other clinical and laboratory parameters evaluated had no prognostic significance. Prior therapy for CLL had no impact on RS survival (P = 0.8) and patients with therapy "naïve" RS and those who had already received chemotherapy prior to developing RS, had the same survival. The addition of rituximab to chemotherapy for RS improved 2 years overall survival from 19% in the chemotherapy alone arm to 42% (P value of 0.001). Although prognosis of patients with RS remains dismal, this retrospective observation provides support for the use of chemo-immunotherapy in DLBCL-RS. © 2014 Wiley Periodicals, Inc.

MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias

PubMed Central

Calin, George Adrian; Liu, Chang-Gong; Sevignani, Cinzia; Ferracin, Manuela; Felli, Nadia; Dumitru, Calin Dan; Shimizu, Masayoshi; Cimmino, Amelia; Zupo, Simona; Dono, Mariella; Dell'Aquila, Marie L.; Alder, Hansjuerg; Rassenti, Laura; Kipps, Thomas J.; Bullrich, Florencia; Negrini, Massimo; Croce, Carlo M.

2004-01-01

Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673–676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253–258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia. PMID:15284443

Trastuzumab-Resistant Luminal B Breast Cancer Cells Show Basal-Like Cell Growth Features Through NF-κB-Activation

PubMed Central

Kanzaki, Hirotaka; Mukhopadhya, Nishit K.; Cui, Xiaojiang; Ramanujan, V. Krishnan

2016-01-01

A major clinical problem in the treatment of breast cancer is mortality due to metastasis. Understanding the molecular mechanisms associated with metastasis should aid in designing new therapeutic approaches for breast cancer. Trastuzumab is the main therapeutic option for HER2+ breast cancer patients; however, the molecular basis for trastuzumab resistance (TZR) and subsequent metastasis is not known. Earlier, we found expression of basal-like molecular markers in TZR tissues from patients with invasive breast cancer.(1) The basal-like phenotype is a particularly aggressive form of breast cancer. This observation suggests that TZR might contribute to an aggressive phenotype. To understand if resistance to TZR can lead to basal-like phenotype, we generated a trastuzumab-resistant human breast cancer cell line (BT-474-R) that maintained human epidermal growth factor receptor 2 (HER2) overexpression and HER2 mediated signaling. Analysis showed that nuclear factor-kappa B (NF-κB) was constitutively activated in the BT-474-R cells, a feature similar to the basal-like tumor phenotype. Pharmacologic inhibition of NF-κB improved sensitivity of BT-474-R cells to trastuzumab. Interestingly, activation of HER2 independent NF-κB is not shown in luminal B breast cancer cells. Our study suggests that by activating the NF-κB pathway, luminal B cells may acquire a HER2+ basal-like phenotype in which NF-κB is constitutively activated; this notion is consistent with the recently proposed “progression through grade” or “evolution of resistance” hypothesis. Furthermore, we identified IKK-α/IKK-β and nuclear accumulation of RelA/p65 as the major determinants in the resistant cells. Thus our study additionally suggests that the nuclear accumulation of p65 may be a useful marker for identifying metastasis-initiating tumor cells and targeting RelA/p65 may limit metastasis of breast and other cancers associated with NF-κB activation. PMID:26871511

ZAP-70 expression in B-cell chronic lymphocytic leukemia: evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgV(H) mutations.

PubMed

Zucchetto, Antonella; Bomben, Riccardo; Bo, Michele Dal; Nanni, Paola; Bulian, Pietro; Rossi, Francesca Maria; Del Principe, Maria Ilaria; Santini, Simone; Del Poeta, Giovanni; Degan, Massimo; Gattei, Valter

2006-07-15

Expression of T cell specific zeta-associated protein 70 (ZAP-70) by B-cell chronic lymphocytic leukemia (B-CLL) cells, as investigated by flow cytometry, has both prognostic relevance and predictive power as surrogate for immunoglobulin heavy chain variable region (IgV(H)) mutations, although a standardization of the cytometric protocol is still lacking. Flow cytometric analyses for ZAP-70 were performed in peripheral blood samples from 145 B-CLL (124 with IgV(H) mutations) by a standard three-color protocol. Identification of ZAP-70(+) cell population was based on an external negative control, i.e., the isotypic control (ISO method) or an internal positive control, i.e., the population of residual normal T/NK cells (TNK method). A comparison between these two approaches was performed. While 86/145 cases were concordant as for ZAP-70 expression according to the two methods (ISO(+)TNK(+) or ISO(-)TNK(-)), 59/145 cases had discordant ZAP-70 expression, mainly (56/59) showing a ISO(+)TNK(-) profile. These latter cases express higher levels of ZAP-70 in their normal T cell component. Moreover, discordant ISO(+)TNK(-) cases had a IgV(H) gene mutation profile similar to that of concordantly positive cases and different from ZAP-70 concordantly negative B-CLL. Analysis of ZAP-70 expression by B-CLL cells by using the ISO method allows to overcome the variability in the expression of ZAP-70 by residual T cells and yields a better correlation with IgV(H) gene mutations. A receiver operating characteristic analysis suggests to employ a higher cut-off than the commonly used 20%. A parallel evaluation of the prognostic value of ZAP-70 expression, as determined according to the ISO and TNK methods, is still needed. (c) 2006 International Society for Analytical Cytology.

Development of the Bruton's tyrosine kinase inhibitor ibrutinib for B cell malignancies.

PubMed

Gayko, Urte; Fung, Mann; Clow, Fong; Sun, Steven; Faust, Elizabeth; Price, Samiyeh; James, Danelle; Doyle, Margaret; Bari, Samina; Zhuang, Sen Hong

2015-11-01

Ibrutinib is a first-in-class oral covalent inhibitor of Bruton's tyrosine kinase that has demonstrated clinical benefit for many patients with B cell malignancies. Positive results in initial trials led the U.S. Food and Drug Administration to grant ibrutinib three breakthrough therapy designations for mantle cell lymphoma (MCL), del17p chronic lymphocytic leukemia (CLL), and Waldenström's macroglobulinemia (WM). Ibrutinib was approved for these three cancers within 14 months of the original U.S. approval. Additionally, ibrutinib is approved for patient subsets with MCL and/or CLL in >45 other countries. Via a unique mechanism of action, ibrutinib inhibits B cell signaling pathways that regulate the survival, proliferation, adhesion, and homing of cancerous cells. This marks a paradigm shift from the conventional cytotoxic chemotherapy approach to treating B cell malignancies. Ibrutinib continues to be evaluated across a range of B cell malignancies, either as single-agent therapy or in combination with other therapies, and continues to transform the lives of these patients. © 2015 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

Coming full circle: 70 years of chronic lymphocytic leukemia cell redistribution, from glucocorticoids to inhibitors of B-cell receptor signaling

PubMed Central

Montserrat, Emili

2013-01-01

Chronic lymphocytic leukemia (CLL) cells proliferate in pseudofollicles within the lymphatic tissues, where signals from the microenvironment and BCR signaling drive the expansion of the CLL clone. Mobilization of tissue-resident cells into the blood removes CLL cells from this nurturing milieu and sensitizes them to cytotoxic drugs. This concept recently gained momentum after the clinical activity of kinase inhibitors that target BCR signaling (spleen tyrosine kinase, Bruton tyrosine kinase, PI3Kδ inhibitors) was established. Besides antiproliferative activity, these drugs cause CLL cell redistribution with rapid lymph node shrinkage, along with a transient surge in lymphocytosis, before inducing objective remissions. Inactivation of critical CLL homing mechanism (chemokine receptors, adhesion molecules), thwarting tissue retention and recirculation into the tissues, appears to be the basis for this striking clinical activity. This effect of BCR-signaling inhibitors resembles redistribution of CLL cells after glucocorticoids, described as early as in the 1940s. As such, we are witnessing a renaissance of the concept of leukemia cell redistribution in modern CLL therapy. Here, we review the molecular basis of CLL cell trafficking, homing, and redistribution and similarities between old and new drugs affecting these processes. In addition, we outline how these discoveries are changing our understanding of CLL biology and therapy. PMID:23264597

Systematic donor blood qualification by flow cytometry would have been able to avoid CLL-type MBL transmission after unrelated hematopoietic stem cell transplantation.

PubMed

Ferrand, Christophe; Garnache-Ottou, Francine; Collonge-Rame, Marie Agnès; Larosa, Fabrice; Blanc, Michel; Behar, Catherine; Giannoli, Catherine; Garnier, Frédérico; Tiberghien, Pierre; Deconinck, Eric; Rohrlich, Pierre Simon

2012-03-01

The current screening for eligibility of unrelated volunteer marrow donors comprises a complete clinical check-up, a blood CBC and serum protein immunoelectrophoresis. This allows to eliminate acute leukemias, myeloproliferative and myelodysplastic disorders, myelomas and MGUS. To date, the risk of transmission of chronic lymphocytic leukemia (CLL) disease is only evaluated by the clinical evaluation and CBC. We report here the case of a CLL-type MBL disease occurring in a 12-year-old boy after unrelated BMT. Deep biological investigations, as Immunophenotyping, cytogenetic and molecular biology allow us to determine the donor origin of the CLL clone. In 2010, 14.2% donor (105/737) for unrelated hematopoietic stem cell transplantation were over 45y. It is currently estimated (USA) that 1 in 210 men and women will be diagnosed with CLL during their lifetime. Given the long asymptomatic phase of CLL, this raises the case for a detection strategy analog to that used for MGUS and myeloma through serum protein electrophoresis. This case-report, to our knowledge, of a CLL-type MBL unrelated donor-to-recipient transmission through BMT raises ethical and practical questions, such as the proper information about disease transmission risk. The cost-effectiveness of a systematic peripheral blood Immunophenotyping in donors elder than 40y at time of stem cell donation should be evaluated. © 2012 John Wiley & Sons A/S.

Mobilization of peripheral blood stem cells in CLL patients after front-line fludarabine treatment.

PubMed

Lysak, D; Koza, V; Steinerova, K; Jindra, P; Vozobulova, V; Schutzova, M

2005-07-01

Autologous peripheral blood stem cell transplantation is performed in an increasing number of chronic lymphocytic leukaemia (CLL) patients who are in the first remission following fludarabine treatment. There are contradictory data about the adverse impact of fludarabine on stem cell harvest. We analysed retrospectively mobilization results in 56 poor-risk CLL patients (median age: 56 years) who underwent first-line treatment with fludarabine and cyclophosphamide. The mobilization, consisting of cyclophosphamide 3 g/m(2) and granulocyte colony-stimulating factor (G-CSF) 10 microg/kg per day, was performed with a median of 77 days following the last fludarabine course. The target yield was >or=2.0x10(6) CD34+ cells/kg. The procedure was successful in 23 (41%) patients. A median of 3.3x10(6) CD34+ cells/kg was collected per patient. The successful mobilization was associated with a longer interval from the last chemotherapy (>2 months). The mobilization result was not influenced by the number of fludarabine cycles. No correlation was found in other parameters such as disease stage at diagnosis, disease status at stimulation or age. The poorly mobilized patients had significantly lower prestimulation blood counts (platelets, WBC and haemoglobin). Our data show that fludarabine does not generally prevent the stem cell mobilization; nevertheless, mechanisms related to the impact of fludarabine on stem cell harvest must be further investigated.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

PubMed

Zwollo, Patty; Ray, Jocelyn C; Sestito, Michael; Kiernan, Elizabeth; Wiens, Gregory D; Kaattari, Steve; StJacques, Brittany; Epp, Lidia

2015-01-01

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative

Lack of autologous mixed lymphocyte reaction in patients with chronic lymphocytic leukemia: evidence for autoreactive T-cell dysfunction not correlated with phenotype, karyotype, or clinical status

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, T.; Bloom, M.L.; Dadey, B.

In the present study, there was a complete lack of autologous MLR between responding T cells or T subsets and unirradiated or irradiated leukemic B cells or monocytes in all 20 patients with CLL, regardless of disease status, stage, phenotype, or karyotype of the disease. The stimulating capacity of unirradiated CLL B cells and CLL monocytes or irradiated CLL B cells was significantly depressed as compared to that of respective normal B cells and monocytes in allogeneic MLR. The responding capacity of CLL T cells was also variably lower than that of normal T cells against unirradiated or irradiated normalmore » allogeneic B cells and monocytes. The depressed allogeneic MLR between CLL B cells or CLL monocytes and normal T cells described in the present study could be explained on the basis of a defect in the stimulating antigens of leukemic B cells or monocytes. The decreased allogeneic MLR of CLL T cells might simply be explained by a defect in the responsiveness of T lymphocytes from patients with CLL. However, these speculations do not adequately explain the complete lack of autologous MLR in these patients. When irradiated CLL B cells or irradiated CLL T cells were cocultured with normal T cells and irradiated normal B cells, it was found that there was no suppressor cell activity of CLL B cells or CLL T cells on normal autologous MLR. Our data suggest that the absence or dysfunction of autoreactive T cells within the Tnon-gamma subset account for the lack of autologous MLR in patients with CLL. The possible significance of the autologous MLR, its relationship to in vivo immunoregulatory mechanisms, and the possible role of breakdown of autoimmunoregulation in the oncogenic process of certain lymphoproliferative and autoimmune diseases in man are discussed.« less

Elevated expression of pleiotrophin in lymphocytic leukemia CD19+ B cells.

PubMed

Du, Chun-Xian; Wang, Lan; Li, Yan; Xiao, Wei; Guo, Qin-Lian; Chen, Fei; Tan, Xin-Ti

2014-10-01

Pleiotrophin (PTN) has been demonstrated to be strongly expressed in many fetal tissues, but seldom in healthy adult tissues. While PTN has been reported to be expressed in many types of tumors as well as at high serum concentrations in patients with many types of cancer, to date, there has been no report that PTN is expressed in leukemia, especially in lymphocytic leukemia. We isolated the CD19(+) subset of B cells from peripheral blood from healthy adults, B-cell acute lymphocytic leukemia (B-ALL) patients, and B-cell chronic lymphocytic leukemia (B-CLL) patients and examined these cells for PTN mRNA and protein expression. We used immunocytochemistry, western blotting, and enzyme-linked immunosorbent assay to show that PTN protein is highly expressed in CD19(+) B cells from B-ALL and B-CLL patients, but barely expressed in B cells from healthy adults. We also examined PTN expression at the nucleic acid level using reverse transcription polymerase chain reaction (RT-PCR) and northern blotting and detected a high levels of PTN transcripts in the CD19(+) B cells from both groups of leukemia patients, but very few in the CD19(+) B cells from the healthy controls. Interestingly, the quantity of the PTN transcripts correlated with the severity of disease. Moreover, suppression of PTN activity with an anti-PTN antibody promoted apoptosis of cells from leukemia patients and cell lines SMS-SB and JVM-2. This effect of the anti-PTN antibody suggests that PTN may be a new target for the treatment of lymphocytic leukemia. © 2014 APMIS. Published by John Wiley & Sons Ltd.

Study of MGUS, Smoldering Myeloma, Early MDS and CLL to Assess Molecular Events of Progression and Clinical Outcome

ClinicalTrials.gov

2017-08-25

Monoclonal Gammopathy of Undetermined Significance (MGUS); Chronic Lymphocytic Leukemia (CLL); Myelodysplastic-Myeloproliferative Diseases; Hematological Malignancies; B-cell Malignancy, Low-grade; Myelodysplastic Syndrome With Low-grade Lesions; IgG Monoclonal Gammopathy of Uncertain Significance; Smoldering Multiple Myeloma; Waldenstrom Macroglobulinemia

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

PubMed Central

Fraietta, Joseph A.; Beckwith, Kyle A.; Patel, Prachi R.; Ruella, Marco; Zheng, Zhaohui; Barrett, David M.; Lacey, Simon F.; Melenhorst, Jan Joseph; McGettigan, Shannon E.; Cook, Danielle R.; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B.; Cogdill, Alexandria P.; Gill, Saar; Porter, David L.; Woyach, Jennifer A.; Long, Meixiao; Johnson, Amy J.; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L.; June, Carl H.; Byrd, John C.

2016-01-01

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. PMID:26813675

FDA Expands Approval of Venetoclax for CLL

Cancer.gov

FDA expanded the approval of venetoclax (Venclexta) for people with chronic lymphocytic leukemia (CLL) to include those whose cancer has progressed after previous treatment, regardless of whether their cancer cells have the deletion 17p gene alteration.

Three newly approved drugs for chronic lymphocytic leukemia (CLL): Incorporating ibrutinib, idelalisib and obinutuzumab into clinical practice

PubMed Central

Sanford, David; Wierda, William G.; Burger, Jan A.; Keating, Michael J.; O’Brien, Susan M.

2016-01-01

Three agents have received FDA approval for treatment of chronic lymphocytic leukemia (CLL) within the last year. Ibrutinib and idelalisib block B-cell receptor signaling through inhibition of BTK and PI3Kδ molecules respectively, interfering with several pathways required for leukemia cell survival. Idelalisib has shown efficacy in in the relapsed setting and is currently approved for use in combination with rituximab. Ibrutinib has been studied in patients with relapsed CLL and as frontline therapy. In the relapsed setting, these agents produce durable remissions, and may be preferable to retreatment with chemoimmunotherapy for many patients. Ibrutinib is also effective treatment for patients with deletion 17p and is approved as frontline therapy in this patient group, although it does not appear to completely abrogate this adverse prognostic factor. These agents have a unique side effect profile and longer follow-up is required to further understand tolerability and rare adverse effects. Obinutuzumab is a type-2 monoclonal anti-CD20 antibody which results in direct and antibody-dependent cell-mediated cytotoxicity of leukemia cells. It is approved in combination with chlorambucil, and has shown efficacy in the frontline setting in patients unfit for more intensive chemoimmunotherapy. It produces increased response rates and minimal residual disease (MRD) negativity in comparison with chlorambucil/rituximab and is associated with an advantage in progression free survival but not yet overall survival. These agents underscore our advancement in the understanding of the biology of CLL and will improve outcomes for many patients with CLL. PMID:25817936

Glycovariant anti-CD37 monospecific protein therapeutic exhibits enhanced effector cell-mediated cytotoxicity against chronic and acute B cell malignancies

PubMed Central

Rafiq, Sarwish; Siadak, Anthony; Butchar, Jonathan P.; Cheney, Carolyn; Lozanski, Gerard; Jacob, Naduparambil K.; Lapalombella, Rosa; McGourty, Jackie; Moledor, Meghan; Lowe, Richard; Setter, Ben; Jones, Jeffrey; Flynn, Joseph M.; Andritsos, Leslie; Devine, Steven; Mo, Xiaokui; Jarjoura, David; Tridandapani, Susheela; Algate, Paul; Byrd, John C.; Muthusamy, Natarajan

2013-01-01

TRU-016 is a SMIPTM (monospecific protein therapeutic) molecule against the tetraspanin transmembrane family protein CD37 that is currently in Phase 2 trials in Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL). In an attempt to enhance the ADCC function of SMIP-016, the chimeric version of TRU-016, SMIP-016GV was engineered with a modification in a glycosylation site in the Fc domain. The wild-type and glycovariant SMIP proteins mediate comparable Type I antibody-like direct cytotoxicity in the presence of anti-human Fc crosslinker and show a similar tyrosine phosphorylation pattern post-treatment. However, NK cells stimulated with the SMIP-016GV exhibit enhanced activation and release 3-fold more interferon-γ compared with SMIP-016. SMIP-016GV shows enhanced ADCC function against cells expressing CD37 with NK cell effectors derived from both normal and CLL-affected individuals. Enhanced ADCC is observed against CLL cells and is sustained at concentrations of SMIP-016GV as low at 5E−6 µg/mL on cells expressing minimal CD37 antigen. In support of the biological relevance of this, SMIP-016GV mediates effective ADCC against primary acute lymphoblastic leukemia (ALL) cells with low surface expression of CD37. Collectively, these data suggest potential use of the novel therapeutic agent SMIP-016GV with enhanced effector function for B cell malignancies, including CLL and ALL therapy. PMID:23883821

Spectrum and immunophenotyping of 653 patients with B-cell chronic lymphoproliferative disorders in China: A single-centre analysis.

PubMed

Miao, Yi; Cao, Lei; Sun, Qian; Li, Xiao-Tong; Wang, Yan; Qiao, Chun; Wang, Li; Wang, Rong; Qiu, Hai-Rong; Xu, Wei; Li, Jian-Yong; Wu, Yu-Jie; Fan, Lei

2018-02-01

The incidence of B-cell chronic lymphoproliferative disorders (B-CLPDs) is significantly lower in China than that in western countries. There have been studies involving small cohorts with conflicting results regarding the spectrum of B-CLPDs in China, and the types and immunophenotyping of B-CLPDs in China remain largely unexplored. We conducted a retrospective analysis of 653 cases of B-CLPDs seen in our centre from 2011 to 2015. Four-colour flow cytometry was used to determine the expression of each immunological marker, and the diagnostic values of the immunological markers were also investigated. Chronic lymphocytic leukaemia (CLL) was the most common type of B-CLPD, which was consistent with that in west countries. However, the proportions of CLL (55.9%), follicular lymphoma (2.6%), and hairy cell leukaemia (0.2%) were lower, while the proportion of lymphoplasmacytic lymphoma/WaldenstrÖm macroglobulinaemia (5.4%) was higher in China, as compared with western countries. With respect to immunophenotypic characteristics, CD23 (31.7%) was more frequently expressed in mantle cell lymphoma (MCL) in our cohort than that in western countries. Immunophenotyping was useful in differentiating MCL from CLL or B-cell prolymphocytic leukaemia and lymphoplasmacytic lymphoma/WaldenstrÖm macroglobulinaemia from splenic marginal zone lymphoma. CD200 was of better diagnostic performance (accuracy: 94.6%) in differentiating CLL from MCL compared with CD23 (accuracy: 93.3%). Some cases of B-CPLDs, however, had no definite diagnoses, which were diagnosed as CD5 + B-CPLDs unclassified (7.7%) and CD5 - B-CPLDs unclassified (15.8%). This is the largest study that systematically explores the spectrum and immunophenotyping of B-CLPDs in Asia, confirming that spectrum of B-CLPDs in China was different from that in western countries. The immunophenotypic features of B-CLPDs were similar between China and western countries, although a few disparities exist. Cases with no definite

Change in IgHV Mutational Status of CLL Suggests Origin From Multiple Clones.

PubMed

Osman, Afaf; Gocke, Christopher D; Gladstone, Douglas E

2017-02-01

Fluorescence in situ hybridization and immunoglobulin (Ig) heavy-chain variable-region (IgHV) mutational status are used to predict outcome in chronic lymphocytic leukemia (CLL). Although DNA aberrations change over time, IgHV sequences and mutational status are considered stable. In a retrospective review, 409 CLL patients, between 2008 and 2015, had IgHV analysis: 56 patients had multiple analyses performed. Seven patients' IgHV results changed: 2 from unmutated to mutated and 5 from mutated to unmutated IgHV sequence. Three concurrently changed their variable heavy-chain sequence. Secondary to allelic exclusion, 2 of the new variable heavy chains produced were biologically nonplausible. The existence of these new nonplausible heavy-chain variable regions suggests either the CLL cancer stem-cell maintains the ability to rearrange a previously silenced IgH allele or more likely that the cancer stem-cell produced at least 2 subclones, suggesting that the CLL cancer stem cell exists before the process of allelic exclusion occurs. Copyright © 2016 Elsevier Inc. All rights reserved.

Resistance of cervical adenocarcinoma cells (HeLa) to venom from the scorpion Centruroides limpidus limpidus

PubMed Central

2013-01-01

Background The venom of Centruroides limpidus limpidus (Cll) is a mixture of pharmacologically active principles. The most important of these are toxic proteins that interact both selectively and specifically with different cellular targets such as ion channels. Recently, anticancer properties of the venom from other scorpion species have been described. Studies in vitro have shown that scorpion venom induces cell death, inhibits proliferation and triggers the apoptotic pathway in different cancer cell lines. Herein, after treating human cervical adenocarcinoma (HeLa) cells with Cll crude venom, their cytotoxic activity and apoptosis induction were assessed. Results Cll crude venom induced cell death in normal macrophages in a dose-dependent manner. However, through viability assays, HeLa cells showed high survival rates after exposure to Cll venom. Also, Cll venom did not induce apoptosis after performing ethidium bromide/acridine orange assays, nor was there any evidence of chromatin condensation or DNA fragmentation. Conclusions Crude Cll venom exposure was not detrimental to HeLa cell cultures. This may be partially attributable to the absence of specific HeLa cell membrane targets for molecules present in the venom of Centruroides limpidus limpidus. Although these results might discourage additional studies exploring the potential of Cll venom to treat human papilloma cervical cancer, further research is required to explore positive effects of crude Cll venom on other cancer cell lines. PMID:24004568

Erufosine, a novel alkylphosphocholine, induces apoptosis in CLL through a caspase-dependent pathway.

PubMed

Königs, Sonja Katharina; Pallasch, Christian Philipp; Lindner, Lars Hartwin; Schwamb, Janine; Schulz, Alexandra; Brinker, Reinhild; Claasen, Julia; Veldurthy, Aditya; Eibl, Hansjoerg; Hallek, Michael; Wendtner, Clemens-Martin

2010-08-01

The alkylphosphocholine (APC) erufosine is a synthetic phospholipid analogue with antineoplastic activity. APC are known to interact with lipid metabolism and modulate cellular signaling pathways, particularly the phosphorylation of Akt. Here, in primary CLL cells induction of apoptosis was detected with an IC50 of 22muM whereas healthy donor PBMC were less sensitive towards erufosine. Treatment with erufosine caused dose-dependent cleavage of PARP, co-incubation with caspase inhibitor z-VAD almost completely abrogated the cytotoxic effect of erufosine indicating a caspase-dependent mechanism of erufosine. Erufosine was shown to induce apoptosis in primary CLL cells and merits further investigation regarding therapeutic options in CLL. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1

PubMed Central

Mraz, Marek; Chen, Liguang; Rassenti, Laura Z.; Ghia, Emanuela M.; Li, Hongying; Jepsen, Kristen; Smith, Erin N.; Messer, Karen; Frazer, Kelly A.; Kipps, Thomas J.

2014-01-01

We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain–associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70–negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3′ untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome. PMID:24787006

miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1.

PubMed

Mraz, Marek; Chen, Liguang; Rassenti, Laura Z; Ghia, Emanuela M; Li, Hongying; Jepsen, Kristen; Smith, Erin N; Messer, Karen; Frazer, Kelly A; Kipps, Thomas J

2014-07-03

We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain-associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70-negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3' untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome. © 2014 by The American Society of Hematology.

Mature B-cell neoplasms in Chernobyl clean-up workers of 1986-1987: summary of cytomorphological and immunocytochemical study in 25 years after Chernobyl accident.

PubMed

Gluzman, D F; Sklyarenko, L M; Nadgornaya, V A; Zavelevich, M P

2011-03-01

The data on the verified cases of mature B-cell neoplasms (chronic lymphocytic leukemia - CLL, B-prolymphocytic leukemia, non-Hodgkin's lymphoma in leukemization phase and multiple myeloma - MM; 146 cases in total) in the consecutive group of Ukrainian clean-up workers within 10-25 years after Chernobyl accident are summarized. B-cell neoplasms represent the most prevalent group among all diagnosed neoplasms of hematopoietic and lymphoid tissues in clean-up worker patients under study (49.4%). MM percentage in the patients of Chernobyl clean-up worker group turned out to be significantly higher than in the patients of the general populations studied at the same period. While the percentage of B-CLL is similar in clean-up worker patients and patients of general population, the trend towards younger age of patients with mature B-cell neoplasms in clean-up worker group is evident. The current concepts on the possible association between mature B-cell neoplasms (mainly B-CLL) and radiation exposure are briefly outlined. Only the precise diagnosis of hematopoietic malignancies combining with large-scale analytical epidemiological studies with careful dose assessment and long-term follow-up may represent the basis for resolving the question whether mature B-cell neoplasms may be radiogenic.

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

PubMed

Fraietta, Joseph A; Beckwith, Kyle A; Patel, Prachi R; Ruella, Marco; Zheng, Zhaohui; Barrett, David M; Lacey, Simon F; Melenhorst, Jan Joseph; McGettigan, Shannon E; Cook, Danielle R; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B; Cogdill, Alexandria P; Gill, Saar; Porter, David L; Woyach, Jennifer A; Long, Meixiao; Johnson, Amy J; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L; June, Carl H; Byrd, John C; Maus, Marcela V

2016-03-03

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. © 2016 by The American Society of Hematology.

Abnormally banded chromosomal regions in doxorubicin-resistant B16-BL6 murine melanoma cells.

PubMed

Slovak, M L; Hoeltge, G A; Ganapathi, R

1986-08-01

B16-BL6 murine melanoma cells were selected for cytogenetic evaluation during the stepwise development of increasing resistance in vitro to the antitumor antibiotic, doxorubicin (DOX). Karyotypic studies demonstrated extensive heteroploidy with both numerical and structural abnormalities which were not present in the parental DOX-sensitive B16-BL6 cells. Trypsin-Giemsa banding revealed the presence of several marker chromosomes containing abnormally banding regions (ABRs) in the 44-fold B16-BL6 DOX-resistant subline. These ABRs appeared to be more homogeneously staining at the higher DOX concentrations. Length measurements (ABR index) in seven banded metaphases indicated a direct correlation with increasing DOX concentration. When the DOX-resistant cells were grown in drug-free medium for 1 yr, the drug-resistant phenotype gradually declined in parallel with the level of resistance and the ABR index. DOX-induced cytogenetic damage examined by sister chromatid exchange methodology in parental B16-BL6 cells indicated a linear sister chromatid exchange:DOX dose-response relationship. However, after continuous treatment of parental B16-BL6 cells with DOX (0.01 microgram/ml) for 30 days, sister chromatid exchange scores were found to return to base-line values. The B16-BL6 resistant cells demonstrated a cross-resistant phenotype with N-trifluoroacetyladriamycin-14-valerate, actinomycin D, and the Vinca alkaloids but not with 1-beta-D-arabinofuranosylcytosine. The results suggest that ABR-containing chromosomes in DOX-resistant sublines may represent cytogenetic alterations of specific amplified genes involved in the expression of DOX resistance. Further studies are required to identify and define the possible gene products and to correlate their relationship to the cytotoxic action of doxorubicin.

Different frequencies and effects of ABCB1 T3435C polymorphism on clinical and laboratory features of B cell chronic lymphocytic leukemia in Kurdish patients.

PubMed

Maroofi, Farzad; Amini, Sabrieh; Roshani, Daem; Ghaderi, Bayazid; Abdi, Mohammad

2015-04-01

Finding the effects of gene polymorphism on cancer pathogenesis is very desirable. The ATP-binding cassette is involved in drug metabolism, and the polymorphism of this gene may be an important risk factor in B cell chronic lymphocytic leukemia (B-CLL) or progression and/or response to chemotherapy agents. For the first time, the present study was aimed to evaluate the probable effects of ABCB1 T3435C polymorphism on clinical and laboratory features of Kurdish patients with B-CLL. This descriptive analytical case-control study was performed on 50 B-CLL patients and 100 healthy subjects. Serum levels of beta-2-microglobulin (B2M) and lactate dehydrogenase (LDH) and blood WBC, RBC, Plt and ESR were measured. The T3435C polymorphism of the ABCB1 gene was determined by PCR-RFLP. Concentration of serum and blood markers was significantly higher in the malignant group than in the benign subjects. The CC genotype had the highest frequency (66%) in the patient groups. There are no significant differences between the genotypes and type of treatment. Our results demonstrate the high frequency of C allele of ABCB1 T3435C in B-CLL patients with Kurdish ethnicity. We also show that this polymorphism has a significant risk factor in B-CLL. However, the effect of this polymorphism on clinical and laboratory characteristics of B-CLL patients was not significant.

Bruton's tyrosine kinase inhibitors in B-cell lymphoma: current experience and future perspectives.

PubMed

Seiler, T; Dreyling, M

2017-08-01

The Bruton tyrosine kinase (BTK) is a central hub in the B cell receptor (BCR) pathway and strongly influences B cell maturation, differentiation and proliferation. Not surprisingly, BTK plays an essential role in the pathogenesis of various B cell lymphomas. Inhibitors of BTK have broadened our therapeutic options in several B cell lymphomas and already are an integral element in the treatment of Mantle Cell Lymphoma (MCL), chronic lymphocytic leukemia (CLL) and Waldenström's marcoglobulinemia. Several second generation BTK inhibitors are in clinical development and might further improve tolerability and efficacy of therapy in advanced stage CLL and MCL. Areas covered: This review illustrates the mechanism of action of BTK inhibitors and provides a comprehensive summary of key clinical trials in the development of BTK inhibitors. Characteristics of second generation BTK-inhibitors are described. Expert opinion: With accumulation of clinical experience after drug approval, longer patient follow-up and larger numbers of treated patients, future development will focus on the identification of intelligent treatment combinations. Individual selection of patients with distinct biologically properties might guide treatment decisions. While BTK inhibitors are moving to earlier treatment lines, the incorporation of these drugs into a comprehensive therapeutic strategy is still difficult to date.

Severe pneumonia associated with ibrutinib monotherapy for CLL and lymphoma.

PubMed

Kreiniz, Natalia; Bejar, Jacob; Polliack, Aaron; Tadmor, Tamar

2018-02-01

In recent years, there have been major advances in the treatment of chronic lymphocytic leukemia (CLL) particularly since the development of novel therapeutic agents, mostly "biological drugs." One of the obvious advantages of these agents is the decreased rate of infectious complications occurring during the course of therapy, compared to the use of standard immuno-chemotherapy regimens. Here, we describe 3 patients with CLL and 1 with mantle cell lymphoma who developed severe life-threatening pneumonias, during monotherapy with ibrutinib. The first case was a 70-year-old woman with relapsed CLL who developed bilateral pneumonia with hypoxia 1 week after starting ibrutinib. She did not respond to broad-spectrum antibiotics and was treated empirically with trimethoprim-sulphamethoxazole and improved. In the second case, we describe a 76-year-old woman with relapsed CLL who developed recurrent pneumonia after 3 years of treatment with ibrutinib. Presuming that ibrutinib was the cause of pneumonitis with secondary infection, it was stopped with subsequent improvement. The third patient a 67 year-old man died because of severe bilateral necrotizing pneumonia due to invasive aspergillosis and mucormycosis with pulmonary hemorrhage. The fourth patient with relapsed mantle cell lymphoma died because of severe bilateral pneumonia, caused by pseudomonas and candida, despite receiving appropriate antibiotics. From this experience, we hypothesize that the etiology of severe pneumonia associated with ibrutinib treatment is probably multifactorial, involving factors like preexisting immune-suppression, drug induced pneumonitis and infections. We suggest that patients with CLL or other lymphoproliferative disorders with suspected pneumonia during monotherapy with ibrutinib should be very carefully evaluated and need to undergo complete diagnostic workup to establish an exact diagnosis. Understanding which patients with CLL or lymphoma treated with kinase inhibitors are at a

Lactoferricin B reverses cisplatin resistance in head and neck squamous cell carcinoma cells through targeting PD-L1.

PubMed

Zhang, Pei; Liu, Jinzhong; Li, Wenlu; Li, Shanshan; Han, Xinguang

2018-05-15

Head and neck squamous cell carcinoma (HNSCC) ranks among the top most common cancers with a poor prognosis. The mechanism of chemoresistance is still not well known. This study is to investigate the programmed death-ligand 1 (PD-L1) expression in HNSCC, and test the effect of lactoferricin B (LfcinB) on chemoresistance and its mechanism. We analyzed 510 HNSCC patients in TCGA database and investigated how CD274 expression was related to patient prognosis. PD-L1 was verified from HNSCC samples at local hospital with immunohistochemistry. PD-L1 expression in the acquired cisplatin-resistant HNSCC cells was examined by PCR and WB in order to test PD-L1-induced chemoresistance. LfcinB inoculation in cisplatin-resistant HNSCC cells and in the nude mice was introduced to test the effect of LfcinB on targeting cisplatin resistance and its mechanism. High CD274 mRNA (>125 FPKM) from TCGA database had a significantly reduced 5-year survival rate, and a lower 5-year survival rate in the chemotherapy and radiotherapy-treated patients (P < .05). PD-L1 overexpression was further supported from analysis of 40 HNSCC specimens. PD-L1 and IL-6 in the established cisplatin-resistant HNSCC cells were shown significantly higher (P < .05). IL-6 and PD-L1 expression were partially inhibited by the anti-IL-6/STAT3 antibody. LfcinB displayed a direct cytotoxic effect on cisplatin-resistant HNSCC cells and HNSCC xenografts of cisplatin-resistant cells in the nude mice displayed significant reduction in tumor volume after LfcinB injection (P < .05). Besides, the increase of IL-6 and PD-L1 in cisplatin-resistant HNSCC cells was abolished in vitro by LfcinB (P < .05). PD-L1 expression in HNSCC cells correlates with poor prognosis and chemoresistance, and LfcinB might provide therapeutic potential in HNSCC patients through modulating IL-6 and PD-L1. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

PubMed

Gautam, S C; Chikkala, N F; Lewis, I; Grabowski, D R; Finke, J H; Ganapathi, R

1992-01-01

Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.

Partial versus Productive Immunoglobulin Heavy Locus Rearrangements in Chronic Lymphocytic Leukemia: Implications for B-Cell Receptor Stereotypy

PubMed Central

Tsakou, Eugenia; Agathagelidis, Andreas; Boudjoghra, Myriam; Raff, Thorsten; Dagklis, Antonis; Chatzouli, Maria; Smilevska, Tatjana; Bourikas, George; Merle-Beral, Helene; Manioudaki-Kavallieratou, Eleni; Anagnostopoulos, Achilles; Brüggemann, Monika; Davi, Frederic; Stamatopoulos, Kostas; Belessi, Chrysoula

2012-01-01

The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level. PMID:21968789

Remission induction using alemtuzumab can permit chemotherapy-refractory chronic lymphocytic leukemia (CLL) patients to undergo allogeneic stem cell transplantation.

PubMed

Knauf, Wolfgang; Rieger, Kathrin; Blau, Wolfgang; Hegenbart, Ute; Von Gruenhagen, Ulrich; Niederwieser, Dietger; Thiel, Eckhard

2004-12-01

The outcome of allogeneic stem cell transplantation depends upon the disease status before transplantation. Patients with refractory disease are at high risk for relapse. To improve the curative potential of the transplant procedure, we treated 3 chemotherapy-refractory CLL patients with alemtuzumab before allogeneic stem cell transplantation. Prior to therapy, all patients suffered from B-symptoms, and had massive adenopathy, splenomegaly, thrombocytopenia, and anemia; two patients had hepatomegaly. Alemtuzumab greatly reduced tumor mass in blood and bone marrow, B-symptoms resolved, and organomegaly improved. Two patients became blood product independent. All patients proceeded to transplantation after conditioning with TBI 2 Gy (n=1) or Treosulfan (n=2) in combination with Fludarabine either from an HLA-matched sibling (n=2) or from an HLA-matched unrelated donor (n=1). All patients engrafted, and are alive and well. Two patients reached complete remission (CR); one patient attained stable partial remission (PR). These heavily pre-treated refractory patients gained substantial clinical benefit from alemtuzumab, and received successful allografts.

Targeting malignant B cells with an immunotoxin against ROR1

PubMed Central

Baskar, Sivasubramanian; Wiestner, Adrian; Wilson, Wyndham H.; Pastan, Ira; Rader, Christoph

2012-01-01

The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the VH and VL fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC50 = 16 pM–16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers. PMID:22531447

Cytological Diagnosis of Small Cell Carcinoma of Urinary Bladder in a Patient with CLL

PubMed Central

Şimşek, Gülçin Güler; Güreşçi, Servet; Oğuz, Ural; Ünsal, Ali

2014-01-01

Small cell carcinoma of the urinary bladder (SCCUB) is an extremely rare bladder malignancy characterized by an aggressive clinical behavior. So, it is important to diagnose this high grade disease by urinary cytology. We report a case of SCCUB in an old man with chronic lymphocytic leukemia (CLL) in remission, while bladder tumor was diagnosed by cytology. With this article, we aimed to review and to update the literature concerning this tumor. PMID:24518979

Methylenetetrahydrofolate reductase (MTHFR) gene 677C>T and 1298A>C polymorphisms are associated with differential apoptosis of leukemic B cells in vitro and disease progression in chronic lymphocytic leukemia.

PubMed

Nückel, H; Frey, U H; Dürig, J; Dührsen, U; Siffert, W

2004-11-01

Methylenetetrahydrofolate reductase (MTHFR) regulates the metabolism of folate and methionine, essential components of DNA synthesis and methylation. We investigated whether the two genetic MTHFR polymorphisms (677C>T and 1298A>C) are associated with an increased risk for chronic lymphocytic leukemia (CLL) or may predict disease progression. Moreover, we measured potential genotype effects on apoptosis of B-CLL cells.Allele frequencies and genotype distributions for both polymorphisms were not significantly different in 111 patients vs 92 healthy controls. While progression-free survival (PFS) was not significantly different in individuals with CLL including all stages, in patients with Binet stage A PFS was significantly longer in patients displaying the MTHFR 677CC (P=0.043) and the MTHFR 1298A/C or CC genotypes (P=0.019). In a multivariate analysis, MTHFR haplotype (677CC plus 1298CC or A/C) was the best independent prognostic factor for PFS compared with other known prognostic factors. Spontaneous apoptosis of B-CLL cells in vitro was significantly increased in the favorable risk group with MTHFR 677CC and MTHFR 1298AC, which may constitute the cellular basis of the observed associations. While MTHFR polymorphisms do not affect the risk for B-CLL, they may be independent prognostic markers that influence the PFS in patients with early-stage B-CLL.

Evolving Understanding of the CLL Genome

PubMed Central

Gruber, Michaela; Wu, Catherine J.

2014-01-01

Over the past few years, massively parallel sequencing technologies have revealed with high resolution the tremendous genetic and epigenetic heterogeneity in chronic lymphocytic leukemia (CLL). We have learned how the molecular architecture differs not only between affected individuals but also within samples and over time. These insights have catalyzed our understanding of the pathobiology of CLL and point to critical signaling pathways in the development and progression of the disease. Several key driver alterations have been identified, which serve to refine prognostic schemata but also inspire the development of new therapeutic strategies. Ongoing advances in technology promise to further elucidate the molecular basis of CLL, and this knowledge is anticipated to aid us in understanding and addressing the clinical challenge presented by the vast variability in the clinical course of patients with CLL. PMID:25048782

Host virus and pneumococcus-specific immune responses in high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia: implications for disease progression

PubMed Central

Criado, Ignacio; Muñoz-Criado, Santiago; Rodríguez-Caballero, Arancha; Nieto, Wendy G.; Romero, Alfonso; Fernández-Navarro, Paulino; Alcoceba, Miguel; Contreras, Teresa; González, Marcos; Orfao, Alberto; Almeida, Julia

2017-01-01

Patients diagnosed with chronic lymphocytic leukemia (CLL) display a high incidence of infections due to an associated immunodeficiency that includes hypogammaglobulinemia. A higher risk of infections has also been recently reported for high-count monoclonal B-cell lymphocytosis, while no information is available in low-count monoclonal B-cell lymphocytosis. Here, we evaluated the status of the humoral immune system in patients with chronic lymphocytic leukemia (n=58), as well as in low- (n=71) and high- (n=29) count monoclonal B-cell lymphocytosis versus healthy donors (n=91). Total free plasma immunoglobulin titers and specific levels of antibodies against cytomegalovirus, Epstein-Barr virus, influenza and S.pneumoniae were measured by nephelometry and ELISA-based techniques, respectively. Overall, our results show that both CLL and high-count monoclonal B-cell lymphocytosis patients, but not low-count monoclonal B-cell lymphocytosis subjects, present with relatively high levels of antibodies specific for the latent viruses investigated, associated with progressively lower levels of S.pneumoniae-specific immunoglobulins. These findings probably reflect asymptomatic chronic reactivation of humoral immune responses against host viruses associated with expanded virus-specific antibody levels and progressively decreased protection against other micro-organisms, denoting a severe humoral immunodeficiency state not reflected by the overall plasma immunoglobulin levels. Alternatively, these results could reflect a potential role of ubiquitous viruses in the pathogenesis of the disease. Further analyses are necessary to establish the relevance of such asymptomatic humoral immune responses against host viruses in the expansion of the tumor B-cell clone and progression from monoclonal B-cell lymphocytosis to CLL. PMID:28385786

STAT3-activated CD36 facilitates fatty acid uptake in chronic lymphocytic leukemia cells

PubMed Central

Rozovski, Uri; Harris, David M.; Li, Ping; Liu, Zhiming; Jain, Preetesh; Ferrajoli, Alessandra; Burger, Jan; Thompson, Phillip; Jain, Nitin; Wierda, William; Keating, Michael J.; Estrov, Zeev

2018-01-01

Although several studies established that unlike normal B cells chronic lymphocytic leukemia (CLL) cells metabolize fatty acids (FA), how CLL cells internalize FA is poorly understood. Because in various cell types CD36 facilitates FA uptake, we wondered whether a similar mechanism is operative CLL. We found that CD36 levels are higher in CLL cells than in normal B cells, and that small interfering RNA, CD36 neutralizing antibodies or sulfosuccinimidyl oleate (SSO) that inhibits CD36 significantly reduced the oxygen consumption of CLL cells incubated with FA. Because CD36 is oeverexpressed and STAT3 is constitutively activated in CLL cells, we wondered whether STAT3 induces CD36 expression. Sequence analysis identified putative STAT3 binding sites in the CD36 gene promoter. Chromatin immunoprecipitation and an electrophoretic mobility shift assay revealed that STAT3 binds to the CD36 gene promoter. A luciferase assay and STAT3-small hairpin RNA, that significantly decreased the levels of CD36 in CLL cells, established that STAT3 activates the transcription of the CD36 gene. Furthermore, SSO induced a dose-dependent apoptosis of CLL cells. Taken together, our data suggest that STAT3 activates CD36 and that CD36 facilitates FA uptake in CLL cells. Whether CD36 inhibition would provide clinical benefits in CLL remains to be determined. PMID:29765537

Low CLL-1 Expression Is a Novel Adverse Predictor in 123 Patients with De Novo CD34+ Acute Myeloid Leukemia.

PubMed

Wang, Yan-Yu; Chen, Wen-Lian; Weng, Xiang-Qin; Sheng, Yan; Wu, Jing; Hao, Jie; Liu, Zhan-Yun; Zhu, Yong-Mei; Chen, Bing; Xiong, Shu-Min; Chen, Yu; Chen, Qiu-Sheng; Sun, Hui-Ping; Li, Jun-Min; Wang, Jin

2017-10-15

Recent reports state that C-type lectin-like molecule-1 (CLL-1) in acute myeloid leukemia (AML) is expressed primarily on myeloid cells, but there is still no investigation about its prognostic significance on leukemic blast compartment. Hence, this study aimed to evaluate the prognostic value of CLL-1 in 123 patients with de novo CD34 + Non-M3 AML. Multiparameter flow cytometry was used to assess the expression of CLL-1 on immature compartment in AML and control groups. We found that CLL-1 expression level on blast compartment was closely linked to clinical characteristics, treatment response, and survival outcome of patients. Decreased expression of CLL-1 was observed on immature compartment from AML patients as compared with controls (62.6% vs. 86.5%, P < 0.05). Logistic model exhibited that CLL-1 low independently predicted low complete remission rate with an odds ratio of 4.57 (2.53-6.61, P < 0.05). Additionally, CLL-1 expression level at diagnosis was inversely correlated to the residual blast cells (residual leukemia cell) after induction chemotherapy (r = -0.423, P < 0.05). Furthermore, multivariate Cox regression model demonstrated that CLL-1 low was still an independent adverse predictor (P < 0.05 for event-free survival, P < 0.05 for overall survival). Notably, CLL-1 low was able to discriminate poor survival patients from intermediate- and favorable-risk groups. Taken together, CLL-1 is a novel prognostic predictor that could be exploited to supplement the current AML prognostic risk stratification system, and potentially optimize the clinical management of AML.

Improved detection rate of cytogenetic abnormalities in chronic lymphocytic leukemia and other mature B-cell neoplasms with use of CpG-oligonucleotide DSP30 and interleukin 2 stimulation.

PubMed

Shi, Min; Cipollini, Matthew J; Crowley-Bish, Patricia A; Higgins, Anne W; Yu, Hongbo; Miron, Patricia M

2013-05-01

Detection of cytogenetic abnormalities requires successful culture of the clonal population to obtain metaphase chromosomes for study, and as such, has been hampered by low mitotic indices of mature B cells in culture. Our study presents data on the improved abnormality detection rate with the use of a CpG-oligonucleotide/interleukin 2 (OL/IL-2) culture protocol for mature B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and non-CLL specimens. The increased detection rate of abnormalities, compared with unstimulated culture and traditional pokeweed mitogen culture, was statistically significant for both CLL and non-CLL neoplasms. For CLL specimens, our data also showed that for cytogenetically visible aberrations, OL/IL-2 was as, if not more, sensitive than detection with interphase fluorescence in situ hybridization (iFISH). Use of OL/IL-2 allowed a number of abnormalities to be detected, which were not covered by specific iFISH panels, especially balanced translocations. Therefore, OL/IL-2 stimulation improves diagnostic sensitivity and increases discovery rate of novel prognostic findings.

The establishment of insulin resistance model in FL83B and L6 cell

NASA Astrophysics Data System (ADS)

Liu, Lanlan; Han, Jizhong; Li, Haoran; Liu, Mengmeng; Zeng, Bin

2017-10-01

The insulin resistance models of mouse liver epithelial and rat myoblasts cells were induced by three kinds of inducers: dexamethasone, high insulin and high glucose. The purpose is to select the optimal insulin resistance model, to provide a simple and reliable TR cell model for the study of the pathogenesis of TR and the improvement of TR drugs and functional foods. The MTT method is used for toxicity screening of three compounds, selecting security and suitable concentration. We performed a Glucose oxidase peroxidase (GOD-POD) method involving FL83B and L6 cell with dexamethasone, high insulin and high glucose-induced insulin resistance. Results suggested that FL83B cells with dexamethasone-induced (0.25uM) were established insulin resistance and L6 cells with high-glucose (30mM) and dexamethasone-induced (0.25uM) were established insulin resistance.

Allogeneic hematopoietic stem cell transplantation for poor-risk CLL: dissecting immune-modulating strategies for disease eradication and treatment of relapse.

PubMed

Hahn, M; Böttcher, S; Dietrich, S; Hegenbart, U; Rieger, M; Stadtherr, P; Bondong, A; Schulz, R; Ritgen, M; Schmitt, T; Tran, T H; Görner, M; Herth, I; Luft, T; Schönland, S; Witzens-Harig, M; Zenz, T; Kneba, M; Ho, A D; Dreger, P

2015-10-01

To elucidate factors contributing to the effectiveness of allogeneic hematopoietic stem cell transplantation (alloHCT) in high-risk CLL, immune interventions, GvHD and clinical outcome of 77 consecutive patients allografted for CLL were analyzed. Immune modulation (immunosuppression tapering, rituximab-augmented donor lymphocyte infusions) was guided by minimal residual disease (MRD) monitoring and commenced at a median of 91 (22-273) days after alloHCT, resulting in a probability of being event free and MRD-negative 1 year after transplant of 57% (84% in those encountering chronic GvHD). Patients who were event free and MRD-negative at the 12-month landmark had a 4-year PFS of 77% and largely remained durably MRD-negative if MRD clearance had occurred subsequent to immune modulation. Three-year overall survival, PFS, relapse incidence and non-relapse mortality of all 77 patients were 69, 57, 26 and 24%, respectively. Survival was not affected by EBMT risk category but by active disease at alloHCT, which could not be overcome by intensification of conditioning. Twenty-three patients who experienced relapse post alloHCT had a survival of 56% at 2 years after CLL recurrence. In conclusion, MRD-guided immune modulation after alloHCT for high-risk CLL can provide durable MRD clearance in more than half of the patients.

Pentostatin and Rituximab Therapy for Previously Untreated B-CLL

PubMed Central

Kay, Neil E.; Wu, Wenting; Kabat, Brian; LaPlant, Betsy; Lin, Thomas S.; Byrd, John C.; Jelinek, Diane F.; Grever, Michael R.; Zent, Clive S.; Call, Timothy G.; Shanafelt, Tait D.

2010-01-01

We have shown that the combination of pentostatin (P), cyclophosphamide (C) and rituximab (R) achieves an overall response (OR) rate >90% with more than 40% complete responses (CR) in patients with untreated CLL. To evaluate if the tolerability of this regimen could be enhanced without sacrificing efficacy, we conducted a phase II trial of P and R without cyclophosphamide, using a higher P dose (4 mg/m2). Among the 33 patients enrolled, 82% were male, median age was 65 (9 patients ≥70 years) and 64% were Rai stage III-IV. The OR rate was 76% with 9 CR (27%), 5 nPR, and 11 PRs. At the time of this analysis, 29/33 patients are still alive and the median follow up for patients still alive is 14 months (range: 1-34.8 months). Four (12%) patients experienced grade 3 or higher hematologic toxicity and 5 (15%) experienced grade 3 or higher non-hematologic toxicity. Comparison of this trial to our previous PCR trial showed that patients treated with PCR had a higher OR rate (91% vs. 76%) and CR rate (41% vs. 27%) compared to patients treated with PR. Median treatment-free survival for all accrued patients was notably longer in PCR treated patients compared to PR (30 vs. 16 months). These findings suggest that increasing the dose of the purine nucleoside analogue does not eliminate the need for cyclophosphamide in chemoimmunotherapy for treatment of CLL. PMID:20187101

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells.

PubMed

Benakanakere, Indira; Johnson, Tyler; Sleightholm, Richard; Villeda, Virgilio; Arya, Monika; Bobba, Ravi; Freter, Carl; Huang, Chunfa

2014-01-01

Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL). MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment.

GIMEMA CLL0809 Study (BendOfa)

ClinicalTrials.gov

2015-01-29

Patients With CLL Relapsing After an Initial Response (CR, PR ≥ 6 Months) Following no More Than Two Prior Treatment Lines; or; Patients With CLL Refractory (SD, PD or CR/PR < 6 Months) Following no More Than Two Prior Treatment Lines

A phase 1 clinical trial of the selective BTK inhibitor ONO/GS-4059 in relapsed and refractory mature B-cell malignancies.

PubMed

Walter, Harriet S; Rule, Simon A; Dyer, Martin J S; Karlin, Lionel; Jones, Ceri; Cazin, Bruno; Quittet, Philippe; Shah, Nimish; Hutchinson, Claire V; Honda, Hideyuki; Duffy, Kevin; Birkett, Joseph; Jamieson, Virginia; Courtenay-Luck, Nigel; Yoshizawa, Toshio; Sharpe, John; Ohno, Tomoya; Abe, Shinichiro; Nishimura, Akihisa; Cartron, Guillaume; Morschhauser, Franck; Fegan, Christopher; Salles, Gilles

2016-01-28

We report the results of a multicenter phase 1 dose-escalation study of the selective Bruton tyrosine kinase (BTK) inhibitor ONO/GS-4059 in 90 patients with relapsed/refractory B-cell malignancies. There were 9 dose-escalation cohorts ranging from 20 mg to 600 mg once daily with twice-daily regimens of 240 mg and 300 mg. Twenty-four of 25 evaluable chronic lymphocytic leukemia (CLL) patients (96%) responded to ONO/GS-4059, with a median treatment duration of 80 weeks; 21 CLL patients remain on treatment. Lymph node responses were rapid and associated with a concurrent lymphocytosis. Eleven of 12 evaluable patients with mantle cell lymphoma (92%) responded (median treatment duration, 40 weeks). Eleven of 31 non-germinal center B-cell diffuse large B-cell lymphoma patients (35%) responded but median treatment duration was 12 weeks due to development of progressive disease. ONO/GS-4059 was very well tolerated with 75% of adverse events (AEs) being Common Toxicity Criteria for Adverse Events version 4.0 grade 1 or grade 2. Grade 3/4 AEs were mainly hematologic and recovered spontaneously during therapy. One CLL patient experienced a grade 3 treatment-related bleeding event (spontaneous muscle hematoma) but no clinically significant diarrhea, cardiac dysrhythmias, or arthralgia were observed. No maximal tolerated dose (MTD) was reached in the CLL cohort. In the non-Hodgkin lymphoma cohort, 4 patients developed a dose-limiting toxicity, yielding an MTD of 480 mg once daily. ONO/GS-4059 has significant activity in relapsed/refractory B-cell malignancies without major drug-related toxicity. The selectivity of ONO/GS-4059 should confer advantages in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT01659255. © 2016 by The American Society of Hematology.

Cell-intrinsic determinants of ibrutinib-induced apoptosis in Chronic Lymphocytic Leukemia

PubMed Central

Amin, Nisar A.; Balasubramanian, Sriram; Saiya-Cork, Kamlai; Shedden, Kerby; Hu, Nan; Malek, Sami N.

2016-01-01

Purpose Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed CLL and CLL with del17p. Mechanistically, ibrutinib interferes with BCR signaling as well as multiple CLL cell to microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted. Experimental Design We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 μM – 5 μM of ibrutinib ex vivo and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted TP53 in HG3, PGA1 and PG-EBV cell lines and measured BCR signaling and ibrutinib responses. Results CLL samples demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib with IC50 values ranging from 0.4 μM – 9.7 μM. Unmutated IGVH status, elevated ZAP70 expression and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/TP53 mutated status. A validation sample of 15 CLL carrying TP53 mutations, of which 13 carried both del17p and a TP53 mutation confirmed substantially less sensitivity to ibrutinib-induced apoptosis. Conclusions This study identifies that CLL harboring del17p/TP53 mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/TP53 wild type cells. PMID:27535981

Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project.

PubMed

Rawstron, Andy C; Kreuzer, Karl-Anton; Soosapilla, Asha; Spacek, Martin; Stehlikova, Olga; Gambell, Peter; McIver-Brown, Neil; Villamor, Neus; Psarra, Katherina; Arroz, Maria; Milani, Raffaella; de la Serna, Javier; Cedena, M Teresa; Jaksic, Ozren; Nomdedeu, Josep; Moreno, Carol; Rigolin, Gian Matteo; Cuneo, Antonio; Johansen, Preben; Johnsen, Hans E; Rosenquist, Richard; Niemann, Carsten Utoft; Kern, Wolfgang; Westerman, David; Trneny, Marek; Mulligan, Stephen; Doubek, Michael; Pospisilova, Sarka; Hillmen, Peter; Oscier, David; Hallek, Michael; Ghia, Paolo; Montserrat, Emili

2018-01-01

The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5-20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for "required" diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. "Recommended" markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as "required" for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus "recommended" panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society. © 2017 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

IgV gene intraclonal diversification and clonal evolution in B-cell chronic lymphocytic leukaemia.

PubMed

Bagnara, Davide; Callea, Vincenzo; Stelitano, Caterina; Morabito, Fortunato; Fabris, Sonia; Neri, Antonino; Zanardi, Sabrina; Ghiotto, Fabio; Ciccone, Ermanno; Grossi, Carlo Enrico; Fais, Franco

2006-04-01

Intraclonal diversification of immunoglobulin (Ig) variable (V) genes was evaluated in leukaemic cells from a B-cell chronic lymphocytic leukaemia (B-CLL) case over a 2-year period at four time points. Intraclonal heterogeneity was analysed by sequencing 305 molecular clones derived from polymerase chain reaction amplification of B-CLL cell IgV heavy (H) and light (C) chain gene rearrangements. Sequences were compared with evaluating intraclonal variation and the nature of somatic mutations. Although IgV intraclonal variation was detected at all time points, its level decreased with time and a parallel emergence of two more represented V(H)DJ(H) clones was observed. They differed by nine nucleotide substitutions one of which only caused a conservative replacement aminoacid change. In addition, one V(L)J(L) rearrangement became more represented over time. Analyses of somatic mutations suggest antigen selection and impairment of negative selection of neoplastic cells. In addition, a genealogical tree representing a model of clonal evolution of the neoplastic cells was created. It is of note that, during the period of study, the patient showed clinical progression of disease. We conclude that antigen stimulation and somatic hypermutation may participate in disease progression through the selection and expansion of neoplastic subclone(s).

BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells.

PubMed

Guarini, Anna; Chiaretti, Sabina; Tavolaro, Simona; Maggio, Roberta; Peragine, Nadia; Citarella, Franca; Ricciardi, Maria Rosaria; Santangelo, Simona; Marinelli, Marilisa; De Propris, Maria Stefania; Messina, Monica; Mauro, Francesca Romana; Del Giudice, Ilaria; Foà, Robert

2008-08-01

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia.

PubMed

Leong, Steven R; Sukumaran, Siddharth; Hristopoulos, Maria; Totpal, Klara; Stainton, Shannon; Lu, Elizabeth; Wong, Alfred; Tam, Lucinda; Newman, Robert; Vuillemenot, Brian R; Ellerman, Diego; Gu, Chen; Mathieu, Mary; Dennis, Mark S; Nguyen, Allen; Zheng, Bing; Zhang, Crystal; Lee, Genee; Chu, Yu-Waye; Prell, Rodney A; Lin, Kedan; Laing, Steven T; Polson, Andrew G

2017-02-02

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML. © 2017 by The American Society of Hematology.

Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression.

PubMed

Dicker, Frank; Schnittger, Susanne; Haferlach, Torsten; Kern, Wolfgang; Schoch, Claudia

2006-11-01

Compared with fluorescence in situ hybridization (FISH), conventional metaphase cytogenetics play only a minor prognostic role in chronic lymphocytic leukemia (CLL) so far, due to technical problems resulting from limited proliferation of CLL cells in vitro. Here, we present a simple method for in vitro stimulation of CLL cells that overcomes this limitation. In our unselected patient population, 125 of 132 cases could be successfully stimulated for metaphase generation by culture with the immunostimulatory CpG-oligonucleotide DSP30 plus interleukin 2. Of 125 cases, 101 showed chromosomal aberrations. The aberration rate is comparable to the rate detected by parallel interphase FISH. In 47 patients, conventional cytogenetics detected additional aberrations not detected by FISH analysis. A complex aberrant karyotype, defined as one having at least 3 aberrations, was detected in 30 of 125 patients, compared with only one such case as defined by FISH. Conventional cytogenetics frequently detected balanced and unbalanced translocations. A significant correlation of the poor-prognosis unmutated IgV(H) status with unbalanced translocations and of the likewise poor-prognosis CD38 expression to balanced translocations and complex aberrant karyotype was found. We demonstrate that FISH analysis underestimates the complexity of chromosomal aberrations in CLL. Therefore, conventional cytogenetics may define subgroups of patients with high risk of progression.

Association of polygenic risk score with the risk of chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis.

PubMed

Kleinstern, Geffen; Camp, Nicola J; Goldin, Lynn R; Vachon, Celine M; Vajdic, Claire M; de Sanjose, Silvia; Weinberg, J Brice; Benavente, Yolanda; Casabonne, Delphine; Liebow, Mark; Nieters, Alexandra; Hjalgrim, Henrik; Melbye, Mads; Glimelius, Bengt; Adami, Hans-Olov; Boffetta, Paolo; Brennan, Paul; Maynadie, Marc; McKay, James; Cocco, Pier Luigi; Shanafelt, Tait D; Call, Timothy G; Norman, Aaron D; Hanson, Curtis; Robinson, Dennis; Chaffee, Kari G; Brooks-Wilson, Angela R; Monnereau, Alain; Clavel, Jacqueline; Glenn, Martha; Curtin, Karen; Conde, Lucia; Bracci, Paige M; Morton, Lindsay M; Cozen, Wendy; Severson, Richard K; Chanock, Stephen J; Spinelli, John J; Johnston, James B; Rothman, Nathaniel; Skibola, Christine F; Leis, Jose F; Kay, Neil E; Smedby, Karin E; Berndt, Sonja I; Cerhan, James R; Caporaso, Neil; Slager, Susan L

2018-06-07

Inherited loci have been found to be associated with risk of chronic lymphocytic leukemia (CLL). A combined polygenic risk score (PRS) of representative single nucleotide polymorphisms (SNPs) from these loci may improve risk prediction over individual SNPs. Herein, we evaluated the association of a PRS with CLL risk and its precursor, monoclonal B-cell lymphocytosis (MBL). We assessed its validity and discriminative ability in an independent sample and evaluated effect modification and confounding by family history (FH) of hematological cancers. For discovery, we pooled genotype data on 41 representative SNPs from 1499 CLL and 2459 controls from the InterLymph Consortium. For validation, we used data from 1267 controls from Mayo Clinic and 201 CLL, 95 MBL, and 144 controls with a FH of CLL from the Genetic Epidemiology of CLL Consortium. We used odds ratios (ORs) to estimate disease associations with PRS and c-statistics to assess discriminatory accuracy. In InterLymph, the continuous PRS was strongly associated with CLL risk (OR, 2.49; P = 4.4 × 10 -94 ). We replicated these findings in the Genetic Epidemiology of CLL Consortium and Mayo controls (OR, 3.02; P = 7.8 × 10 -30 ) and observed high discrimination (c-statistic = 0.78). When jointly modeled with FH, PRS retained its significance, along with FH status. Finally, we found a highly significant association of the continuous PRS with MBL risk (OR, 2.81; P = 9.8 × 10 -16 ). In conclusion, our validated PRS was strongly associated with CLL risk, adding information beyond FH. The PRS provides a means of identifying those individuals at greater risk for CLL as well as those at increased risk of MBL, a condition that has potential clinical impact beyond CLL.

Safety and activity of the anti-CD79B antibody-drug conjugate polatuzumab vedotin in relapsed or refractory B-cell non-Hodgkin lymphoma and chronic lymphocytic leukaemia: a phase 1 study.

PubMed

Palanca-Wessels, Maria Corinna A; Czuczman, Myron; Salles, Gilles; Assouline, Sarit; Sehn, Laurie H; Flinn, Ian; Patel, Manish R; Sangha, Randeep; Hagenbeek, Anton; Advani, Ranjana; Tilly, Herve; Casasnovas, Olivier; Press, Oliver W; Yalamanchili, Sreeni; Kahn, Robert; Dere, Randall C; Lu, Dan; Jones, Surai; Jones, Cheryl; Chu, Yu-Waye; Morschhauser, Franck

2015-06-01

Patients with relapsed or refractory B-cell non-Hodgkin lymphoma (NHL) have an unfavourable prognosis with few treatment options. Polatuzumab vedotin is an antibody-drug conjugate containing an anti-CD79B monoclonal antibody conjugated to the microtubule-disrupting agent monomethyl auristatin E. We aimed to assess the safety and clinical activity of polatuzumab vedotin in relapsed or refractory B-cell NHL and chronic lymphocytic leukaemia (CLL). In this phase 1, multicentre, open-label study, we enrolled patients with documented NHL or CLL expected to express CD79B (confirmation of CD79B expression was not required) and for whom no suitable therapy of curative intent or higher priority existed from 13 centres. The primary endpoints of the study were to assess safety and tolerability, determine the maximum tolerated dose, and identify the recommended phase 2 dose of polatuzumab vedotin as a single agent and in combination with rituximab. A 3 + 3 dose-escalation design was used in which we treated patients with polatuzumab vedotin (0·1-2·4 mg/kg every 21 days) in separate dose-escalation cohorts for NHL and CLL. After determination of the recommended phase 2 dose, we enrolled patients with relapsed or refractory diffuse large B-cell lymphoma and relapsed or refractory indolent NHL into indication-specific cohorts. We also enrolled patients with relapsed or refractory NHL into an additional cohort to assess the feasibility of the combination of polatuzumab vedotin and rituximab 375 mg/m(2). Patients who received any dose of polatuzumab vedotin were available for safety analyses. This study is registered with ClinicalTrials.gov, number NCT01290549. Between March 21, 2011, and Nov 30, 2012, we enrolled 95 patients (34 to the NHL dose-escalation cohort, 18 to the CLL dose-escalation cohort, 34 with NHL to the expansion cohort at the recommended phase 2 dose, and nine with NHL to the rituximab combination cohort; no expansion cohort of CLL was started due to lack of

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells

PubMed Central

2014-01-01

Background Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL). Methods MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. Results We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Conclusion Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment. PMID:25401046

Strategic Therapeutic Targeting to Overcome Venetoclax Resistance in Aggressive B-cell Lymphomas.

PubMed

Pham, Lan V; Huang, Shengjian; Zhang, Hui; Zhang, Jun; Bell, Taylor; Zhou, Shouhao; Pogue, Elizabeth; Ding, Zhiyong; Lam, Laura; Westin, Jason; Davis, R Eric; Young, Ken H; Medeiros, L Jeffrey; Ford, Richard J; Nomie, Krystle; Zhang, Leo; Wang, Michael

2018-04-17

Purpose: B-cell lymphoma-2 (BCL-2), an antiapoptotic protein often dysregulated in B-cell lymphomas, promotes cell survival and provides protection from stress. A recent phase I first-in-human study of the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma showed an overall response rate of 44%. These promising clinical results prompted our examination of the biological effects and mechanism of action underlying venetoclax activity in aggressive B-cell lymphoma, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Experimental Design: MCL and DLBCL cell lines, primary patient samples, and in vivo patient-derived xenograft (PDX) models were utilized to examine venetoclax efficacy. Furthermore, the mechanisms underlying venetoclax response and the development of venetoclax resistance were evaluated using proteomics analysis and Western blotting. Results: Potential biomarkers linked to venetoclax activity and targeted combination therapies that can augment venetoclax response were identified. We demonstrate that DLBCL and MCL cell lines, primary patient samples, and PDX mouse models expressing high BCL-2 levels are extremely sensitive to venetoclax treatment. Proteomics studies showed that venetoclax substantially alters the expression levels and phosphorylation status of key proteins involved in cellular processes, including the DNA damage response, cell metabolism, cell growth/survival, and apoptosis. Short- and long-term exposure to venetoclax inhibited PTEN expression, leading to enhanced AKT pathway activation and concomitant susceptibility to PI3K/AKT inhibition. Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K/AKT inhibition. Conclusions: These findings demonstrate the on-target effect of venetoclax and offer potential mechanisms to overcome acquired and intrinsic venetoclax resistance through PI3K/AKT inhibition. Clin Cancer Res; 1-14. ©2018 AACR. ©2018 American Association for

The Phospholipase Cγ2 Mutants R665W and L845F Identified in Ibrutinib-resistant Chronic Lymphocytic Leukemia Patients Are Hypersensitive to the Rho GTPase Rac2 Protein.

PubMed

Walliser, Claudia; Hermkes, Elisabeth; Schade, Anja; Wiese, Sebastian; Deinzer, Julia; Zapatka, Marc; Désiré, Laurent; Mertens, Daniel; Stilgenbauer, Stephan; Gierschik, Peter

2016-10-14

Mutations in the gene encoding phospholipase C-γ 2 (PLCγ 2 ) have been shown to be associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Bruton's tyrosine kinase inhibitor ibrutinib. The fact that two of these mutations, R665W and L845F, imparted upon PLCγ 2 an ∼2-3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca 2+ following ligation of the B cell antigen receptor (BCR) led to the assumption that the two mutants exhibit constitutively enhanced intrinsic activity. Here, we show that the two PLCγ 2 mutants are strikingly hypersensitive to activation by Rac2 such that even wild-type Rac2 suffices to activate the mutant enzymes upon its introduction into intact cells. Enhanced "basal" activity of PLCγ 2 in intact cells is shown using the pharmacologic Rac inhibitor EHT 1864 and the PLCγ 2 F897Q mutation mediating Rac resistance to be caused by Rac-stimulated rather than by constitutively enhanced PLCγ 2 activity. We suggest that R665W and L845F be referred to as allomorphic rather than hypermorphic mutations of PLCG2 Rerouting of the transmembrane signals emanating from BCR and converging on PLCγ 2 through Rac in ibrutinib-resistant CLL cells may provide novel drug treatment strategies to overcome ibrutinib resistance mediated by PLCG2 mutations or to prevent its development in ibrutinib-treated CLL patients. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Progressive but Previously Untreated CLL Patients with Greater Array CGH Complexity Exhibit a Less Durable Response to Chemoimmunotherapy

PubMed Central

Kay, Neil E.; Eckel-Passow, Jeanette E.; Braggio, Esteban; VanWier, Scott; Shanafelt, Tait D.; Van Dyke, Daniel L.; Jelinek, Diane F.; Tschumper, Renee C.; Kipps, Thomas; Byrd, John C.; Fonseca, Rafael

2010-01-01

To better understand the implications of genomic instability and outcome in B-cell CLL, we sought to address genomic complexity as a predictor of chemosensitivity and ultimately clinical outcome in this disease. We employed array-based comparative genomic hybridization (aCGH), using a one-million probe array and identified gains and losses of genetic material in 48 patients treated on a chemoimmunotherapy (CIT) clinical trial. We identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. Higher genomic complexity, as a mechanism favoring clonal selection, was associated with shorter progression-free survival and predicted a poor response to treatment. Of interest, CLL cases with loss of p53 surveillance showed more complex genomic features and were found both in patients with a 17p13.1 deletion and in the more favorable genetic subtype characterized by the presence of 13q14.1 deletion. This aCGH study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. PMID:21156228

Analysis of IgV gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery.

PubMed

Degan, Massimo; Bomben, Riccardo; Bo, Michele Dal; Zucchetto, Antonella; Nanni, Paola; Rupolo, Maurizio; Steffan, Agostino; Attadia, Vincenza; Ballerini, Pier Ferruccio; Damiani, Daniela; Pucillo, Carlo; Poeta, Giovanni Del; Colombatti, Alfonso; Gattei, Valter

2004-07-01

Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M) IgV(H) genes have a better prognosis than unmutated (UM) cases. We analysed the IgV(H) mutational status of B-CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B-CLLs, 124 cases were examined for IgV(H) gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity-determining regions as evidence of antigen-driven selection; this identified three B-CLL subsets: significantly mutated (sM), with evidence of antigen-driven selection, not significantly mutated (nsM) and UM, without such evidence and IgV(H) gene per cent mutations above or below the 2% cut-off. sM B-CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgV(H) genes. sM, nsM and UM B-CLL were also characterized for the biased usage of IgV(H) families, intraclonal IgV(H) gene diversification, preference of mutations to target-specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase zeta and eta and activation-induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B-CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B-CLL.

Defective immunoregulatory T-cell function in chronic lymphocytic leukemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, T.; Ozer, H.; Henderson, E.S.

Chronic lymphocytic leukemia (CLL) of B-cell origin results in the malignant proliferation of small immunoglobulin-bearing lymphocytes. There is currently a controversy in the literature regarding both the ability of this leukemic population to differentiate into mature plasma cells, as well as the ability of apparently normal T cells from these patients to regulate allogeneic B-cell differentiation. In the present study we have examined the lymphocytes of CLL patients in various clinical stages of their disease and with different surface phenotypes of their leukemic B-cell population. Our results show that leukemic CLL B cells from all 20 patients (including one patientmore » with a monoclonal IgM paraprotein and another with a monoclonal IgG paraprotein) are incapable of further differentiation even in the absence of suppressor T cells and the presence of helper T lymphocytes. This lack of capacity to differentiate is unaffected by clinical stage, by therapy, or by the phenotype of the malignant population. Since the leukemic B population did not suppress normal allogeneic B-cell differentiation, the maturation deficit is evidently intrinsic to the leukemic clone rather than a result of activity of non-T suppressor cells. T helper function was also variably depressed in the blood of some patients with CLL, and this depression did not correlate with clinical stage, with therapy, or with the degree of lymphocytosis. Dysfunction of radiosensitive T suppressor cells was found to be the most consistent regulatory deficit of CLL T cells. Each of 11 patients whose leukemic cell population was of the ..mu..delta, ..mu cap alpha.., or ..mu.. phenotype had both helper and suppressor cell defects.« less

Inflammation Causes Resistance to Anti-CD20-Mediated B Cell Depletion.

PubMed

Laws, L H; Parker, C E; Cherala, G; Koguchi, Y; Waisman, A; Slifka, M K; Oberbarnscheidt, M H; Obhrai, J S; Yeung, M Y; Riella, L V

2016-11-01

B cells play a central role in antibody-mediated rejection and certain autoimmune diseases. However, B cell-targeted therapy such as anti-CD20 B cell-depleting antibody (aCD20) has yielded mixed results in improving outcomes. In this study, we investigated whether an accelerated B cell reconstitution leading to aCD20 depletion resistance could account for these discrepancies. Using a transplantation model, we found that antigen-independent inflammation, likely through toll-like receptor (TLR) signaling, was sufficient to mitigate B cell depletion. Secondary lymphoid organs had a quicker recovery of B cells when compared to peripheral blood. Inflammation altered the pharmacokinetics (PK) and pharmacodynamics (PD) of aCD20 therapy by shortening drug half-life and accelerating the reconstitution of the peripheral B cell pool by bone marrow-derived B cell precursors. IVIG (intravenous immunoglobulin) coadministration also shortened aCD20 drug half-life and led to accelerated B cell recovery. Repeated aCD20 dosing restored B cell depletion and delayed allograft rejection, especially B cell-dependent, antibody-independent allograft rejection. These data demonstrate the importance of further clinical studies of the PK/PD of monoclonal antibody treatment in inflammatory conditions. The data also highlight the disconnect between B cell depletion on peripheral blood compared to secondary lymphoid organs, the deleterious effect of IVIG when given with aCD20 and the relevance of redosing of aCD20 for effective B cell depletion in alloimmunity. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation.

PubMed

Kochenderfer, James N; Dudley, Mark E; Carpenter, Robert O; Kassim, Sadik H; Rose, Jeremy J; Telford, William G; Hakim, Frances T; Halverson, David C; Fowler, Daniel H; Hardy, Nancy M; Mato, Anthony R; Hickstein, Dennis D; Gea-Banacloche, Juan C; Pavletic, Steven Z; Sportes, Claude; Maric, Irina; Feldman, Steven A; Hansen, Brenna G; Wilder, Jennifer S; Blacklock-Schuver, Bazetta; Jena, Bipulendu; Bishop, Michael R; Gress, Ronald E; Rosenberg, Steven A

2013-12-12

New treatments are needed for B-cell malignancies persisting after allogeneic hematopoietic stem cell transplantation (alloHSCT). We conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. T cells for genetic modification were obtained from each patient's alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. These results show for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies resistant to standard DLIs without causing GVHD.

[Reduced intensity conditioning allogeneic hematopoietic stem cell transplantation in chronic lymphocytic leukemia (CLL) patients with the aberration of p53 gene].

PubMed

Wang, Li; Miao, Kourong; Fan, Lei; Xu, Ji; Wu, Hanxin; Li, Jianyong; Xu, Wei

2016-04-01

To investigate the effectiveness and safety of reduced intensity conditioning allogeneic hematopoietic stem cell transplantation (RIC allo-HSCT) in ultra high risk chronic lymphocytic leukemia (CLL) patients with the deletion of p53 to deepen the understanding of allo-HSCT in the treatment of CLL. In this retrospective study, a total of 4 ultra high risk CLL patients with the deletion of p53 in our center between July 2012 and Jan 2014 were enrolled. The RIC regimen was administered and the hematopoietic reconstitution, transplantation related mortality (TRM), overall survival (OS), progress free survival (PFS) were evaluated. We registered 4 patients with the median age of 56 years (49-61 years), including 3 males and 1 female. The median mononuclear cells (MNC) and CD34(+) cells were 6.54 (2.85-14.7) × 10(8)/kg (recipient body weight) and 5.81 (2.85-7.79) × 10(6)/kg (recipient body weight), respectively. The median time of the neutrophil recovery was 11 days (range of 9-12 days), and the median time of the platelet recovery 5.5 days (range of 0-11 days). Three patients (75%) attained a full donor chimerism at day 28 after transplantation and one (25%) got a mixed chimerism of donor and recipient. During the follow-up at a median time of 26.5 months (range of 21-39 months), 2 (50%) patients developed acute graft versus host disease (aGVHD) grade I and 2 (50%) patients got CMV infection. One patient got herpes zoster virus and EB virus infections. No transplantation related mortality was found in the 4 patients. One patient who was in partial response status progressed 5 months after transplantation, and the other 3 patients remained in durable remission after allo-HSCT. These results suggested that RIC allo-HSCT showed durable remission, good tolerance and acceptable toxicity, which could be a better option for the treatment of ultra high risk CLL patients with the deletion of p53 and was worth to be investigated and applied widely in future.

The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo

PubMed Central

Ponader, Sabine; Chen, Shih-Shih; Buggy, Joseph J.; Balakrishnan, Kumudha; Gandhi, Varsha; Wierda, William G.; Keating, Michael J.; O'Brien, Susan; Chiorazzi, Nicholas

2012-01-01

B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of several B-cell malignancies, including chronic lymphocytic leukemia (CLL), and can be targeted by inhibitors of BCR-associated kinases, such as Bruton tyrosine kinase (Btk). PCI-32765, a selective, irreversible Btk inhibitor, is a novel, molecularly targeted agent for patients with B-cell malignancies, and is particularly active in patients with CLL. In this study, we analyzed the mechanism of action of PCI-32765 in CLL, using in vitro and in vivo models, and performed correlative studies on specimens from patients receiving therapy with PCI-32765. PCI-32765 significantly inhibited CLL cell survival, DNA synthesis, and migration in response to tissue homing chemokines (CXCL12, CXCL13). PCI-32765 also down-regulated secretion of BCR-dependent chemokines (CCL3, CCL4) by the CLL cells, both in vitro and in vivo. In an adoptive transfer TCL1 mouse model of CLL, PCI-32765 affected disease progression. In this model, PCI-32765 caused a transient early lymphocytosis, and profoundly inhibited CLL progression, as assessed by weight, development, and extent of hepatospenomegaly, and survival. Our data demonstrate that PCI-32765 effectively inhibits CLL cell migration and survival, possibly explaining some of the characteristic clinical activity of this new targeted agent. PMID:22180443

Variables affecting the quantitation of CD22 in neoplastic B cells.

PubMed

Jasper, Gregory A; Arun, Indu; Venzon, David; Kreitman, Robert J; Wayne, Alan S; Yuan, Constance M; Marti, Gerald E; Stetler-Stevenson, Maryalice

2011-03-01

Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory for diagnosis, prognosis, and assessment of patients receiving antibody-based therapy. ABC values and the effect of technical variables on CD22 quantitation in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FCL), hairy cell leukemia (HCL) and normal B cells were studied. The QuantiBrite System® was used to determine the level of CD22 expression (mean antibody bound per cell, ABC) by malignant and normal B cells. The intra-assay variability, number of cells required for precision, effect of delayed processing as well as shipment of peripheral blood specimens (delayed processing and exposure to noncontrolled environments), and the effect of paraformaldehyde fixation on assay results were studied. The QuantiBRITE method of measuring CD22 ABC is precise (median CV 1.6%, 95% confidence interval, 1.2-2.3%) but a threshold of 250 malignant cells is required for reliable CD22 ABC values. Delayed processing and overnight shipment of specimens resulted in significantly different ABC values whereas fixation for up to 12 h had no significant effect. ABC measurements determined that CD22 expression is lower than normal in ALL, CLL, FCL, and MCL but higher than normal in HCL. CD22 expression was atypical in the hematolymphoid malignancies studied and may have diagnostic utility. Technical variables such as cell number analyzed and delayed processing or overnight shipment of specimens impact significantly on the measurement of antigen expression by QFCM in the clinical laboratory. Published 2010 Wiley-Liss, Inc.

Genomic characterization of chronic lymphocytic leukemia (CLL) in radiation-exposed Chornobyl cleanup workers.

PubMed

Ojha, Juhi; Dyagil, Iryna; Finch, Stuart C; Reiss, Robert F; de Smith, Adam J; Gonseth, Semira; Zhou, Mi; Hansen, Helen M; Sherborne, Amy L; Nakamura, Jean; Bracci, Paige M; Gudzenko, Nataliya; Hatch, Maureen; Babkina, Nataliya; Little, Mark P; Chumak, Vadim V; Walsh, Kyle M; Bazyka, Dimitry; Wiemels, Joseph L; Zablotska, Lydia B

2018-05-02

Chronic lymphocytic leukemia (CLL) was the predominant leukemia in a recent study of Chornobyl cleanup workers from Ukraine exposed to radiation (UR-CLL). Radiation risks of CLL significantly increased with increasing bone marrow radiation doses. Current analysis aimed to clarify whether the increased risks were due to radiation or to genetic mutations in the Ukrainian population. A detailed characterization of the genomic landscape was performed in a unique sample of 16 UR-CLL patients and age- and sex-matched unexposed general population Ukrainian-CLL (UN-CLL) and Western-CLL (W-CLL) patients (n = 28 and 100, respectively). Mutations in telomere-maintenance pathway genes POT1 and ATM were more frequent in UR-CLL compared to UN-CLL and W-CLL (both p < 0.05). No significant enrichment in copy-number abnormalities at del13q14, del11q, del17p or trisomy12 was identified in UR-CLL compared to other groups. Type of work performed in the Chornobyl zone, age at exposure and at diagnosis, calendar time, and Rai stage were significant predictors of total genetic lesions (all p < 0.05). Tumor telomere length was significantly longer in UR-CLL than in UN-CLL (p = 0.009) and was associated with the POT1 mutation and survival. No significant enrichment in copy-number abnormalities at CLL-associated genes was identified in UR-CLL compared to other groups. The novel associations between radiation exposure, telomere maintenance and CLL prognosis identified in this unique case series provide suggestive, though limited data and merit further investigation.

Monoclonal B lymphocytes with the characteristics of "indolent" chronic lymphocytic leukemia are present in 3.5% of adults with normal blood counts.

PubMed

Rawstron, Andy C; Green, Michael J; Kuzmicki, Anita; Kennedy, Ben; Fenton, James A L; Evans, Paul A S; O'Connor, Sheila J M; Richards, Stephen J; Morgan, Gareth J; Jack, Andrew S; Hillmen, Peter

2002-07-15

Molecular and cellular markers associated with malignant disease are frequently identified in healthy individuals. The relationship between these markers and clinical disease is not clear, except where a neoplastic cell population can be identified as in myeloma/monoclonal gammopathies of undetermined significance (MGUS). We have used the distinctive phenotype of chronic lymphocytic leukemia (CLL) cells to determine whether low levels of these cells can be identified in individuals with normal complete blood counts. CLL cells were identified by 4-color flow cytometric analysis of CD19/CD5/CD79b/CD20 expression in 910 outpatients over 40 years old. These outpatients were age- and sex-matched to the general population with normal hematologic parameters and no evident history of malignant disease. CLL phenotype cells were detectable in 3.5% of individuals at low level (median, 0.013; range, 0.002- 1.458 x 10(9) cells/L), and represented a minority of B lymphocytes (median, 11%; range, 3%-95%). Monoclonality was demonstrated by immunoglobulin light-chain restriction in all cases with CLL phenotype cells present and confirmed in a subset of cases by consensus-primer IgH-polymerase chain reaction. As in clinical disease, CLL phenotype cells were detected with a higher frequency in men (male-to-female ratio, 1.9:1) and elderly individuals (2.1% of 40- to 59-year-olds versus 5.0% of 60- to 89-year-olds, P =.01). The neoplastic cells were identical to good-prognosis CLL, being CD5+23+20(wk)79b(wk)11a(-)22(wk)sIg(wk)CD38-, and where assessed had a high degree (4.8%-6.6%) of IgH somatic hypermutation. The monoclonal CLL phenotype cells present in otherwise healthy individuals may represent a very early stage of indolent CLL and should be useful in elucidating the mechanisms of leukemogenesis.

Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project

PubMed Central

Kreuzer, Karl‐Anton; Soosapilla, Asha; Spacek, Martin; Stehlikova, Olga; Gambell, Peter; McIver‐Brown, Neil; Villamor, Neus; Psarra, Katherina; Arroz, Maria; Milani, Raffaella; de la Serna, Javier; Cedena, M. Teresa; Jaksic, Ozren; Nomdedeu, Josep; Moreno, Carol; Rigolin, Gian Matteo; Cuneo, Antonio; Johansen, Preben; Johnsen, Hans E.; Rosenquist, Richard; Niemann, Carsten Utoft; Kern, Wolfgang; Westerman, David; Trneny, Marek; Mulligan, Stephen; Doubek, Michael; Pospisilova, Sarka; Hillmen, Peter; Oscier, David; Hallek, Michael; Ghia, Paolo; Montserrat, Emili

2018-01-01

The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as “required” or “recommended” for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate “required” markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5–20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for “required” diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. “Recommended” markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as “required” for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus “recommended” panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society PMID:29024461

Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia.

PubMed

Herling, Carmen D; Abedpour, Nima; Weiss, Jonathan; Schmitt, Anna; Jachimowicz, Ron Daniel; Merkel, Olaf; Cartolano, Maria; Oberbeck, Sebastian; Mayer, Petra; Berg, Valeska; Thomalla, Daniel; Kutsch, Nadine; Stiefelhagen, Marius; Cramer, Paula; Wendtner, Clemens-Martin; Persigehl, Thorsten; Saleh, Andreas; Altmüller, Janine; Nürnberg, Peter; Pallasch, Christian; Achter, Viktor; Lang, Ulrich; Eichhorst, Barbara; Castiglione, Roberta; Schäfer, Stephan C; Büttner, Reinhard; Kreuzer, Karl-Anton; Reinhardt, Hans Christian; Hallek, Michael; Frenzel, Lukas P; Peifer, Martin

2018-02-20

Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.

MiR-20a-5p promotes radio-resistance by targeting Rab27B in nasopharyngeal cancer cells.

PubMed

Huang, Dabing; Bian, Geng; Pan, Yueyin; Han, Xinghua; Sun, Yubei; Wang, Yong; Shen, Guodong; Cheng, Min; Fang, Xiang; Hu, Shilian

2017-01-01

MicroRNAs (miRNAs) was reported to be involved in cancer radio-resistance, which remains a major obstacle for effective cancer therapy. The differently expressed miRNAs were detected by RNA-seq experiment in nasopharyngeal cancer (NPC) cells. MiR-20a-5p was selected as our target, which was subject to finding its target gene Rab27B via bioinformatics analysis. The qRT-PCR, western blot and the luciferase reporter assays were performed to confirm Rab27B as the target of miR-20a-5p. In addition, the roles of miR-20a-5p in NPC radio-resistance were detected by transfection of either miR-20a-5p-mimic or miR-20a-5p-antagomiR. The involvement of Rab27B with NPC radio-resistance was also detected by the experiments with siRNA-mediated repression of Rab27B or over-expression of GFP-Rab27B. Wound healing and invasion assays were performed to detect the roles of both miR-20a-5p and Rab27B. MiR-20a-5p promotes NPC radio-resistance. We identified that its target gene Rab27B negatively correlates with miR-20a-5p-mediated NPC radio-resistance by systematic studies of a radio-sensitive (CNE-2) and resistant (CNE-1) NPC cell lines. Repression of Rab27B by siRNA suppresses cell apoptosis and passivates CNE-2 cells, whereas over-expression of Rab27B triggered cell apoptosis and sensitizes CNE-1 cells. MiR-20a-5p and its target gene Rab27B might be involved in the NPC radio-resistance. Thus the key players and regulators involved in this pathway might be the potential targets for developing effective therapeutic strategies against NPC.

Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation

PubMed Central

Dudley, Mark E.; Carpenter, Robert O.; Kassim, Sadik H.; Rose, Jeremy J.; Telford, William G.; Hakim, Frances T.; Halverson, David C.; Fowler, Daniel H.; Hardy, Nancy M.; Mato, Anthony R.; Hickstein, Dennis D.; Gea-Banacloche, Juan C.; Pavletic, Steven Z.; Sportes, Claude; Maric, Irina; Feldman, Steven A.; Hansen, Brenna G.; Wilder, Jennifer S.; Blacklock-Schuver, Bazetta; Jena, Bipulendu; Bishop, Michael R.; Gress, Ronald E.; Rosenberg, Steven A.

2013-01-01

New treatments are needed for B-cell malignancies persisting after allogeneic hematopoietic stem cell transplantation (alloHSCT). We conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. T cells for genetic modification were obtained from each patient’s alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. These results show for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies resistant to standard DLIs without causing GVHD. This trial was registered at www.clinicaltrials.gov as #NCT01087294. PMID:24055823

Salvianolic acid B reverses multidrug resistance in nude mice bearing human colon cancer stem cells.

PubMed

Guo, Piaoting; Wang, Jianchao; Gao, Wencang; Liu, Xia; Wu, Shaofei; Wan, Boshun; Xu, Lei; Li, Yanhua

2018-05-29

Salvianolic acid B (SalB) is a water‑soluble phenolic compound, extractable from Salvia miltiorrhiza, and has previously been demonstrated to reverse tumor multidrug resistance (MDR) in colon cancer cells. Cancer stem cells (CSCs) are closely associated with drug resistance. Therefore, establishing a nude mouse model bearing human colon CSCs is important for the study of the mechanisms underlying colon cancer drug resistance as well as the reversal of drug resistance. The present study aimed to establish a nude mouse model bearing human colon CSCs and to investigate the effects of SalB on the drug resistance exhibited by the nude mouse model as well as determine its underlying mechanism. Cells from two colon cancer cell lines (LoVo and HCT‑116) were cultured in serum‑free medium to obtain CSCs‑enriched spheroid cells. Following this, nude mice were transplanted with LoVo and HCT‑116 colon CSCs to establish the CSC nude mouse model, which was subsequently demonstrated to exhibit MDR. The results of the present study revealed that following treatment with SalB, the chemotherapeutic drug resistance of xenografts was reversed to a certain extent. Western blot analysis was performed to investigate the expression levels of cluster of differentiation (CD)44, CD133, transcription factor sox‑2 (SOX2) and ATP‑binding cassette sub‑family G member 2 (ABCG2) proteins, and the results demonstrated that treatment with SalB downregulated the expression of CD44, SOX2 and ABCG2 proteins in both LoVo and HCT‑116 colon CSCs xenografts. In conclusion, the results of the present study revealed that a serum‑free suspension method can be performed to successfully isolate colon CSCs. In addition, a nude mice bearing colon CSCs animal model was successfully established, and associated tumors were confirmed to exhibit MDR. Furthermore, SalB was demonstrated to successfully reverse MDR in nude mice bearing LoVo and HCT‑116 colon CSCs, as well as suppress the expression

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

PubMed

Rawstron, A C; Fazi, C; Agathangelidis, A; Villamor, N; Letestu, R; Nomdedeu, J; Palacio, C; Stehlikova, O; Kreuzer, K-A; Liptrot, S; O'Brien, D; de Tute, R M; Marinov, I; Hauwel, M; Spacek, M; Dobber, J; Kater, A P; Gambell, P; Soosapilla, A; Lozanski, G; Brachtl, G; Lin, K; Boysen, J; Hanson, C; Jorgensen, J L; Stetler-Stevenson, M; Yuan, C; Broome, H E; Rassenti, L; Craig, F; Delgado, J; Moreno, C; Bosch, F; Egle, A; Doubek, M; Pospisilova, S; Mulligan, S; Westerman, D; Sanders, C M; Emerson, R; Robins, H S; Kirsch, I; Shanafelt, T; Pettitt, A; Kipps, T J; Wierda, W G; Cymbalista, F; Hallek, M; Hillmen, P; Montserrat, E; Ghia, P

2016-04-01

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells.

PubMed

Zaborsky, Nadja; Gassner, Franz Josef; Asslaber, Daniela; Reinthaler, Petra; Denk, Ursula; Flenady, Sabine; Hofbauer, Josefina Piñón; Danner, Barbara; Rebhandl, Stefan; Harrer, Andrea; Geisberger, Roland; Greil, Richard; Egle, Alexander

2016-08-02

Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vβ7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.

Characterization in vitro and in vivo of progressively adriamycin-resistant B16-BL6 mouse melanoma cells.

PubMed

Ganapathi, R; Grabowski, D; Schmidt, H; Bell, D; Melia, M

1987-07-01

Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.

IKK phosphorylation of NF-κB at serine 536 contributes to acquired cisplatin resistance in head and neck squamous cell cancer

PubMed Central

Li, Zhipeng; Yang, Zejia; Lapidus, Rena G; Liu, Xuefeng; Cullen, Kevin J; Dan, Han C

2015-01-01

Current treatment methods for advanced head and neck squamous cell carcinoma (HNSCC) include surgery, radiation therapy and chemotherapy. For recurrent and metastatic HNSCC, cisplatin is the most common treatment option, but most of patients will eventually develop cisplatin resistance. Therefore, it is imperative to define the mechanisms involved in cisplatin resistance and find novel therapeutic strategies to overcome this deadly disease. In order to determine the role of nuclear factor-kappa B (NF-κB) in contributing to acquired cisplatin resistance in HNSCC, the expression and activity of NF-κB and its upstream kinases, IKKα and IKKβ, were evaluated and compared in three pairs of cisplatin sensitive and resistant HNSCC cell lines, including a pair of patient derived HNSCC cell line. The experiments revealed that NF-κB p65 activity was elevated in cisplatin resistant HNSCC cells compared to that in their parent cells. Importantly, the phosphorylation of NF-κB p65 at serine 536 and the phosphorylation of IKKα and IKKβ at their activation loops were dramatically elevated in the resistant cell lines. Furthermore, knockdown of NF-κB or overexpression of p65-S536 alanine (p65-S536A) mutant sensitizes resistant cells to cisplatin. Additionally, the novel IKKβ inhibitor CmpdA has been shown to consistently block the phosphorylation of NF-κB at serine 536 while also dramatically improving the efficacy of cisplatin in inhibition of cell proliferation and induction of apoptosis in the cisplatin resistant cancer cells. These results indicated that IKK/NF-κB plays a pivotal role in controlling acquired cisplatin resistance and that targeting the IKK/NF-κB signaling pathway may provide a possible therapeutic method to overcome the acquired resistance to cisplatin in HNSCC. PMID:26693062

AID induces intraclonal diversity and genomic damage in CD86+ chronic lymphocytic leukemia cells

PubMed Central

Huemer, Michael; Rebhandl, Stefan; Zaborsky, Nadja; Gassner, Franz J; Hainzl, Stefan; Weiss, Lukas; Hebenstreit, Daniel; Greil, Richard; Geisberger, Roland

2014-01-01

The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sμ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL. PMID:25179679

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia

PubMed Central

2011-01-01

Background The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as

Possible role of CD22, CD79b and CD20 expression in distinguishing small lymphocytic lymphoma from chronic lymphocytic leukemia.

PubMed

Jovanovic, Danijela; Djurdjevic, Predrag; Andjelkovic, Nebojsa; Zivic, Ljubica

2014-01-01

Flow cytometry has an important role in diagnosis and classification of B-cell lymphoproliferative disorders (BCLPDs). However, in distinguishing chronic lymphocytic leukemia (CLL) from small lymphocytic lymphoma (SLL) only clinical criteria are available so far. Aim of the study was to determine differences in the expression of common B cell markers (CD22, CD79b and CD20) on the malignant lymphocytes in the peripheral blood samples of CLL and SLL patients. Peripheral blood samples of 56 CLL and 11 SLL patients were analyzed by 5-color flow cytometry on the CD45/CD19/CD5 gate for CD22, CD79b and CD20. In the samples collected from the CLL patients, CD22 expression was detected in only 20% of patients in the low pattern, while in SLL patients the expression was medium and present in 90.9% of patients (p < 0.0001). For CD79b expression, statistical significance is reached both in the expression pattern, which was low/medium for CLL and high for SLL, and expression level (p = 0.006). The expression of CD20 was counted as the CD20/CD19 ratio. The average ratio was 0.512 in the CLL patients vs. 0.931 in the SLL patients (p = 0.0001). The pattern of expression and expression level of CD22, CD79b and CD20 in peripheral blood could be used for distinguishing SLL from CLL patients.

Natural killer cells inhibit oxaliplatin-resistant colorectal cancer by repressing WBSCR22 via upregulating microRNA-146b-5p.

PubMed

Zhao, Haiyan; Su, Wuyun; Kang, Qingmei; Xing, Ze; Lin, Xue; Wu, Zhongjun

2018-01-01

Natural killer (NK) cells have exhibited promising efficacy in inhibiting cancer growth. We aimed to explorer the effect of NK cells on oxaliplatin-resistant colorectal cancer and the underlying molecular mechanism. Oxaliplatin-resistant colorectal cancer cell lines were co-cultured with NK cells to evaluate the effect on viability, proliferation, migration and invasion in vitro . Oxaliplatin-resistant colorectal cancer cells were also co-injected with NK cells into mice to establish xenograft tumor model, to assess the in vivo effect of NK cells on tumorigenesis of the oxaliplatin-resistant colorectal cancer cells. Expression of WBSCR22 gene was assessed in the oxaliplatin-resistant colorectal cancer cells following NK cell treatment to elucidate the mechanism. NK cell treatment significantly reduces growth of oxaliplatin-resistant colorectal cancer cells both in vitro and in vivo , as well as reduced WBSCR22 expression. MicroRNAs potentially targeting WBSCR22 were analyzed, and microRNA-146b-5p was found to be significantly upregulated following NK cell treatment. MicroRNA-146b-5p directly targeted WBSCR22 mRNA 3'-UTR to inhibit its expression, which was required for NK cell-induced inhibition of oxaliplatin-resistant colorectal cancer cell lines. NK cells inhibit oxaliplatin-resistant colorectal cancer by repressing WBSCR22 via upregulating microRNA-146b-5p, both of which could serve as candidates for targeted therapy against oxaliplatin-resistant colorectal cancer.

DSP30 and interleukin-2 as a mitotic stimulant in B-cell disorders including those with a low disease burden.

PubMed

Dun, Karen A; Riley, Louise A; Diano, Giuseppe; Adams, Leanne B; Chiu, Eleanor; Sharma, Archna

2018-05-01

Chromosome abnormalities detected during cytogenetic investigations for B-cell malignancy offer prognostic information that can have wide ranging clinical impacts on patients. These impacts may include monitoring frequency, treatment type, and disease staging level. The use of the synthetic oligonucleotide DSP30 combined with interleukin 2 (IL2) has been described as an effective mitotic stimulant in B-cell disorders, not only in chronic lymphocytic leukemia (CLL) but also in a range of other B-cell malignancies. Here, we describe the comparison of two B-cell mitogens, lipopolysaccharide (LPS), and DSP30 combined with IL2 as mitogens in a range of common B-cell disorders excluding CLL. The results showed that DSP30/IL2 was an effective mitogen in mature B-cell disorders, revealing abnormal cytogenetic results in a range of B-cell malignancies. The abnormality rate increased when compared to the use of LPS to 64% (DSP30/IL2) from 14% (LPS). In a number of cases the disease burden was proportionally very low, less than 10% of white cells. In 37% of these cases, the DSP30 culture revealed abnormal results. Importantly, we also obtained abnormal conventional cytogenetics results in 3 bone marrow cases in which immunophenotyping showed an absence of an abnormal B-cell clone. In these cases, the cytogenetics results correlated with the provisional diagnosis and altered their staging level. The use of DSP30 and IL2 is recommended for use in many B-cell malignancies as an effective mitogen and their use has been shown to enable successful culture of the malignant clone, even at very low levels of disease. © 2018 Wiley Periodicals, Inc.

Novel BAFF-Receptor Antibody to Natively Folded Recombinant Protein Eliminates Drug-Resistant Human B-cell Malignancies In Vivo.

PubMed

Qin, Hong; Wei, Guowei; Sakamaki, Ippei; Dong, Zhenyuan; Cheng, Wesley A; Smith, D Lynne; Wen, Feng; Sun, Han; Kim, Kunhwa; Cha, Soungchul; Bover, Laura; Neelapu, Sattva S; Kwak, Larry W

2018-03-01

Purpose: mAbs such as anti-CD20 rituximab are proven therapies in B-cell malignancies, yet many patients develop resistance. Novel therapies against alternative targets are needed to circumvent resistance mechanisms. We sought to generate mAbs against human B-cell-activating factor receptor (BAFF-R/TNFRSF13C), which has not yet been targeted successfully for cancer therapy. Experimental Design: Novel mAbs were generated against BAFF-R, expressed as a natively folded cell surface immunogen on mouse fibroblast cells. Chimeric BAFF-R mAbs were developed and assessed for in vitro and in vivo monotherapy cytotoxicity. The chimeric mAbs were tested against human B-cell tumor lines, primary patient samples, and drug-resistant tumors. Results: Chimeric antibodies bound with high affinity to multiple human malignant B-cell lines and induced potent antibody-dependent cellular cytotoxicity (ADCC) against multiple subtypes of human lymphoma and leukemia, including primary tumors from patients who had relapsed after anti-CD20 therapy. Chimeric antibodies also induced ADCC against ibrutinib-resistant and rituximab-insensitive CD20-deficient variant lymphomas, respectively. Importantly, they demonstrated remarkable in vivo growth inhibition of drug-resistant tumor models in immunodeficient mice. Conclusions: Our method generated novel anti-BAFF-R antibody therapeutics with remarkable single-agent antitumor effects. We propose that these antibodies represent an effective new strategy for targeting and treating drug-resistant B-cell malignancies and warrant further development. Clin Cancer Res; 24(5); 1114-23. ©2017 AACR . ©2017 American Association for Cancer Research.

Improving therapy of chronic lymphocytic leukemia with chimeric antigen receptor T cells.

PubMed

Fraietta, Joseph A; Schwab, Robert D; Maus, Marcela V

2016-04-01

Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and redirecting the immune system against cancer. This review will briefly summarize T-cell therapies in development for CLL disease. We discuss the role of T-cell function and phenotype, T-cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. Copyright © 2016 Elsevier Inc. All rights reserved.

An immunophenotypic and molecular diagnosis of composite hairy cell leukaemia and chronic lymphocytic leukaemia.

PubMed

Liptrot, Stuart; O' Brien, David; Langabeer, Stephen E; Quinn, Fiona; Mackarel, A Jill; Elder, Patrick; Vandenberghe, Elisabeth; Hayden, Patrick J

2013-12-01

Hairy cell leukaemia (HCL) and chronic lymphocytic leukaemia (CLL) are distinct clinicopathological B cell chronic lymphoproliferative disorders (B-CLPD). Both diseases have characteristic immunophenotypic and molecular features. The co-existence of two B-CLPD is perhaps more common than previously thought but a composite HCL and CLL has been rarely documented. A case is reported in which the morphology, integrated with an extensive immunophenotyping panel, and incorporation of the recently described HCL-associated BRAF V600E mutation, enabled the prompt diagnosis of composite HCL and CLL thus allowing appropriate treatment selection. This case serves to highlight the benefit of a multidisciplinary approach to the diagnosis of bi-clonal B-CLPD.

B cell receptor inhibition as a target for CLL therapy.

PubMed

Jeyakumar, Deepa; O'Brien, Susan

2016-03-01

Inhibitors of the B cell receptor (BCR) represent an attractive therapeutic option for patients with chronic lymphocytic leukemia. Recently approved inhibitors of Bruton's tyrosine kinase (ibrutinib) and phosphatidylinositol 3-kinase (idelalisib), are promising agents because they are generally well tolerated and highly effective. These agents may be particularly important in the treatment of older patients who are less able to tolerate the myelosuppression (and infections) associated with chemoimmunotherapy. As a class of medications, BCR inhibitors have some unique side effects including redistribution lymphocytosis. Ibrutinib has specific toxicities including increased risk for bleeding and atrial fibrillation. Idelalisib also has some unique toxicities consisting of transaminitis, diarrhea and pneumonitis. Ongoing clinical trials are evaluating these agents in combination with antibodies, chemotherapy and other small molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bendamustine added to allogeneic conditioning improves long-term outcomes in patients with CLL.

PubMed

Khouri, I F; Sui, D; Jabbour, E J; Samuels, B I; Turturro, F; Alatrash, G; Anderlini, P; Ahmed, S; Oran, B; Ciurea, S O; Marin, D; Olson, A; Patel, K K; Popat, U R; Ledesma, C; Kadia, T M; Ferrajoli, A; Burger, J A; Jorgensen, J L; Medeiros, L J; Bassett, R L; Gulbis, A M

2017-01-01

Bendamustine has shown a favorable safety profile when included in chemotherapy regimens for several types of lymphoma, including CLL. This study investigated the long-term effect of adding bendamustine to a conditioning regimen on survival, rate of engraftment, immune recovery and GvHD after allogeneic stem cell transplantation (alloSCT) in CLL patients. These outcomes were compared with the fludarabine, cyclophosphamide and rituximab (FCR) conditioning regimen. We reviewed the data for 89 CLL patients treated on three trials at our institution. Twenty-six (29%) patients received bendamustine, fludarabine and rituximab (BFR) and 63 (71%) received FCR. Patient characteristics were similar in both groups. Ten (38%) BFR-treated patients vs only two (3%) FCR-treated patients did not experience severe neutropenia (P=<0.001). The 3-year overall survival estimates for the BFR and FCR groups were 82 and 51% (P=0.03), and the 3-year PFS estimates were 63% and 27% (P=0.001), respectively. The 2-year treatment-related mortality was 8 and 23% and the incidence of grade 3 or 4 GvHD was 4% and 10%, respectively. This study is the first to report that addition of bendamustine to alloSCT conditioning for CLL patients is associated with improved survival and lower mortality, myelosuppression, and GvHD.

Comparative Assessment of Clinically Utilized CD20-directed Antibodies in Chronic Lymphocytic Leukemia Cells Reveals Divergent NK cell, Monocyte and Macrophage Properties

PubMed Central

Rafiq, Sarwish; Butchar, Jonathon P.; Cheney, Carolyn; Mo, Xiaokui; Trotta, Rossana; Caligiuri, Michael; Jarjoura, David; Tridandapani, Susheela; Muthusamy, Natarajan; Byrd, John C.

2013-01-01

CD20 is a widely validated, B cell specific target for therapy in B cell malignancies. Rituximab is an anti-CD20 antibody that when combined with chemotherapy prolongs survival of CLL patients. Ofatumumab and GA101 (obinutuzumab) are CD20-directed antibodies now being developed as alternative agents to rituximab in CLL based upon different properties of enhanced direct cell death (DCD), NK cell-mediated antibody dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). Despite wide spread study, ofatumumab and GA101 have not been directly compared to one another, nor studied for interaction with monocytes and macrophages that are critical to CD20-mediated antibody efficacy in murine models. In CLL cells, we show that DCD is greatest with GA101 and CDC with ofatumumab. GA101 promotes enhanced NK cell activation and ADCC at high antibody concentrations. Ofatumumab has superior antibody dependent cellular phagocytosis (ADCP) with monocyte derived macrophages (MDM). GA101 demonstrated reduced activation of monocytes with diminished pERK, TNF-α release, and FcγRIIa recruitment to lipid rafts. These data demonstrate GA101 and ofatumumab are superior to rituximab against CLL cells via different mechanisms of potential tumor elimination. These findings bear relevance to potential combination strategies with each of these anti-CD20 antibodies in the treatment of CLL. PMID:23418626

Overexpressed BAG3 is a potential therapeutic target in chronic lymphocytic leukemia.

PubMed

Zhu, Huayuan; Wu, Wei; Fu, Yuan; Shen, Wenyi; Miao, Kourong; Hong, Min; Xu, Wei; Young, Ken H; Liu, Peng; Li, Jianyong

2014-03-01

Bcl-2-associated athanogene 3 (BAG3), a member of BAG family, is shown to sustain cell survival and underlie resistance to chemotherapy in human neoplastic cells. We aimed to determine the exact role and underlying mechanisms of BAG3 in human chronic lymphocytic leukemia (CLL). One hundred human CLL samples and 20 normal B-cell samples from healthy controls were collected. We measured the BAG3 expression in these cells and explored its relationship with known prognostic factors for CLL. The roles of BAG3 in cell apoptosis and migration were evaluated by small interfering RNA-mediated knockdown of BAG3 in primary CLL cells. We showed that BAG3 expression level was increased in CLL cells compared with normal B cells. Moreover, BAG3 expression was particularly upregulated in CD38 positive, unmutated immunoglobulin heavy-chain patients and those with lymphadenopathy and/or splenomegaly. Importantly, patients with increased BAG3 expression level have poor overall survival in subgroups with positive ZAP-70 or those without any "p53 abnormality". In addition, knocking down of BAG3 expression resulted in increased apoptotic ratio and decreased migration in primary CLL cells. Our data indicate that BAG3 is a marker of poor prognostic in specific subgroups of CLL patients and may be a potential therapeutic target for this disease.

Hyperdiploidy in CLL/SLL: A Rare Cytogenetic Event Associated with Poor Prognosis.

PubMed

DeNicola, Matthew; Pullarkat, Sheeja; Yea, Steven; Rao, Nagesh; Yang, Lynn; Tirado, Carlos A

2014-01-01

Hyperdiploidy has been described in a variety of malignancies including acute lymphoblastic leukemia and plasma cell myeloma, in which the abnormality is associated with a very good prognosis. Herein, we describe a 61-year-old female that was diagnosed with atypical chronic lymphocytic leukemia (CLL). Initial chromosome analysis of a lymph node specimen showed an abnormal karyotype described as 46-48,XX,add(3)(q12),+16,+mar[cp3]/46,XX[1]. Chromosome analysis of the bone marrow a week later showed a pseudodiploid and normal diploid clone described as: 46,X,-X,-3,-6,+7,+9,-14,-15,+16,+17,+17,+20,-22[1]/46,XX[19]. Concurrent FISH studies of peripheral blood samples using the CLL FISH panel showed nuclei with an extra copy of chromosome 13 and an extra copy of the short arm of chromosome 17. FISH for t(11;14) was negative. These results suggest the presence of an underlying complex hyperdiploid karyotype. Hyperdiploidy is a rare event in SLL/CLL and is usually associated with a poor prognosis.

Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.

PubMed

Amigo-Jiménez, Irene; Bailón, Elvira; Ugarte-Berzal, Estefanía; Aguilera-Montilla, Noemí; García-Marco, José A; García-Pardo, Angeles

2014-01-01

Matrix metalloproteinase-9 (MMP-9) contributes to chronic lymphocytic leukemia (CLL) pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO) and fludarabine as examples of cytotoxic drugs. We used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test. In response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2) and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9. Our study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This is a novel role for MMP-9 contributing to CLL progression

Matrix Metalloproteinase-9 Is Involved in Chronic Lymphocytic Leukemia Cell Response to Fludarabine and Arsenic Trioxide

PubMed Central

Amigo-Jiménez, Irene; Bailón, Elvira; Ugarte-Berzal, Estefanía; Aguilera-Montilla, Noemí; García-Marco, José A.; García-Pardo, Angeles

2014-01-01

Background Matrix metalloproteinase-9 (MMP-9) contributes to chronic lymphocytic leukemia (CLL) pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO) and fludarabine as examples of cytotoxic drugs. Methods We used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test. Results In response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2) and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9. Conclusions Our study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This is a novel role for

AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody–Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML

PubMed Central

Pereira, Daniel S.; Guevara, Claudia I.; Jin, Liqing; Mbong, Nathan; Verlinsky, Alla; Hsu, Ssucheng J.; Aviña, Hector; Karki, Sher; Abad, Joseph D.; Yang, Peng; Moon, Sung-Ju; Malik, Faisal; Choi, Michael Y.; An, Zili; Morrison, Kendall; Challita-Eid, Pia M.; Doñate, Fernando; Joseph, Ingrid B.J.; Kipps, Thomas J.; Dick, John E.; Stover, David R.

2015-01-01

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody–drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent mono-methyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34+CD38− leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML. PMID:25934707

Present and future of personalized medicine in CLL.

PubMed

Montserrat, Emili; Bauman, Tycho; Delgado, Julio

2016-03-01

Medicine has been 'personalized' (i.e. centred in persons) since its foundation. Recently, however, the term 'personalized medicine' (or, better, 'precision medicine') has been introduced to define 'a form of medicine that uses information about a person's genes, proteins, and environment to prevent, diagnose, and treat disease'. This concept has gained momentum thanks to next-generation-sequencing (NGS) techniques that allow identification of molecular characteristics unique to the patient and to the tumour. It is hoped that NGS will not only contribute to a better understanding of chronic lymphocytic leukaemia (CLL), but will identify disease subsets that could benefit from specific treatment interventions. Recent advances in diagnosis (e.g. high-resolution immunophenotyping, markers of genetic abnormalities), prognosis (e.g. biomarkers), response predictors [e.g. del(17p)/TP53 mutations even at subclonal level], treatment (e.g. BCR signalling inhibitors, BCL2 antagonists, CAR-T cells) and methods to evaluate minimal residual disease constitute good examples of tools facilitating 'personalized' management of patients with CLL. Copyright © 2016. Published by Elsevier Ltd.

Ibrutinib synergizes with MDM-2 inhibitors in promoting cytotoxicity in B chronic lymphocytic leukemia.

PubMed

Voltan, Rebecca; Rimondi, Erika; Melloni, Elisabetta; Rigolin, Gian Matteo; Casciano, Fabio; Arcidiacono, Maria Vittoria; Celeghini, Claudio; Cuneo, Antonio; Zauli, Giorgio; Secchiero, Paola

2016-10-25

The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models. The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model. Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model. Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL.

Ibrutinib synergizes with MDM-2 inhibitors in promoting cytotoxicity in B chronic lymphocytic leukemia

PubMed Central

Melloni, Elisabetta; Rigolin, Gian Matteo; Casciano, Fabio; Arcidiacono, Maria Vittoria; Celeghini, Claudio; Cuneo, Antonio; Zauli, Giorgio; Secchiero, Paola

2016-01-01

Objective The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models. Methods The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model. Results Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model. Conclusions Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL. PMID:27661115

Adoptive immunotherapy utilizing anti-CD19 chimeric antigen receptor T-cells for B-cell malignancies.

PubMed

Oh, Iekuni; Oh, Yukiko; Ohmine, Ken

2016-01-01

Genetically modified T-cells with forced expression of anti-CD19 chimeric antigen receptor (CD19 CAR) have demonstrated promising clinical results for relapsed and refractory B cell malignancies in early clinical trial settings. The first beneficial tumor regressions were identified among approximately half of CLL patients in 2011. Similarly, CD19 CAR T-cells achieved remissions in about 80% of aggressive B-cell lymphomas in 2012. Furthermore, in 2013 this cellular therapy showed an extremely high rate of efficacy against refractory CD19 positive acute lymphoid leukemia, which had been regarded as the most difficult to treat hematologic disease. Recently, despite the absence of CD19 expression by neoplastic plasma cells, patients with refractory multiple myeloma achieved stringent complete remission after this therapy coupled with high dose chemotherapy and autologous stem cell transplantation. However, there are significant toxicities. Cytokine releasing syndrome and neurotoxicity are recognized as life-threatening adverse events. Although phase I/II clinical trials have just started in Japan, given the exciting results obtained to date, this cellular therapy is expected to be a novel breakthrough immunotherapy for treating refractory B-cell malignancies.

Overexpression of B7-H3 augments anti-apoptosis of colorectal cancer cells by Jak2-STAT3.

PubMed

Zhang, Ting; Jiang, Bo; Zou, Shi-Tao; Liu, Fen; Hua, Dong

2015-02-14

To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms. SW620 cells that highly overexpressed B7-H3 (SW620-B7-H3-EGFP) and HCT8 cells stably transfected with B7-H3 shRNA (HCT8-shB7-H3) were previously constructed in our laboratory. Cells transfected with pIRES2-EGFP were used as negative controls (SW620-NC and HCT8-NC). Real-time PCR and western blotting analysis were used to detect the mRNA and protein expressions of the apoptosis regulator proteins Bcl-2, Bcl-xl and Bax. A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells. The effect of drug resistance was detected by a cell cycle assay. Active caspase-3 western blotting was used to reflect the anti-apoptotic ability of cells. Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490, a Jak2 specific inhibitor, in B7-H3 overexpressing cells. The data were analyzed by GraphPad Prism 6 using a non-paired t-test. Whether by overexpression in SW620 cells or downregulation in HCT8, B7-H3 significantly affected the expression of anti- and pro-apoptotic proteins, at both the transcriptional and translational levels, compared with the negative control (P < 0.05). A cell proliferation assay revealed that B7-H3 overexpression increased the drug resistance of cells and resulted in a higher survival rate (P < 0.05). In addition, the results of cell cycle and active caspase-3 western blotting proved that B7-H3 overexpression inhibited apoptosis in colorectal cancer cell lines (P < 0.05). B7-H3 overexpression improved Jak2 and STAT3 phosphorylation and, in turn, increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-xl, based on western blotting (P < 0.05). After treating B7-H3 overexpressing cells with the Jak2

Development of venetoclax for therapy of lymphoid malignancies.

PubMed

Zhu, Huayuan; Almasan, Alexandru

2017-01-01

B-cell lymphoma-2 (BCL-2) family dysfunction and impairment of apoptosis are common in most B-cell lymphoid malignancies. Venetoclax (Venclexta™, formerly ABT-199, GDC-0199) is a highly selective BCL-2 inhibitor, which mimics its BCL-2 homology 3-domain to induce apoptosis. It was approved for treatment of previously treated chronic lymphocytic leukemia (CLL) patients with 17p deletion early in 2016. It has also been in clinical trials for other B-cell lymphoid malignancies. Unlike the other recently approved targeted agents idelalisib and ibrutinib, so far there has been no relapse reported in some patients. Also, unlike the other targeted agents, it is effective against tumor cells that reside in the blood marrow. Despite its promising outcome in CLL, preclinical data have already uncovered mechanistic insights underlying venetoclax resistance, such as upregulation of MCL-1 or BCL-xL expression and protective signaling from the microenvironment. In this review, we describe the role of the BCL-2 family in the pathogenesis of B-cell lymphoid malignancies, the development of venetoclax, and its current clinical outcome in CLL and other B-cell malignancies. We also discuss the resistance mechanisms that develop following venetoclax therapy, potential strategies to overcome them, and how this knowledge can be translated into clinical applications.

Development of venetoclax for therapy of lymphoid malignancies

PubMed Central

Zhu, Huayuan; Almasan, Alexandru

2017-01-01

B-cell lymphoma-2 (BCL-2) family dysfunction and impairment of apoptosis are common in most B-cell lymphoid malignancies. Venetoclax (Venclexta™, formerly ABT-199, GDC-0199) is a highly selective BCL-2 inhibitor, which mimics its BCL-2 homology 3-domain to induce apoptosis. It was approved for treatment of previously treated chronic lymphocytic leukemia (CLL) patients with 17p deletion early in 2016. It has also been in clinical trials for other B-cell lymphoid malignancies. Unlike the other recently approved targeted agents idelalisib and ibrutinib, so far there has been no relapse reported in some patients. Also, unlike the other targeted agents, it is effective against tumor cells that reside in the blood marrow. Despite its promising outcome in CLL, preclinical data have already uncovered mechanistic insights underlying venetoclax resistance, such as upregulation of MCL-1 or BCL-xL expression and protective signaling from the microenvironment. In this review, we describe the role of the BCL-2 family in the pathogenesis of B-cell lymphoid malignancies, the development of venetoclax, and its current clinical outcome in CLL and other B-cell malignancies. We also discuss the resistance mechanisms that develop following venetoclax therapy, potential strategies to overcome them, and how this knowledge can be translated into clinical applications. PMID:28331288

Clinical Practice Guidelines for Diagnosis and Treatment of Chronic Lymphocytic Leukemia (CLL) in The Netherlands.

PubMed

Kersting, Sabina; Neppelenbroek, Suzanne I M; Visser, Hein P J; van Gelder, Michel; Levin, Mark-David; Mous, Rogier; Posthuma, Ward; van der Straaten, Hanneke M; Kater, Arnon P

2018-01-01

In recent years, considerable progress has been made in the treatment of patients with chronic lymphocytic leukemia (CLL), and new potent drugs have become available. Therefore, the CLL working party revised the Dutch guidelines. Not only efficacy but also quality of life and socio-economic impact were taken into account in the formulation of treatment recommendations. The working party discussed a set of questions regarding diagnostic tests and treatment and wrote the draft guideline. This was evidence-based whenever possible, but in cases of low evidence, an expert-based recommendation was formulated with input of the entire working party. The draft guideline was sent to all hematologists in the Netherlands for comment and was subsequently approved. Recommendations were formulated on diagnostic tests and work-up before treatment. Also, recommendations were made for treatment with fludarabine-cyclophosphamide-rituximab, bendamustine-rituximab, chlorambucil with anti-CD20 antibody, ibrutinib, idelalisib-rituximab, venetoclax, and allogeneic stem cell transplantation. In the revised Dutch CLL guidelines, chemo-immunotherapy is still the cornerstone of CLL treatment with novel targeted drugs for specific risk groups. Copyright © 2017 Elsevier Inc. All rights reserved.

What is the best frontline therapy for patients with CLL and 17p deletion?

PubMed

Badoux, Xavier C; Keating, Michael J; Wierda, William G

2011-03-01

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with significant variation in disease progression, response to therapy, and survival outcome. Deletions of 17p or mutations of TP53 have been identified as one of the poorest prognostic factors, being predictive of short time for disease progression, lack of response to therapy, short response duration, and short overall survival. The treatment of patients with CLL has improved significantly with the development of chemoimmunotherapy, but this benefit was not pronounced in patients with 17p deletion. We compare various treatment strategies used in these patients, including FCR-like chemoimmunotherapy, alemtuzumab, other antibody combinations, or novel targeted therapies with promising results. Allogeneic stem cell transplantation offers the possibility for long-term disease control in these patients and should be considered early in younger, transplant-eligible patients. The current state of therapy is far from optimal and resources should be applied to studying therapeutic options for patients who have CLL with loss of p53 function.

Horizontal transfer of miR-106a/b from cisplatin resistant hepatocarcinoma cells can alter the sensitivity of cervical cancer cells to cisplatin.

PubMed

Raji, Grace R; Sruthi, T V; Edatt, Lincy; Haritha, K; Sharath Shankar, S; Sameer Kumar, V B

2017-10-01

Recent studies indicate that horizontal transfer of genetic material can act as a communication tool between heterogenous populations of tumour cells, thus altering the chemosensitivity of tumour cells. The present study was designed to check whether the horizontal transfer of miRNAs released by cisplatin resistant (Cp-r) Hepatocarcinoma cells can alter the sensitivity of cervical cancer cells. For this exosomes secreted by cisplatin resistant and cisplatin sensitive HepG2 cells (EXres and EXsen) were isolated and characterised. Cytotoxicity analysis showed that EXres can make Hela cells resistant to cisplatin. Analysis of miR-106a/b levels in EXres and EXsen showed that their levels vary. Mechanistic studies showed that miR-106a/b play an important role in EXsen and EXres mediated change in chemosensitivity of Hela cells to cisplatin. Further SIRT1 was identified as a major target of miR-106a/b using in silico tools and this was proved by experimentation. Also the effect of miR-106a/b in chemosensitivity was seen to be dependent on regulation of SIRT1 by miR-106a/b. In brief, this study brings into light, the SIRT1 dependent mechanism of miR-106a/b mediated regulation of chemosensitivity upon the horizontal transfer from one cell type to another. Copyright © 2017 Elsevier Inc. All rights reserved.

Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia

PubMed Central

Porter, David L.; Hwang, Wei-Ting; Frey, Noelle V.; Lacey, Simon F.; Shaw, Pamela A.; Loren, Alison W.; Bagg, Adam; Marcucci, Katherine T.; Shen, Angela; Gonzalez, Vanessa; Ambrose, David; Grupp, Stephan A.; Chew, Anne; Zheng, Zhaohui; Milone, Michael C.; Levine, Bruce L.; Melenhorst, Jan J.; June, Carl H.

2018-01-01

Patients with multiply relapsed or refractory chronic lymphocytic leukemia (CLL) have a poor prognosis. Chimeric antigen receptor (CAR)–modified T cells targeting CD19 have the potential to improve on the low complete response rates with conventional therapies by inducing sustained remissions in patients with refractory B cell malignancies. We previously reported preliminary results on three patients with refractory CLL. We report the mature results from our initial trial using CAR-modified T cells to treat 14 patients with relapsed and refractory CLL. Autologous T cells transduced with a CD19-directed CAR (CTL019) lentiviral vector were infused into patients with relapsed/refractory CLL at doses of 0.14 × 108 to 11 × 108 CTL019 cells (median, 1.6 × 108 cells). Patients were monitored for toxicity, response, expansion, and persistence of circulating CTL019 T cells. The overall response rate in these heavily pretreated CLL patients was 8 of 14 (57%), with 4 complete remissions (CR) and 4 partial remissions (PR). The in vivo expansion of the CAR T cells correlated with clinical responses, and the CAR T cells persisted and remained functional beyond 4 years in the first two patients achieving CR. No patient in CR has relapsed. All responding patients developed B cell aplasia and experienced cytokine release syndrome, coincident with T cell proliferation. Minimal residual disease was not detectable in patients who achieved CR, suggesting that disease eradication may be possible in some patients with advanced CLL. PMID:26333935

CD79B limits response of diffuse large B cell lymphoma to ibrutinib.

PubMed

Kim, Joo Hyun; Kim, Won Seog; Ryu, Kyungju; Kim, Seok Jin; Park, Chaehwa

2016-01-01

Blockage of B cell receptor signaling with ibrutinib presents a promising clinical approach for treatment of B-cell malignancies. However, many patients show primary resistance to the drug or develop secondary resistance. In the current study, cDNA microarray and Western blot analyses revealed CD79B upregulation in the activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) that display differential resistance to ibrutinib. CD79B overexpression was sufficient to induce resistance to ibrutinib and enhanced AKT and MAPK activation, indicative of an alternative mechanism underlying resistance. Conversely, depletion of CD79B sensitized primary refractory cells to ibrutinib and led to reduced phosphorylation of AKT or MAPK. Combination of the AKT inhibitor or the MAPK inhibitor with ibrutinib resulted in circumvention of both primary and acquired resistance in ABC-DLBCL. Our data collectively indicate that CD79B overexpression leading to activation of AKT/MAPK is a potential mechanism underlying primary ibrutinib resistance in ABC-DLBCL, and support its utility as an effective biomarker to predict therapeutic response to ibrutinib.

ATP11B mediates platinum resistance in ovarian cancer

PubMed Central

Moreno-Smith, Myrthala; Halder, J.B.; Meltzer, Paul S.; Gonda, Tamas A.; Mangala, Lingegowda S.; Rupaimoole, Rajesha; Lu, Chunhua; Nagaraja, Archana S.; Gharpure, Kshipra M.; Kang, Yu; Rodriguez-Aguayo, Cristian; Vivas-Mejia, Pablo E.; Zand, Behrouz; Schmandt, Rosemarie; Wang, Hua; Langley, Robert R.; Jennings, Nicholas B.; Ivan, Cristina; Coffin, Jeremy E.; Armaiz, Guillermo N.; Bottsford-Miller, Justin; Kim, Sang Bae; Halleck, Margaret S.; Hendrix, Mary J.C.; Bornman, William; Bar-Eli, Menashe; Lee, Ju-Seog; Siddik, Zahid H.; Lopez-Berestein, Gabriel; Sood, Anil K.

2013-01-01

Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance. PMID:23585472

B-cell receptor signalling and its crosstalk with other pathways in normal and malignant cells.

PubMed

Seda, Vaclav; Mraz, Marek

2015-03-01

The physiology of B cells is intimately connected with the function of their B-cell receptor (BCR). B-cell lymphomas frequently (dys)regulate BCR signalling and thus take advantage of this pre-existing pathway for B-cell proliferation and survival. This has recently been underscored by clinical trials demonstrating that small molecules (fosfamatinib, ibrutinib, idelalisib) inhibiting BCR-associated kinases (SYK, BTK, PI3K) have an encouraging clinical effect. Here we describe the current knowledge of the specific aspects of BCR signalling in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukaemia (CLL) and normal B cells. Multiple factors can contribute to BCR pathway (dys)regulation in these malignancies and the activation of 'chronic' or 'tonic' BCR signalling. In lymphoma B cells, the balance of initiation, amplitude and duration of BCR activation can be influenced by a specific immunoglobulin structure, the expression and mutations of adaptor molecules (like GAB1, BLNK, GRB2, CARD11), the activity of kinases (like LYN, SYK, PI3K) or phosphatases (like SHIP-1, SHP-1 and PTEN) and levels of microRNAs. We also discuss the crosstalk of BCR with other signalling pathways (NF-κB, adhesion through integrins, migration and chemokine signalling) to emphasise that the 'BCR inhibitors' target multiple pathways interconnected with BCR, which might explain some of their clinical activity. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Prolonging microtubule dysruption enhances the immunogenicity of chronic lymphocytic leukaemia cells

PubMed Central

Shaha, S P; Tomic, J; Shi, Y; Pham, T; Mero, P; White, D; He, L; Baryza, J L; Wender, P A; Booth, J W; Spaner, D E

2009-01-01

Cytotoxic chemotherapies do not usually mediate the expression of an immunogenic gene programme in tumours, despite activating many of the signalling pathways employed by highly immunogenic cells. Concomitant use of agents that modulate and complement stress-signalling pathways activated by chemotherapeutic agents may then enhance the immunogenicity of cancer cells, increase their susceptibility to T cell-mediated controls and lead to higher clinical remission rates. Consistent with this hypothesis, the microtubule inhibitor, vincristine, caused chronic lymphocytic leukaemia (CLL) cells to die rapidly, without increasing their immunogenicity. Protein kinase C (PKC) agonists (such as bryostatin) delayed the death of vincristine-treated CLL cells and made them highly immunogenic, with increased stimulatory abilities in mixed lymphocyte responses, production of proinflammatory cytokines, expression of co-stimulatory molecules and activation of c-Jun N-terminal kinase (JNK), p38 and nuclear factor kappa B (NF-κB) signalling pathways. This phenotype was similar to the result of activating CLL cells through Toll-like receptors (TLRs), which communicate ‘danger’ signals from infectious pathogens. Use of PKC agonists and microtubule inhibitors to mimic TLR-signalling, and increase the immunogenicity of CLL cells, has implications for the design of chemo-immunotherapeutic strategies. PMID:19737143

Construction of a 780-kb PAC, BAC, and cosmid contig encompassing the minimal critical deletion involved in B cell lymphocytic leukemia at 13q14.3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouyge-Moreau, I.; Rondeau, G.; Andre, M.T.

A putative tumor suppressor gene involved in B cell chronic lymphocytic leukemia (B-CLL) was mapped to human chromosome 13q14.3 close to the genetic markers D13S25 and D13S319. We constructed a 780-kb-long contig composed of cosmids, bacterial artificial chromosomes, and bacteriophage PI-derived artificial chromosomes that provides essential information and tools for the positional cloning of this gene. The contig contains both flanking markers as well as several additional genetic markers, three ESTs, and one potential CpG island. In addition, using one B-CLL patient, we characterized a small internal deleted region of 550 kb. Comparing this deletion with other recently published deletionsmore » narrows the minimally deleted area to less than 100 kb in our physical map. This deletion core region should contain all or part of the disrupted in B cell malignancies tumor suppressor gene. 27 refs., 3 figs.« less

Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System

PubMed Central

Taniguchi, Miki; Tasaka-Fujita, Megumi; Nakagawa, Mina; Watanabe, Takako; Kawai-Kitahata, Fukiko; Otani, Satoshi; Goto, Fumio; Nagata, Hiroko; Kaneko, Shun; Nitta, Sayuri; Murakawa, Miyako; Nishimura-Sakurai, Yuki; Azuma, Seishin; Itsui, Yasuhiro; Mori, Kenichi; Yagi, Shintaro; Kakinuma, Sei; Asahina, Yasuhiro; Watanabe, Mamoru

2016-01-01

Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment. PMID:27047766

Fucosterol activates the insulin signaling pathway in insulin resistant HepG2 cells via inhibiting PTP1B.

PubMed

Jung, Hyun Ah; Bhakta, Himanshu Kumar; Min, Byung-Sun; Choi, Jae Sue

2016-10-01

Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. This study investigated the modulatory effects of fucosterol on the insulin signaling pathway in insulin-resistant HepG2 cells by inhibiting protein tyrosine phosphatase 1B (PTP1B). In addition, molecular docking simulation studies were performed to predict binding energies, the specific binding site of fucosterol to PTP1B, and to identify interacting residues using Autodock 4.2 software. Glucose uptake was determined using a fluorescent D-glucose analogue and the glucose tracer 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose, and the signaling pathway was detected by Western blot analysis. We found that fucosterol enhanced insulin-provoked glucose uptake and conjointly decreased PTP1B expression level in insulin-resistant HepG2 cells. Moreover, fucosterol significantly reduced insulin-stimulated serine (Ser307) phosphorylation of insulin receptor substrate 1 (IRS1) and increased phosphorylation of Akt, phosphatidylinositol-3-kinase, and extracellular signal- regulated kinase 1 at concentrations of 12.5, 25, and 50 µM in insulin-resistant HepG2 cells. Fucosterol inhibited caspase-3 activation and nuclear factor kappa B in insulin-resistant hepatocytes. These results suggest that fucosterol stimulates glucose uptake and improves insulin resistance by downregulating expression of PTP1B and activating the insulin signaling pathway. Thus, fucosterol has potential for development as an anti-diabetic agent.

Late relapse of progressive multifocal leucoencephalopathy postallogenic transplant in a young patient with CLL.

PubMed

Sanchez-Quintana, Ana; Breña-Atienza, Joaquín; Marrero-Santos, Carmen; Alvarez-Acosta, Luis

2013-08-05

We describe a case of progressive multifocal leucoencephalopathy (PML) in a 39-year-old patient diagnosed with chronic lymphocytic leukaemia (CLL) who underwent two allogenic matched-sibling stem cell transplantations. PML was confirmed just after the first transplantation with cerebral MRI and by PCR in the cerebrospinal fluid. After immunosuppression withdrawal and cidofovir treatment, he achieved a reversal of clinical symptoms, John Cunningham (JC) virus positivity and MRI lesions regression. He remained asymptomatic for 5 years with no signs of infection activity, even though he received three new chemotherapy regimens due to a CLL relapse. However, after the second stem cell transplantation, new neurological symptoms began and a reactivation of the JC virus infection was detected. This time, treatment with mefloquine was started, but he experienced a progressive neurological deterioration and died 1 month after the symptoms began.

Concurrent chronic lymphocytic leukemia cutis and acute myelogenous leukemia cutis in a patient with untreated CLL.

PubMed

Miller, M K; Strauchen, J A; Nichols, K T; Phelps, R G

2001-08-01

Patients who have chronic lymphocytic leukemia (CLL) are known to have a high frequency of second malignant neoplasms. However, acute myelogenous leukemia (AML) occurring concurrent with or after a diagnosis of CLL is extremely rare. In this article we report a case of AML developing in a 55-year-old male with a 6-year history of untreated CLL. The diagnosis was facilitated by touch preparation of a skin punch biopsy specimen. The patient presented with a two-week history of fever, weakness, anasarca, and a skin rash. Physical examination revealed pink to skin-colored firm papules, which coalesced into indurated plaques on his trunk, upper extremities, and face. The lesions, in combination with generalized edema, produced a leonine facies. Touch prep of the biopsy showed medium to large blasts, large monocytoid cells, and numerous small mature lymphocytes, providing the preliminary diagnosis of a second, previously undiagnosed myelomonocytic malignancy in this patient. The initial diagnosis was subsequently confirmed by histologic, cytochemical, immunohistochemical and flow cytometry studies. This is the first reported case of CLL with concurrent AML in which rapid touch prep of a skin punch biopsy facilitated diagnosis.

TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia

PubMed Central

Catakovic, Kemal; Gassner, Franz Josef; Ratswohl, Christoph; Zaborsky, Nadja; Rebhandl, Stefan; Schubert, Maria; Steiner, Markus; Gutjahr, Julia Christine; Pleyer, Lisa; Egle, Alexander; Hartmann, Tanja Nicole; Greil, Richard; Geisberger, Roland

2018-01-01

ABSTRACT While research on T cell exhaustion in context of cancer particularly focuses on CD8+ cytotoxic T cells, the role of inhibitory receptors on CD4+ T-helper cells have remained largely unexplored. TIGIT is a recently identified inhibitory receptor on T cells and natural killer (NK) cells. In this study, we examined TIGIT expression on T cell subsets from CLL patients. While we did not observe any differences in TIGIT expression in CD8+ T cells of healthy controls and CLL cells, we found an enrichment of TIGIT+ T cells in the CD4+ T cell compartment in CLL. Intriguingly, CLL patients with an advanced disease stage displayed elevated numbers of CD4+ TIGIT+ T cells compared to low risk patients. Autologous CLL-T cell co-culture assays revealed that depleting CD4+ TIGIT+ expressing T cells from co-cultures significantly decreased CLL viability. Accordingly, a supportive effect of TIGIT+CD4+ T cells on CLL cells in vitro could be recapitulated by blocking the interaction of TIGIT with its ligands using TIGIT-Fc molecules, which also impeded the T cell specific production of CLL-prosurvival cytokines. Our data reveal that TIGIT+CD4+T cells provide a supportive microenvironment for CLL cells, representing a potential therapeutic target for CLL treatment. PMID:29296521

Outcomes for Patients with Chronic Lymphocytic Leukemia (CLL) and Acute Leukemia or Myelodysplastic Syndrome

PubMed Central

Tambaro, Francesco Paolo; Garcia-Manero, Guillermo; O’Brien, Susan M.; Faderl, Stefan H.; Ferrajoli, Alessandra; Burger, Jan A.; Pierce, Sherry; Wang, Xuemei; Do, Kim-Anh; Kantarjian, Hagop M.; Keating, Michael J.; Wierda, William G.

2016-01-01

Acute leukemia (AL) and myelodysplastic syndrome (MDS) are uncommon in CLL. We retrospectively identified 95 patients with CLL also diagnosed with AL (n=38) or MDS (n=57), either concurrently (n=5) or subsequent (n=90) to CLL diagnosis and report their outcomes. Median number of CLL treatments prior to AL and MDS was 2(0–9) and 1(0–8), respectively; the most common regimen was purine analogue combined with alkylating agent±CD20 mAb. Twelve had no prior CLL treatment. Among 38 with AL, 33 had AML, 3 had ALL (1Ph+), 1 had biphenotypic, and 1 had extramedullary (bladder) AML. Unfavorable AML karyotype was noted in 26, intermediate-risk in 7. There was no association between survival from AL and number of prior CLL regimens or karyotype. Expression of CD7 on blasts was associated with shorter survival. Among MDS cases, all IPSS were represented; karyotype was unfavorable in 36, intermediate in 6, and favorable in 12 patients; 10 experienced transformation to AML. Shorter survival from MDS correlated with higher-risk IPSS, poor-risk karyotype, and increased number of prior CLL treatments. Overall, outcomes for patients with CLL subsequently diagnosed with AL or MDS were poor; AL/MDS occurred without prior CLL treatment. Effective therapies for these patients are desperately needed. PMID:26290497

A novel role for BDNF-TrkB in the regulation of chemotherapy resistance in head and neck squamous cell carcinoma.

PubMed

Lee, Junegoo; Jiffar, Tilahun; Kupferman, Michael E

2012-01-01

Mechanisms of resistance for HNSCC to cisplatin (CDDP), the foundational chemotherapeutic agent in the treatment of this disease, remain poorly understood. We previously demonstrated that cisplatin resistance (CR) can be overcome by targeting Trk receptor. In the current study, we explored the potential mechanistic role of the BDNF-TrkB signaling system in the development of CDDP resistance in HNSCC. Utilizing an in vitro system of acquired CR, we confirmed a substantial up-regulation of both BDNF and TrkB at the protein and mRNA levels in CR cells, suggesting an autocrine pathway dysregulation in this system. Exogenous BDNF stimulation led to an enhanced expression of the drug-resistance and anti-apoptotic proteins MDR1 and XiAP, respectively, in a dose-dependently manner, demonstrating a key role for BDNF-TrkB signaling in modulating the response to cytotoxic agents. In addition, modulation of TrkB expression induced an enhanced sensitivity of cells to CDDP in HNSCC. Moreover, genetic suppression of TrkB resulted in changes in expression of Bim, XiAP, and MDR1 contributing to HNSCC survival. To elucidate intracellular signaling pathways responsible for mechanisms underlying BDNF/TrkB induced CDDP-resistance, we analyzed expression levels of these molecules following inhibition of Akt. Inhibition of Akt eliminated BDNF effect on MDR1 and Bim expression in OSC-19P cells as well as modulated expressions of MDR1, Bim, and XiAP in OSC-19CR cells. These results suggest BDNF/TrkB system plays critical roles in CDDP-resistance development by utilizing Akt-dependent signaling pathways.

Tectorigenin sensitizes paclitaxel-resistant human ovarian cancer cells through downregulation of the Akt and NFκB pathway.

PubMed

Yang, Yeong-In; Lee, Kyung-Tae; Park, Hee-Juhn; Kim, Tae Jin; Choi, Youn Seok; Shih, Ie-Ming; Choi, Jung-Hye

2012-12-01

Paclitaxel (Taxol) is currently used as the front-line chemotherapeutic agent for several cancers including ovarian carcinoma; however, the drug frequently induces drug resistance through multiple mechanisms. The new strategy of using natural compounds in combination therapies is highly attractive because those compounds may enhance the efficacy of chemotherapy. In this study, we found that tectorigenin, an isoflavonoid isolated from flower of Pueraria thunbergiana, enhanced the growth-inhibitory effect of paclitaxel in paclitaxel-resistant ovarian cancer cells (MPSC1(TR), A2780(TR) and SKOV3(TR)) as well as their naive counterparts. The combination of tectorigenin with paclitaxel resulted in a synergistic apoptosis compared with either agent alone through activation of caspases-3, -8 and -9. Treatment with tectorigenin inhibited the nuclear translocation of NFκB and the expression of NFκB-dependent genes such as FLIP, XIAP, Bcl-2, Bcl-xL and COX-2, which are known to be associated with chemoresistance. In addition, the tectorigenin-paclitaxel combination inhibited the phosphorylation of IκB and IKK and the activation of Akt in paclitaxel-resistant cancer cells. Moreover, tectorigenin-paclitaxel-induced cell growth inhibition was enhanced by pretreatment with the Akt inhibitor LY294002 or overexpression of the dominant negative Akt (Akt-DN), but reduced by overexpression of constitutively activated Akt (Akt-Myr). Furthermore, we found that Akt-Myr, at least in part, reversed tectorigenin-paclitaxel-induced nuclear translocation of NFκB and the phosphorylation of IκB and IKK. These data suggest that tectorigenin could sensitize paclitaxel-resistant human ovarian cancer cells through inactivation of the Akt/IKK/IκB/NFκB signaling pathway, and promise a new intervention to chemosensitize paclitaxel-induced cytotoxicity in ovarian cancer.

T-cell receptor signaling activates an ITK/NF-κB/GATA-3 axis in T-cell lymphomas facilitating resistance to chemotherapy

PubMed Central

Wang, Tianjiao; Lu, Ye; Polk, Avery; Chowdhury, Pinki; Zamalloa, Carlos Murga; Fujiwara, Hiroshi; Suemori, Koichiro; Beyersdorf, Niklas; Hristov, Alexandra C.; Lim, Megan S.; Bailey, Nathanael G.; Wilcox, Ryan A.

2016-01-01

Purpose T-cell lymphomas are a molecularly heterogeneous group of non-Hodgkin lymphomas (NHL) that account for a disproportionate number of NHL disease-related deaths due to their inherent and acquired resistance to standard multiagent chemotherapy regimens. Despite their molecular heterogeneity and frequent loss of various T-cell specific receptors, the T-cell antigen receptor is retained in the majority of these lymphomas. As T-cell receptor (TCR) engagement activates a number of signaling pathways and transcription factors that regulate T-cell growth and survival, we examined the TCR’s role in mediating resistance to chemotherapy. Experimental Design Genetic and pharmacologic strategies were utilized to determine the contribution of tyrosine kinases and transcription factors activated in conventional T cells following T-cell receptor (TCR) engagement in acquired chemotherapy resistance in primary T-cell lymphoma cells and patient-derived cell lines. Results Here we report that TCR signaling activates a signaling axis that includes ITK, NF-κB, and GATA-3, and promotes chemotherapy resistance. Conclusions These observations have significant therapeutic implications, as pharmacologic inhibition of ITK prevented activation of this signaling axis and overcame chemotherapy resistance. PMID:27780854

Curcumin regulates gene expression of insulin like growth factor, B-cell CLL/lymphoma 2 and antioxidant enzymes in streptozotocin induced diabetic rats

PubMed Central

2013-01-01

Background The effects of curcumin on the activities and gene expression of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (G-ST), B-cell CLL/lymphoma 2 (Bcl-2) and insulin like growth factor-1 (IGF-1) in diabetic rats were studied. Methods Twenty four rats were assigned to three groups (8 rats for each). Rats of first group were non diabetic and rats of the second group were rendered diabetic by streptozotocin (STZ). Both groups received vehicle, corn oil only (5 ml/kg body weight) and served as negative and positive controls, respectively. Rats of the third group were rendered diabetic and received oral curcumin dissolved in corn oil at a dose of 15 mg/5 ml/kg body weight for 6 weeks. Results Diabetic rats showed significant increase of blood glucose, thiobarbituric acid reactive substances (TBARS) and activities of all antioxidant enzymes with significant reduction of reduced glutathione (GSH) compare to the control non diabetic group. Gene expression of Bcl2, SOD, CAT, GPX and GST was increased significantly in diabetic untreated rats compare to the control non diabetic group. The administration of curcumin to diabetic rats normalized significantly their blood sugar level and TBARS values and increased the activities of all antioxidant enzymes and GSH concentration. In addition, curcumin treated rats showed significant increase in gene expression of IGF-1, Bcl2, SOD and GST compare to non diabetic and diabetic untreated rats. Conclusion Curcumin was antidiabetic therapy, induced hypoglycemia by up-regulation of IGF-1 gene and ameliorate the diabetes induced oxidative stress via increasing the availability of GSH, increasing the activities and gene expression of antioxidant enzymes and Bcl2. Further studies are required to investigate the actual mechanism of action of curcumin regarding the up regulation of gene expression of examined parameters. PMID:24364912

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells.

PubMed

Chen, Lisa S; Redkar, Sanjeev; Bearss, David; Wierda, William G; Gandhi, Varsha

2009-11-05

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-X(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells

PubMed Central

Chen, Lisa S.; Redkar, Sanjeev; Bearss, David; Wierda, William G.

2009-01-01

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-XL, XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction. PMID:19734450

CD5+ true SLL/CLL with plasmacytic differentiation and an unusual 1p36 translocation: case report and review of the literature.

PubMed

Evans, H L; Polski, J M; Deshpande, V; Dunphy, C H

2000-11-01

Lymphoplasmacytic lymphoma (LPL) and small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL)are distinct clinicopathologic entities. Although some cases of SLL/CLL may show plasmacytic differentiation and be associated with monoclonal immunoglobulin in serum, such cases appear to be very rare, and if plasma cell differentiation were marked, differentiation of SLL/CLL from LPL could be difficult. We report a rare case of true CD5-positive small lymphocytic lymphoma/chronic lymphocytic leukemia with unequivocal plasmacytic differentiation. This case also showed an abnormality of chromosome 1p36 not previously described in small lymphocytic lymphoma/chronic lymphocytic leukemia.

Lymphomagenic CARD11/BCL10/MALT1 signaling drives malignant B-cell proliferation via cooperative NF-κB and JNK activation.

PubMed

Knies, Nathalie; Alankus, Begüm; Weilemann, Andre; Tzankov, Alexandar; Brunner, Kristina; Ruff, Tanja; Kremer, Marcus; Keller, Ulrich B; Lenz, Georg; Ruland, Jürgen

2015-12-29

The aggressive activated B cell-like subtype of diffuse large B-cell lymphoma is characterized by aberrant B-cell receptor (BCR) signaling and constitutive nuclear factor kappa-B (NF-κB) activation, which is required for tumor cell survival. BCR-induced NF-κB activation requires caspase recruitment domain-containing protein 11 (CARD11), and CARD11 gain-of-function mutations are recurrently detected in human diffuse large B-cell lymphoma (DLBCL). To investigate the consequences of dysregulated CARD11 signaling in vivo, we generated mice that conditionally express the human DLBCL-derived CARD11(L225LI) mutant. Surprisingly, CARD11(L225LI) was sufficient to trigger aggressive B-cell lymphoproliferation, leading to early postnatal lethality. CARD11(L225LI) constitutively associated with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) to simultaneously activate the NF-κB and c-Jun N-terminal kinase (JNK) signaling cascades. Genetic deficiencies of either BCL10 or MALT1 completely rescued the phenotype, and pharmacological inhibition of JNK was, similar to NF-κB blockage, toxic to autonomously proliferating CARD11(L225LI)-expressing B cells. Moreover, constitutive JNK activity was observed in primary human activated B cell-like (ABC)-DLBCL specimens, and human ABC-DLBCL cells were also sensitive to JNK inhibitors. Thus, our results demonstrate that enforced activation of CARD11/BCL10/MALT1 signaling is sufficient to drive transformed B-cell expansion in vivo and identify the JNK pathway as a therapeutic target for ABC-DLBCL.

Functional characterization of BTK(C481S) mutation that confers ibrutinib resistance: exploration of alternative kinase inhibitors.

PubMed

Cheng, S; Guo, A; Lu, P; Ma, J; Coleman, M; Wang, Y L

2015-04-01

The Bruton tyrosine kinase (BTK) inhibitor, ibrutinib, has produced remarkable clinical response in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. We previously reported the identification of BTK(C481S) mutation in a CLL patient who progressed following 21-month ibrutinib therapy. Initial characterization at structural and biochemical levels revealed that the mutation disrupts the covalent binding of ibrutinib to BTK, reduces its binding affinity and diminishes its ability to inhibit the BTK enzymatic activity. Herein, we further characterized the functional consequences of BTK(C481S) in terms of molecular signaling, gene expression and cellular behavior in the patient, as well as in lymphoma cells transfected with either the wild-type or the mutant BTK constructs. Further, using an in vitro CLL proliferation model, alternative kinase inhibitors that have the potential to overcome ibrutinib resistance were explored.

PI3Kδ-selective and PI3Kα/δ-combinatorial inhibitors in clinical development for B-cell non-Hodgkin lymphoma.

PubMed

Lampson, Benjamin L; Brown, Jennifer R

2017-11-01

The efficacy of the prototypical phosphatidylinositol-3-kinase (PI3K) inhibitor idelalisib for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin lymphoma (iNHL) has led to development of multiple compounds targeting this pathway. Areas Covered: We review the hypothesized therapeutic mechanisms of PI3K inhibitors, including abrogation of B cell receptor signaling, blockade of microenvironmental pro-survival signals, and enhancement of anti-tumor immunity. We examine toxicities of idelalisib, including bacterial infections (possibly secondary to drug-induced neutropenia), opportunistic infections (possibly attributable to on-target inhibition of T cell function), and organ toxicities such as transaminitis and enterocolitis (possibly autoimmune, secondary to on-target inhibition of p110δ in regulatory T cells). We evaluate PI3K inhibitors that have entered trials for the treatment of lymphoma, focusing on agents with selectivity for PI3Kα and PI3Kδ. Expert Opinion: PI3K inhibitors, particularly those that target p110δ, have robust efficacy in the treatment of CLL and iNHL. However, idelalisib has infectious and autoimmune toxicities that limit its use. Outside of trials, idelalisib should be restricted to CLL patients with progression on ibrutinib or iNHL patients with progression on two prior therapies. Whether newer PI3K inhibitors will demonstrate differentiated toxicity profiles in comparable patient populations while retaining efficacy remains to be seen.

Recombinant antibodies encoded by IGHV1-69 react with pUL32, a phosphoprotein of cytomegalovirus and B-cell superantigen

PubMed Central

Steininger, Christoph; Widhopf, George F.; Ghia, Emanuela M.; Morello, Christopher S.; Vanura, Katrina; Sanders, Rebecca; Spector, Deborah; Guiney, Don; Jäger, Ulrich

2012-01-01

Leukemia cells from patients with chronic lymphocytic leukemia (CLL) express a highly restricted immunoglobulin heavy variable chain (IGHV) repertoire, suggesting that a limited set of antigens reacts with leukemic cells. Here, we evaluated the reactivity of a panel of different CLL recombinant antibodies (rAbs) encoded by the most commonly expressed IGHV genes with a panel of selected viral and bacterial pathogens. Six different CLL rAbs encoded by IGHV1-69 or IGHV3-21, but not a CLL rAb encoded by IGHV4-39 genes, reacted with a single protein of human cytomegalovirus (CMV). The CMV protein was identified as the large structural phosphoprotein pUL32. In contrast, none of the CLL rAbs bound to any other structure of CMV, adenovirus serotype 2, Salmonella enterica serovar Typhimurium, or of cells used for propagation of these microorganisms. Monoclonal antibodies or humanized rAbs of irrelevant specificity to pUL32 did not react with any of the proteins present in the different lysates. Still, rAbs encoded by a germ line IGHV1-69 51p1 allele from CMV-seropositive and -negative adults also reacted with pUL32. The observed reactivity of multiple different CLL rAbs and natural antibodies from CMV-seronegative adults with pUL32 is consistent with the properties of a superantigen. PMID:22234695

Appearance and Disappearance of Chronic Myeloid Leukemia (CML) in Patient with Chronic Lymphocytic Leukemia (CLL).

PubMed

Payandeh, Mehrdad; Sadeghi, Edris; Khodarahmi, Reza; Sadeghi, Masoud

2014-10-01

Chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML) are the most common leukemias of the elderly (>43 year). However, the sequential occurrence of CML followed by CLL in the same patient is extremely rare. In our report, a 52-year-old female was diagnosed with CLL (type of bone marrow (BM) infiltration was nodular and interstitial) and was treated with chlorambucil. 64 months after the diagnosis of CLL, she developed CML. She was treated with imatinib (400mg/day). After a few months, signs of CML were disappeared and CLL became dominant. This is first reported case.

Three-year follow-up of treatment-naïve and previously treated patients with CLL and SLL receiving single-agent ibrutinib.

PubMed

Byrd, John C; Furman, Richard R; Coutre, Steven E; Burger, Jan A; Blum, Kristie A; Coleman, Morton; Wierda, William G; Jones, Jeffrey A; Zhao, Weiqiang; Heerema, Nyla A; Johnson, Amy J; Shaw, Yun; Bilotti, Elizabeth; Zhou, Cathy; James, Danelle F; O'Brien, Susan

2015-04-16

Ibrutinib is an orally administered inhibitor of Bruton tyrosine kinase that antagonizes B-cell receptor, chemokine, and integrin-mediated signaling. In early-phase studies, ibrutinib demonstrated high response rates and prolonged progression-free survival (PFS) in chronic lymphocytic leukemia (CLL). The durable responses observed with ibrutinib relate in part to a modest toxicity profile that allows the majority of patients to receive continuous therapy for an extended period. We report on median 3-year follow-up of 132 patients with symptomatic treatment-naïve and relapsed/refractory CLL or small lymphocytic lymphoma. Longer treatment with ibrutinib was associated with improvement in response quality over time and durable remissions. Toxicity with longer follow-up diminished with respect to occurrence of grade 3 or greater cytopenias, fatigue, and infections. Progression remains uncommon, occurring primarily in some patients with relapsed del(17)(p13.1) and/or del(11)(q22.3) disease. Treatment-related lymphocytosis remains largely asymptomatic even when persisting >1 year and does not appear to alter longer-term PFS and overall survival compared with patients with partial response or better. Collectively, these data provide evidence that ibrutinib controls CLL disease manifestations and is well tolerated for an extended period; this information can help direct potential treatment options for different subgroups to diminish the long-term risk of relapse. © 2015 by The American Society of Hematology.

Three-year follow-up of treatment-naïve and previously treated patients with CLL and SLL receiving single-agent ibrutinib

PubMed Central

Furman, Richard R.; Coutre, Steven E.; Burger, Jan A.; Blum, Kristie A.; Coleman, Morton; Wierda, William G.; Jones, Jeffrey A.; Zhao, Weiqiang; Heerema, Nyla A.; Johnson, Amy J.; Shaw, Yun; Bilotti, Elizabeth; Zhou, Cathy; James, Danelle F.; O'Brien, Susan

2015-01-01

Ibrutinib is an orally administered inhibitor of Bruton tyrosine kinase that antagonizes B-cell receptor, chemokine, and integrin-mediated signaling. In early-phase studies, ibrutinib demonstrated high response rates and prolonged progression-free survival (PFS) in chronic lymphocytic leukemia (CLL). The durable responses observed with ibrutinib relate in part to a modest toxicity profile that allows the majority of patients to receive continuous therapy for an extended period. We report on median 3-year follow-up of 132 patients with symptomatic treatment-naïve and relapsed/refractory CLL or small lymphocytic lymphoma. Longer treatment with ibrutinib was associated with improvement in response quality over time and durable remissions. Toxicity with longer follow-up diminished with respect to occurrence of grade 3 or greater cytopenias, fatigue, and infections. Progression remains uncommon, occurring primarily in some patients with relapsed del(17)(p13.1) and/or del(11)(q22.3) disease. Treatment-related lymphocytosis remains largely asymptomatic even when persisting >1 year and does not appear to alter longer-term PFS and overall survival compared with patients with partial response or better. Collectively, these data provide evidence that ibrutinib controls CLL disease manifestations and is well tolerated for an extended period; this information can help direct potential treatment options for different subgroups to diminish the long-term risk of relapse. PMID:25700432

T-cell Receptor Signaling Activates an ITK/NF-κB/GATA-3 axis in T-cell Lymphomas Facilitating Resistance to Chemotherapy.

PubMed

Wang, Tianjiao; Lu, Ye; Polk, Avery; Chowdhury, Pinki; Zamalloa, Carlos Murga; Fujiwara, Hiroshi; Suemori, Koichiro; Beyersdorf, Niklas; Hristov, Alexandra C; Lim, Megan S; Bailey, Nathanael G; Wilcox, Ryan A

2017-05-15

Purpose: T-cell lymphomas are a molecularly heterogeneous group of non-Hodgkin lymphomas (NHL) that account for a disproportionate number of NHL disease-related deaths due to their inherent and acquired resistance to standard multiagent chemotherapy regimens. Despite their molecular heterogeneity and frequent loss of various T cell-specific receptors, the T-cell antigen receptor is retained in the majority of these lymphomas. As T-cell receptor (TCR) engagement activates a number of signaling pathways and transcription factors that regulate T-cell growth and survival, we examined the TCR's role in mediating resistance to chemotherapy. Experimental Design: Genetic and pharmacologic strategies were utilized to determine the contribution of tyrosine kinases and transcription factors activated in conventional T cells following TCR engagement in acquired chemotherapy resistance in primary T-cell lymphoma cells and patient-derived cell lines. Results: Here, we report that TCR signaling activates a signaling axis that includes ITK, NF-κB, and GATA-3 and promotes chemotherapy resistance. Conclusions: These observations have significant therapeutic implications, as pharmacologic inhibition of ITK prevented the activation of this signaling axis and overcame chemotherapy resistance. Clin Cancer Res; 23(10); 2506-15. ©2016 AACR . ©2016 American Association for Cancer Research.

Fludarabine plus alemtuzumab (FA) front-line treatment in young patients with chronic lymphocytic leukemia (CLL) and an adverse biologic profile.

PubMed

Mauro, Francesca R; Molica, Stefano; Laurenti, Luca; Cortelezzi, Agostino; Carella, Angelo M; Zaja, Francesco; Chiarenza, Annalisa; Angrilli, Francesco; Nobile, Francesco; Marasca, Roberto; Musolino, Caterina; Brugiatelli, Maura; Piciocchi, Alfonso; Vignetti, Marco; Fazi, Paola; Gentile, Giuseppe; De Propris, Maria S; Della Starza, Irene; Marinelli, Marilisa; Chiaretti, Sabina; Del Giudice, Ilaria; Nanni, Mauro; Albano, Francesco; Cuneo, Antonio; Guarini, Anna; Foà, Robin

2014-02-01

In 45, ≤ 60 years old patients with CLL and an adverse biologic profile, a front-line treatment with Fludarabine and Campath (Alemtuzumab(®)) was given. The overall response rate was 75.5%, the complete response rate (CR) 24.4% with the lowest CR rates, 16.7% and 8.3%, in 11q and 17p deleted cases. The 3-year progression-free survival (PFS) and overall survival were 42.5% and 79.9%, respectively. PFS was significantly influenced by CLL duration, beta2-microglobulin, and improved by post-remissional stem cell transplantation. Front-line fludarabine and alemtuzumab showed a manageable safety profile and evidence of a benefit in a small series of CLL patients with adverse biologic features. Copyright © 2013 Elsevier Ltd. All rights reserved.

A critical appraisal of ibrutinib in the treatment of mantle cell lymphoma and chronic lymphocytic leukemia

PubMed Central

Tucker, David L; Rule, Simon A

2015-01-01

Although chemo-immunotherapy remains at the forefront of first-line treatment for mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), small molecules, such as ibrutinib, are beginning to play a significant role, particularly in patients with multiply relapsed or chemotherapy-refractory disease and where toxicity is an overriding concern. Ibrutinib is a first-in-class, oral inhibitor of Bruton’s tyrosine kinase, which functions by irreversible inhibition of the downstream signaling pathway of the B-cell receptor, which normally promotes cell survival and proliferation. Early clinical trials have demonstrated excellent tolerability and a modest side-effect profile even in elderly and multiply pretreated patient cohorts. Although the majority of disease responses tend to be partial, efficacy data have also been encouraging with more than two-thirds of patients with CLL and MCL demonstrating a durable response, even in the high-risk disease setting. Resistance mechanisms are only partially understood and appear to be multifactorial, including the binding site mutation C481S, and escape through other common cell-signaling pathways. This article appraises the currently available data on safety and efficacy from clinical trials of ibrutinib in the management of MCL and CLL, both as a single agent and in combination with other therapies, and considers how this drug is likely to be used in future clinical practice. PMID:26150724

Leukemia cell proliferation and death in chronic lymphocytic leukemia patients on therapy with the BTK inhibitor ibrutinib.

PubMed

Burger, Jan A; Li, Kelvin W; Keating, Michael J; Sivina, Mariela; Amer, Ahmed M; Garg, Naveen; Ferrajoli, Alessandra; Huang, Xuelin; Kantarjian, Hagop; Wierda, William G; O'Brien, Susan; Hellerstein, Marc K; Turner, Scott M; Emson, Claire L; Chen, Shih-Shih; Yan, Xiao-Jie; Wodarz, Dominik; Chiorazzi, Nicholas

2017-01-26

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water ( 2 H 2 O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

The impact of trisomy 12, retinoblastoma gene and P53 in prognosis of B-cell chronic lymphocytic leukemia.

PubMed

AbdelSalam, M; El Sissy, A; Samra, M A; Ibrahim, S; El Markaby, D; Gadallah, F

2008-06-01

Routine cytogenetic analysis frequently fails to identify an abnormal clone in B-cell lymphocytic leukaemia (B-CLL) due to poor response to mitogen stimulation. Fluorescence in situ hybridization (FISH) suggest that chromosomal abnormalities occur more frequently, most commonly trisomy 12, retinoblastoma gene deletion (Rb1 gene) and P53 gene deletion. 30 patients with B-CLL were enrolled in the trial from two centers in Cairo, Egypt during the period May 2000 to January 2002. Karyotyping and FISH assessment for possible chromosomal abnormalities (trisomy 12, Rb1 gene and P53 gene) were done at initial diagnosis. Results of cytogenetic abnormalities were correlated with clinical picture and survival. The median age was 57.4 years (range 40-75). Karyotyping technique showed that no metaphase could be detected in 30%, metaphase with normal karyotyping was observed in 63% and cytogenetic abnormalities were detected in two cases (one trisomy 12 and one deletion in chromosome 13). FISH examination of interphase and metaphase nuclei revealed cytogenetic abnormalities in 15 cases (50%), trisomy 12 in 9 (30%), Rb1 gene deletion in 5 (17%) and P53 gene deletion in 3. At diagnosis, patients with trisomy 12 were significantly associated with advanced stage and absolute lymphocyte count of >or=30,000/mm(3). Univariate analysis showed that absolute lymphocyte count >or=30,000/mm(3) (p=0.004) and trisomy 12 (p=0.024) were associated with poor progression free survival. Interphase and metaphase FISH studies improve the cytogenetic diagnosis of chromosomal abnormalities in B-CLL. Lymphocytosis and trisomy 12 may be a good indicator of poor prognosis.

The targeted inhibition of mitochondrial Hsp90 overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Chunlan; Department of Physiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058; Oh, Joon Seok

Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantlymore » inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis. Highlights: ► G-TPP binds to mitochondrial Hsp90. ► G-TPP induces apoptosis in U937 human leukemia cancer cells. ► G-TPP induces combination of death pathways in Hep3B cell. ► G-TPP overcomes the resistance conferred by Bcl-2 in Hep3B cells via necroptosis. ► G-TPP triggers Beclin 1

B cell-deficient mice display markedly enhanced resistance to the intracellular bacterium Brucella abortus.

PubMed

Goenka, Radhika; Parent, Michelle A; Elzer, Philip H; Baldwin, Cynthia L

2011-04-15

Brucella species are facultative intracellular bacteria that cause lifelong infections in humans and livestock. Here we evaluated the contribution of B cells in control of murine brucellosis in the more susceptible BALB/c and the more resistant C57BL/6 mice by infecting B cell-deficient mice. Strikingly, in the absence of B cells in both C57BL/6 and BALB/c mice, 99% and 99.5% of the infection found in wild type mice was cleared, respectively. This augmented clearance was not reversed in either strain by passive transfer of immune serum. In C57BL/6 mice, the clearance of infection coincided with an increase in interferon γ (IFN-γ)-producing CD4 and CD8 T cells and a reduction in interleukin 10 (IL-10)-producing cells. In BALB/c mice, this clearance was IFN-γ-dependent, as B cell/IFN-γ dual knockout mice were unable to clear the infection, and was inversely related to the levels of transforming growth factor β (TGF-β). Furthermore, B cells were found to produce TGF-β and IL-10 during early stages of infection in BALB/c wild-type and C57BL/6 wild-type mice, respectively. Thus, we demonstrate that the establishment of the high plateau phase of infection is dependent on non-antibody-mediated B cell effector mechanisms, including B regulatory functions, during murine brucellosis. © The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.

Using prognostic models in CLL to personalize approach to clinical care: Are we there yet?

PubMed

Mina, Alain; Sandoval Sus, Jose; Sleiman, Elsa; Pinilla-Ibarz, Javier; Awan, Farrukh T; Kharfan-Dabaja, Mohamed A

2018-03-01

Four decades ago, two staging systems were developed to help stratify CLL into different prognostic categories. These systems, the Rai and the Binet staging, depended entirely on abnormal exam findings and evidence of anemia and thrombocytopenia. Better understanding of biologic, genetic, and molecular characteristics of CLL have contributed to better appreciating its clinical heterogeneity. New prognostic models, the GCLLSG prognostic index and the CLL-IPI, emerged. They incorporate biologic and genetic information related to CLL and are capable of predicting survival outcomes and cases anticipated to need therapy earlier in the disease course. Accordingly, these newer models are helping develop better informed surveillance strategies and ultimately tailor treatment intensity according to presence (or lack thereof) of certain prognostic markers. This represents a step towards personalizing care of CLL patients. We anticipate that as more prognostic factors continue to be identified, the GCLLSG prognostic index and CLL-IPI models will undergo further revisions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeting BCL2 With BH3 Mimetics: Basic Science and Clinical Application of Venetoclax in Chronic Lymphocytic Leukemia and Related B Cell Malignancies.

PubMed

Roberts, A W; Huang, Dcs

2017-01-01

The intracellular protein B-cell-lymphoma-2 (BCL2) has been considered an attractive target for cancer therapy since the discovery of its function as a major promoter of cell survival (an anti-apoptotic) in the late 1980s. However, the challenges of targeting a protein-protein interaction delayed the discovery of fit-for-purpose molecules until the mid-2000s. Since then, a series of high affinity small organic molecules that inhibits the interaction of BCL2 with the apoptotic machinery, the so-called BH3-mimetics, have been developed. Venetoclax (formerly ABT-199) is the first to achieve US Food and Drug Administration approval, with an indication for treatment of patients with previously treated chronic lymphocytic leukemia (CLL) bearing deletion of the long arm of chromosome 17. Here, we review key aspects of the science underpinning the clinical application of BCL2 inhibitors and explore both our current knowledge and unresolved questions about its clinical utility, both in CLL and in other B-cell malignancies that highly express BCL2. © 2016 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Development of tumor-reactive T cells after nonmyeloablative allogeneic hematopoietic stem cell transplant for chronic lymphocytic leukemia.

PubMed

Nishida, Tetsuya; Hudecek, Michael; Kostic, Ana; Bleakley, Marie; Warren, Edus H; Maloney, David; Storb, Rainer; Riddell, Stanley R

2009-07-15

Allogeneic nonmyeloablative hematopoietic stem cell transplant (NM-HSCT) can result in durable remission of chronic lymphocytic leukemia (CLL). It is thought that the efficacy of NM-HSCT is mediated by recognition of tumor cells by T cells in the donor stem cell graft. We evaluated the development of CTLs specific for CLL after NM-HSCT to determine if their presence correlated with antitumor efficacy. Peripheral blood mononuclear cells obtained from 12 transplant recipients at intervals after NM-HSCT were stimulated in vitro with CLL cells. Polyclonal T-cell lines and CD8(+) T-cell clones were derived from these cultures and evaluated for lysis of donor and recipient target cells including CLL. The presence and specificity of responses was correlated with clinical outcomes. Eight of the 12 patients achieved remission or a major antitumor response and all 8 developed CD8(+) and CD4(+) T cells specific for antigens expressed by CLL. A clonal analysis of the CD8(+) T-cell response identified T cells specific for multiple minor histocompatibility (H) antigens expressed on CLL in six of the responding patients. A significant fraction of the CD8(+) T-cell response in some patients was also directed against nonshared tumor-specific antigens. By contrast, CLL-reactive T cells were not detected in the four patients who had persistent CLL after NM-HSCT, despite the development of graft-versus-host disease. The development of a diverse T-cell response specific for minor H and tumor-associated antigens expressed by CLL predicts an effective graft-versus-leukemia response after NM-HSCT.

Inter- and intra-cellular mechanism of NF-kB-dependent survival advantage and clonal expansion of radio-resistant cancer cells.

PubMed

Yu, Hui; Aravindan, Natarajan; Xu, Ji; Natarajan, Mohan

2017-02-01

Understanding the underlying mechanism by which cancer cells acquire resistance to radiation and favorably selected for its clonal expansion will provide molecular insight into tumor recurrence at the treatment site. In the present study, we investigated the molecular mechanisms prompted in MCF-7 breast cancer cells in response to clinical radiation and the associated coordination of intra- and inter-cellular signaling that orchestrate radio-resistance and tumor relapse/recurrence. Our findings showed that 2 or 10Gy of 137 Cs γ-rays at a dose rate of 1.03Gy/min trigger the activation of nuclear factor kappa B (NF-κB), its DNA-binding activity and recycles its own transcription. NF-κB DNA-binding kinetic analysis demonstrated both sustained and dual phase NF-κB activation with radiation. Gene manipulation approach revealed that radiation triggered NF-κB-mediated TNF-α transcriptional activity. TNF-α blocking approach confirmed that the de novo synthesis and secretion of TNF-α serves as a pre-requisite for the second phase of NF-κB activation and sustained maintenance. Radiation-associated NF-κB-dependent secretion of TNF-α from irradiated cells, in parallel, activates NF-κB in the non-targeted un-irradiated bystander cells. Together, these findings demonstrated that radiation-triggered NF-κB-dependent TNFα secretion is critical for self-sustenance of NF-κB (through autocrine positive feedback signaling) and for coordinating bystander response (through inter-cellular paracrine mechanism) after radiation exposure. Further, the data suggest that this self-sustained NF-κB in the irradiated cells determines radio-resistance, survival advantage and clonal expansion of the tumor cells at the treatment site. Parallel maintenance of NF-ΚB-TNF-α-NF-κB feedback-cycle in the un-irradiated non-targeted bystander cells initiates supportive mechanism for the promotion and progression of surviving tumor cells. Intervening this molecular pathway would help us to

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

PubMed Central

Knödgen, Eva; Göckeritz, Elisa; Vondey, Verena; Neumann, Lars; Herter, Sylvia; Klein, Christian; Hallek, Michael

2018-01-01

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism. PMID:29750146

p53 mutation, but not in vitro predictor genes of therapeutic efficacy of cisplatin, is clinically relevant in comparing partial and complete responder cases of maxillary squamous cell carcinoma.

PubMed

Kudo, Itsuhiro; Esumi, Mariko; Kida, Akihiro; Ikeda, Minoru

2010-10-01

To predict the efficacy of cisplatin and radiation therapy for maxillary squamous cell carcinoma, we examined the mRNA expression of 14 cisplatin-resistant genes and p53 mutation in specimens biopsied from patients prior to initiation of therapy. Five of 10 patients had mutations in the p53 gene, of whom four had residual tumors pathologically following chemoradiotherapy (p=0.0476). Of 14 genes examined, the mRNA expression of ATP7B was significantly lower in cases that were resistant to chemoradiotherapy. Six genes including multidrug resistance protein 1 (MDR-1), multidrug resistance associated protein 1 (MRP-1), Cu++ transporting, beta polypeptide (ATP7B), xeroderma pigmentosum, complementation group A (XPA), excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC-1) and B-cell CLL/lymphoma 2 (BCL2) were down-regulated in cases of recurrent cancers. These results show that the evaluation of p53 mutation provides the most useful predictor of therapeutic effects. In responder cases, the drug-resistant genes that were determined in cell lines by culture do not necessarily translate into clinical relevance.

Safety and tolerability of ibrutinib monotherapy in Japanese patients with relapsed/refractory B cell malignancies.

PubMed

Tobinai, Kensei; Ogura, Michinori; Ishizawa, Kenichi; Suzuki, Tatsuya; Munakata, Wataru; Uchida, Toshiki; Aoki, Tomohiro; Morishita, Takanobu; Ushijima, Yoko; Takahara, Satoko

2016-01-01

In this phase I dose-escalation study we evaluated the safety, tolerability, pharmacokinetics, and antitumor activity of ibrutinib, an oral covalent inhibitor of Bruton's tyrosine kinase (BTK, in Japanese patients with relapsed/refractory B cell malignancies (RRBCM). Fifteen patients aged 42-78 years were enrolled to one of three cohorts. Cohort 1 (n = 3) consisted of two phases, a single-dose (140 and 280 mg) phase and a multiple-dose (420 mg) phase of ibrutinib; cohort 2 (n = 6) included multiple doses of ibrutinib 560 mg; and cohort 3 (n = 6) included only patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) dosed at ibrutinib 420 mg. One patient (CLL/SLL cohort) experienced grade 3 pneumonia and sepsis, which were considered dose-limiting toxicities. No deaths were reported. The most common (≥ 20% patients) adverse events were neutropenia, anemia, nasopharyngitis, increased bilirubin, and rash. Dose-dependent increase in maximum plasma concentration and area under the concentration from 0 to the last quantifiable time was observed, while time to reach maximum plasma concentration and elimination half-life was similar between doses. The overall response rate was 73.3% (11/15) for all cohorts combined. Overall, ibrutinib (420 and 560 mg) was tolerable with acceptable safety profiles and effective for Japanese patients with RRBCM including CLL/SLL. NCT01704963.

Comparing efficacy of reduced-toxicity allogeneic hematopoietic cell transplantation with conventional chemo-(immuno) therapy in patients with relapsed or refractory CLL: a Markov decision analysis.

PubMed

Kharfan-Dabaja, M A; Pidala, J; Kumar, A; Terasawa, T; Djulbegovic, B

2012-09-01

Despite therapeutic advances, relapsed/refractory CLL, particularly after fludarabine-based regimens, remains a major challenge for which optimal therapy is undefined. No randomized comparative data exist to suggest the superiority of reduced-toxicity allogeneic hematopoietic cell transplantation (RT-allo-HCT) over conventional chemo-(immuno) therapy (CCIT). By using estimates from a systematic review and by meta-analysis of available published evidence, we constructed a Markov decision model to examine these competing modalities. Cohort analysis demonstrated superior outcome for RT-allo-HCT, with a 10-month overall life expectancy (and 6-month quality-adjusted life expectancy (QALE)) advantage over CCIT. Although the model was sensitive to changes in base-case assumptions and transition probabilities, RT-allo-HCT provided superior overall life expectancy through a range of values supported by the meta-analysis. QALE was superior for RT-allo-HCT compared with CCIT. This conclusion was sensitive to change in the anticipated state utility associated with the post-allogeneic HCT state; however, RT-allo-HCT remained the optimal strategy for values supported by existing literature. This analysis provides a quantitative comparison of outcomes between RT-allo-HCT and CCIT for relapsed/refractory CLL in the absence of randomized comparative trials. Confirmation of these findings requires a prospective randomized trial, which compares the most effective RT-allo-HCT and CCIT regimens for relapsed/refractory CLL.

The pan phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) blocks survival, adhesion and proliferation of primary chronic lymphocytic leukemia cells.

PubMed

Thijssen, R; Ter Burg, J; van Bochove, G G W; de Rooij, M F M; Kuil, A; Jansen, M H; Kuijpers, T W; Baars, J W; Virone-Oddos, A; Spaargaren, M; Egile, C; van Oers, M H J; Eldering, E; Kersten, M J; Kater, A P

2016-02-01

The phosphoinositide 3-kinases (PI3Ks) are critical components of the B-cell receptor (BCR) pathway and have an important role in the pathobiology of chronic lymphocytic leukemia (CLL). Inhibitors of PI3Kδ block BCR-mediated cross-talk between CLL cells and the lymph node microenvironment and provide significant clinical benefit to CLL patients. However, the PI3Kδ inhibitors applied thus far have limited direct impact on leukemia cell survival and thus are unlikely to eradicate the disease. The use of inhibitors of multiple isoforms of PI3K might lead to deeper remissions. Here we demonstrate that the pan-PI3K/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) was more pro-apoptotic to CLL cells--irrespective of their ATM/p53 status--than PI3Kα or PI3Kδ isoform selective inhibitors. Furthermore, SAR245409 blocked CLL survival, adhesion and proliferation. Moreover, SAR245409 was a more potent inhibitor of T-cell-mediated production of cytokines, which support CLL survival. Taken together, our in vitro data provide a rationale for the evaluation of a pan-PI3K inhibitor in CLL patients.

Duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocytic leukemia cells to venetoclax (ABT-199)

PubMed Central

Patel, Viralkumar M.; Balakrishnan, Kumudha; Douglas, Mark; Tibbitts, Thomas; Xu, Ethan Y.; Kutok, Jeffery L.; Ayers, Mary; Sarkar, Aloke; Guerrieri, Renato; Wierda, William G.; O’Brien, Susan; Jain, Nitin; Stern, Howard M.; Gandhi, Varsha

2017-01-01

Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin’s lymphoma (iNHL). In CLL, duvelisib monotherapy is associated with high iwCLL and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene-expression studies (RNA seq, Nanostring, Affymetrix array, and real time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes. In concert with induction of transcript levels, reverse phase protein arrays and immunoblots confirmed increase at the protein level. The BCL2 inhibitor venetoclax induced greater apoptosis in ex-vivo cultured CLL cells obtained from patients on duvelisib compared to pre-treatment CLL cells from the same patients. In vitro combination of duvelisib and venetoclax resulted in enhanced apoptosis even in CLL cells cultured under conditions that simulate the tumor microenvironment. These data provide a mechanistic rationale for testing the combination of duvelisib and venetoclax in the clinic. Such combination regimen (NCT02640833) is being evaluated for patients with B-cell malignancies including CLL. PMID:28017967

Duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocytic leukemia cells to venetoclax (ABT-199).

PubMed

Patel, V M; Balakrishnan, K; Douglas, M; Tibbitts, T; Xu, E Y; Kutok, J L; Ayers, M; Sarkar, A; Guerrieri, R; Wierda, W G; O'Brien, S; Jain, N; Stern, H M; Gandhi, V

2017-09-01

Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin's lymphoma. In CLL, duvelisib monotherapy is associated with high iwCLL (International Workshop on Chronic Lymphocytic Leukemia) and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene expression studies (RNAseq, Nanostring, Affymetrix array and real-time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes. In concert with induction of transcript levels, reverse phase protein arrays and immunoblots confirmed increase at the protein level. The BCL2 inhibitor venetoclax induced greater apoptosis in ex vivo-cultured CLL cells obtained from patients on duvelisib compared with pre-treatment CLL cells from the same patients. In vitro combination of duvelisib and venetoclax resulted in enhanced apoptosis even in CLL cells cultured under conditions that simulate the tumor microenvironment. These data provide a mechanistic rationale for testing the combination of duvelisib and venetoclax in the clinic. Such combination regimen (NCT02640833) is being evaluated for patients with B-cell malignancies including CLL.

Adoptive cellular therapy for chronic lymphocytic leukemia and B cell malignancies. CARs and more.

PubMed

Castro, Januario E; Kipps, Thomas J

2016-03-01

Treatment of patients with chronic lymphocytic leukemia and other B cell malignancies is evolving very rapidly. We have observed the quick transition during the last couple of years, from chemo-immunotherapy based treatments to oral targeted therapies based on B cell receptor signaling and Bcl-2 inhibitors, as well as the increasing use of second generation glyco-engineered antibodies. The next wave of revolution in the treatment for this conditions is approaching and it will be based on strategies that harness the power of the immune system to fight cancer. In the center of this biotechnological revolution is cellular engineering, the field that had made possible to redirect the immune system effector cells to achieve a more effective and targeted adoptive cellular therapy. In this chapter, we will review the historical context of these scientific developments, the most recent basic and clinical research in the field and some opinions regarding the future of adoptive cellular therapy in CLL and other B cell malignancies. Copyright © 2016 Elsevier Ltd. All rights reserved.

ARResT/AssignSubsets: a novel application for robust subclassification of chronic lymphocytic leukemia based on B cell receptor IG stereotypy.

PubMed

Bystry, Vojtech; Agathangelidis, Andreas; Bikos, Vasilis; Sutton, Lesley Ann; Baliakas, Panagiotis; Hadzidimitriou, Anastasia; Stamatopoulos, Kostas; Darzentas, Nikos

2015-12-01

An ever-increasing body of evidence supports the importance of B cell receptor immunoglobulin (BcR IG) sequence restriction, alias stereotypy, in chronic lymphocytic leukemia (CLL). This phenomenon accounts for ∼30% of studied cases, one in eight of which belong to major subsets, and extends beyond restricted sequence patterns to shared biologic and clinical characteristics and, generally, outcome. Thus, the robust assignment of new cases to major CLL subsets is a critical, and yet unmet, requirement. We introduce a novel application, ARResT/AssignSubsets, which enables the robust assignment of BcR IG sequences from CLL patients to major stereotyped subsets. ARResT/AssignSubsets uniquely combines expert immunogenetic sequence annotation from IMGT/V-QUEST with curation to safeguard quality, statistical modeling of sequence features from more than 7500 CLL patients, and results from multiple perspectives to allow for both objective and subjective assessment. We validated our approach on the learning set, and evaluated its real-world applicability on a new representative dataset comprising 459 sequences from a single institution. ARResT/AssignSubsets is freely available on the web at http://bat.infspire.org/arrest/assignsubsets/ nikos.darzentas@gmail.com. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Y-box-binding protein-1 (YB-1) promotes cell proliferation, adhesion and drug resistance in diffuse large B-cell lymphoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, Xiaobing; Wu, Yaxun; Wang, Yuchan

YB-1 is a multifunctional protein, which has been shown to correlate with resistance to treatment of various tumor types. This study investigated the expression and biologic function of YB-1 in diffuse large B-cell lymphoma (DLBCL). Immunohistochemical analysis showed that the expression statuses of YB-1 and pYB-1{sup S102} were reversely correlated with the clinical outcomes of DLBCL patients. In addition, we found that YB-1 could promote the proliferation of DLBCL cells by accelerating the G1/S transition. Ectopic expression of YB-1 could markedly increase the expression of cell cycle regulators cyclin D1 and cyclin E. Furthermore, we found that adhesion of DLBCLmore » cells to fibronectin (FN) could increase YB-1 phosphorylation at Ser102 and pYB-1{sup S102} nuclear translocation. In addition, overexpression of YB-1 could increase the adhesion of DLBCL cells to FN. Intriguingly, we found that YB-1 overexpression could confer drug resistance through cell-adhesion dependent and independent mechanisms in DLBCL. Silencing of YB-1 could sensitize DLBCL cells to mitoxantrone and overcome cell adhesion-mediated drug resistance (CAM-DR) phenotype in an AKT-dependent manner. - Highlights: • The expression statuses of YB-1 and pYB-1{sup S102} are reversely correlated with outcomes of DLBCL patients. • YB-1 promotes cell proliferation by accelerating G1/S transition in DLBCL. • YB-1 confers drug resistance to mitoxantrone in DLBCL.« less

Changes in T-cell subpopulations and cytokine network during early period of ibrutinib therapy in chronic lymphocytic leukemia patients: the significant decrease in T regulatory cells number.

PubMed

Podhorecka, Monika; Goracy, Aneta; Szymczyk, Agnieszka; Kowal, Malgorzata; Ibanez, Blanca; Jankowska-Lecka, Olga; Macheta, Arkadiusz; Nowaczynska, Aleksandra; Drab-Urbanek, Elzbieta; Chocholska, Sylwia; Jawniak, Dariusz; Hus, Marek

2017-05-23

B cell receptor (BCR) stimulation signal plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), and kinase inhibitors directed toward the BCR pathway are now the promising anti-leukemic drugs. Ibrutinib, a Bruton tyrosine kinase inhibitor, demonstrates promising clinical activity in CLL. It is reported that ibrutinib, additionally to directly targeting leukemic cells, also inhibits the interactions of these cells with T cells, macrophages and accessory cells. Assessment of these mechanisms is important because of their non -direct anti-leukemic effects and to identify possible side effects connected with long-term drug administration.The aim of this study was to assess the in vivo effects of ibrutinib on T-cell subpopulations and cytokine network in CLL. The analysis was performed on a group of 19 patients during first month of ibrutinib therapy. The standard multicolor flow cytometry and cytometric bead array methods were used for assessment of T-cell subsets and cytokines/chemokines, respectively.The data obtained indicates that Ibrutinib treatment results in changes in T-cell subpopulations and cytokine network in CLL patients. Particularly, a significant reduction of T regulatory cells in peripheral blood was observed. By targeting these populations of T cells Ibrutinib can stimulate rejection of tumor cells by the immune system.

How I treat CLL patients with ibrutinib.

PubMed

Brown, Jennifer R

2018-01-25

Ibrutinib is a transformative therapy for high-risk and relapsed refractory chronic lymphocytic leukemia (CLL) patients. In clinical trials in relatively healthy younger patients, ibrutinib has been well tolerated. As its use has become more widespread in the community, however, its full adverse event profile has emerged and proven more challenging than was initially anticipated. Reports of community-based use have estimated discontinuation rates as high as 40% in the first year of therapy. This article therefore reviews my approach to the evaluation and management of a CLL patient starting on ibrutinib, with the goal of minimizing and managing toxicity to maintain patients on ibrutinib. Key topics discussed include bleeding risk; cardiac complications, particularly atrial fibrillation; drug interactions; and infections. © 2018 by The American Society of Hematology.

CAR T-cells merge into the fast lane of cancer care.

PubMed

Frey, Noelle V; Porter, David L

2016-01-01

Chimeric antigen receptors (CARs) can be introduced into T-cells redirecting them to target specific tumor antigens. CAR-modified T cells targeting CD19 have shown remarkable activity against CD19+ malignancies including B cell acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphomas (NHL). Complete remission rates as high as 90% have been observed for patients with relapsed and refractory ALL and greater than 50% response rates have been seen in heavily pre-treated CLL and NHL. Excitingly, some remissions have been durable without any additional therapy, a finding which correlates with in-vivo T-cell persistence and B-cell aplasia. The major treatment related toxicities include B-cell aplasia, neurologic toxicities, and a potentially severe cytokine release syndrome. This review summarizes outcomes for patients treated with CD19-CAR T-cells while exploring the field's challenges and future directions. © 2015 Wiley Periodicals, Inc.

Targeting SYK kinase-dependent anti-apoptotic resistance pathway in B-lineage acute lymphoblastic leukaemia (ALL) cells with a potent SYK inhibitory pentapeptide mimic.

PubMed

Uckun, Fatih M; Ek, Rauf O; Jan, Shyi-Tai; Chen, Chun-Lin; Qazi, Sanjive

2010-05-01

The present study found that the pentapeptide mimic C-61, targeting the substrate binding P-site of SYK tyrosine kinase acted as a potent inducer of apoptosis in chemotherapy-resistant SYK-expressing primary leukemic B-cell precursors taken directly from relapsed B-precursor leukaemia (BPL) patients (but not SYK-deficient infant pro-B leukaemia cells), exhibited favourable pharmacokinetics in mice and non-human primates, and eradicated in vivo clonogenic leukaemia cells in severe combined immunodeficient mouse xenograft models of chemotherapy-resistant human BPL at dose levels non-toxic to mice and non-human primates. These in vitro and in vivo findings provide proof of principle for effective treatment of chemotherapy-resistant BPL by targeting SYK-dependent anti-apoptotic blast cell survival machinery with a SYK P-Site inhibitor. Further development of C-61 may provide the foundation for therapeutic innovation against chemotherapy-resistant BPL.

Positive regulation of deoxycytidine kinase activity by phosphorylation of Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes.

PubMed

Smal, Caroline; Van Den Neste, Eric; Maerevoet, Marie; Poiré, Xavier; Théate, Ivan; Bontemps, Françoise

2007-08-08

Deoxycytidine kinase (dCK) activates several antileukaemic nucleoside analogues. We have recently reported that the activity of dCK, overexpressed in HEK 293T cells, correlates with its phosphorylation level on Ser-74. Here, we show that dCK from B-cell chronic lymphocytic leukaemia (B-CLL) lymphocytes can be detected by an anti-phospho-Ser-74 antibody and that interindividual variability in dCK activity is related to its phosphorylation level on Ser-74. Moreover, pharmacological intervention modified Ser-74 phosphorylation, in close parallel with changes in dCK activity. These results suggest that activation of dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogues.

Tenascin-C induces resistance to apoptosis in pancreatic cancer cell through activation of ERK/NF-κB pathway.

PubMed

Shi, Meiyan; He, Xiaodan; Wei, Wei; Wang, Juan; Zhang, Ti; Shen, Xiaohong

2015-06-01