B-cell Chronic Lymphocytic Leukemia Patients Research Articles

Enrichment of T-Lymphocytes from Leukemic Blood using Inertial Microfluidics towards Improved Chimeric Antigen Receptor-T Cell Manufacturing

Chimeric antigen receptor (CAR-T) cell therapy is a successful immunotherapy for treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (CLL) patient's samples. In healthy donors used to optimise the process, the lymphocyte purity was enriched from 65% (SD±0.2) to 91% (SD±0.06) and T-cell purity was enriched from 45% (SD±0.1) to 73% (SD±0.02). Leukemic samples had higher starting B-cell compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells was achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD±0.09), 89% (SD±0.1) and 74% (SD±0.09) respectively, and a 70% (SD±0.1) T-cell recovery. CLL samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the CAR-T manufacturing workflow.

Bcl-2 Family (Bcl-2, Mcl-1, Bim and Bax) and Btk Expression in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Lymphocytes in Different Lymphoid Compartments and Changes during Btk Inhibitor Therapies (ibrutinib or acalabrutinib)

B-cell chronic lymphocytic leukemia (B-CLL) is characterized with variable infiltration by B -CLL lymphocytes of various lymphoid compartments, i.e peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) and lymphoid organs. Interactions with various microenvironments may result in different disease activity and resistance to apoptosis. Novel agents targeting Btk and Bcl-2 show remarkable activity in B-CLL. However, they may have different activity in different lymphoid compartments while Btk inhibitors also may cause significant B-CLL lymphocyte redistribution between compartments. Aim of this study was to evaluate: 1) is there a different expression of Bcl-2 family of anti- and pro-apoptotic proteins (Bcl-2, Bax, Bim, mcl-1) and Btk in B-CLL lymphocytes from different lymphoid compartments; 2) relationship of observed expressions to disease parameters (TTM, TD, stage, B2MG, LDH); 3) changes during treatment with Btk inhibitors. We have included 28 B-CLL patients (18/10 M/F, median age 71 yr, range 48-85) treated with Btk inhibitors (ibrutinib 24, acalabrutinib 4). There were 15 previously treated patients and 13 treatment naïve patients. All evaluated patients had samples from PB and BM before treatment while 19 patients had also LN samples. At least one follow-up sample from PB had 20 patients. Median TTM score was 10.2 (range 3.4-23.9) and TD score was 0.68. Rai stage III/IV had 8 patients. Expression of Bcl-2, Mcl-1, Bim, Bax and Btk was determined by flow cytometry in CD19+CD5+ lymphocytes from fresh samples taken simultaneously by venipuncture (PB) or fine needle aspiration (BM and LN). There is higher expression of Bcl-2, Mcl-1 in LN compared to PB and BM (p<0.05), while there is no change in follow-up PB during ibrutinib/acalbrutinib treatment. There is no significant difference in Bim expression between compartments, while there is trend for higher expression in PB follow-up sample (p=0.054). There is no significant difference in Bax expression between PB, BM and LN compartments, while there is significant drop in Bax expression in PB follow-up sample during treatment (p<0.05). p-Btk has higher expression in LN compared to PB and BM (p<0.05). Follow-up PB p-Btk expression is comparable to pretreatment expression in PB (despite redistribution). We haven't found significant correlation of Bcl-2 family expression and p-Btk expression to the parameters of tumor mass (TTM) and tumor distribution (TD). In conclusion, there is a different pattern of expression of anti-apoptotic and pro-apoptotic Bcl-2 family members and Btk between lymphoid compartments. Our results may indicate higher BCR signaling and higher antiapoptotic activity in B-CLL lymphocytes in lymph node microenvironment. With redistribution during Btk inhibitor treatment B-CLL lymphocytes assume PB phenotype with downregulation of Btk and anti-apoptotic Bcl-2 and Mcl-1 proteins. Downregulation of pro-apoptotic Bax and possible upregulation of Bim protein indicate that there may be more complex interplay between various proteins resulting in different disease course. These results again emphasize the role of LN microenvironment in pathogenesis of B-CLL and may provide insights for treatment strategies combining targeted agents (Btk inhibitors and Bcl-2 antagonists).

Risk factors for iron metabolic disorders in newly diagnosed B-chronic lymphocytic leukemia patients

Due to the importance of increasing the incidence conditions of B cell chronic lymphocytic leukemia (B-CLL) that requiring accurate knowledge of the biology behind the pathogenesis and risks of this disease of each individual patient, this study was designed to investigate if hormonal alterations such as Hepcidin, Erythropoietin, and oxidant-antioxidant status may be useful in the understanding of B-CLL development in addition the relationship of these parameters with anemia in advance clinical stage in newly diagnosed patients with B-CLL. This study was conducted on 60 newly diagnosed patients with B-CLL and 30 control group .The socio-demographic information including (age and sex) and clinical data were collected for the period from 20 February until 25 December  2021. The result of the present study showed that out of the 60 newly diagnosed B-CLL patients, 39 was male and 21 was females, with the mean age ( 64.04 ±  5.6 ) years and out of the 30 controls group ,12 was males and 18 was females with the mean age of ( 63.33 ± 4.70) years .therefore, it was a higher prevalence p value ≤0.05 of newly diagnosed B-CLL patients in males (65.0 %) compare to females (35.0 % ). 

Abstract 2749: iPSC-derived natural killer cells as the front-runner program of the EVOcells Oncology platform: From inception to translational validation using patient samples

Abstract Cancer immunotherapy has transformed the oncology landscape and shown promising therapeutic results in the clinic but it is also clearly facing multiple limitations. So far, the focus of immunotherapies has mainly been on the modulation of T-cells. Natural killer (NK) cells offer promising perspectives for immunotherapies as they directly kill tumor cells and produce pro-inflammatory cytokines to activate and recruit T-cells and other immune cells. Thus, NK cells are considered key players in the anti-tumor immune response that can make a “cold” tumor “hot”. However, in the tumor microenvironment (TME) of cancer patients, the low numbers of NK cells present and their exhaustion due to local immunosuppression often limit tumor cell killing. The development of NK based cell therapy products can overcome these limitations, either as single therapy or in combination with biologics such as e.g. NK cell engagers or monoclonal antibodies for boosting ADCC (antibody dependent cell cytotoxicity). To overcome the main hurdle of current autologous cell therapies, the EVOcells Oncology platform aims at developing a streamlined manufacturing process for iPSC-derived immune effector cells. The goal is to develop “off-the-shelf” cell therapy products for clinical use in large patient populations. We are currently establishing a multi-asset portfolio combining multiple immune effector cell types with different genetic editing strategies that serve to optimize this approach further. iPSC-derived NK cells (iNK cells) offer an attractive option for improving both therapeutic efficacy and safety of CAR cell therapies. Our robust, feeder-free production protocol produces iNK cells that can be reliably frozen with good recovery rates. We have generated multiple genetically modified iNK cells and have validated that the genetic modifications boost iNK function. At the end of the production process, iNK cells show phenotypic properties and single cell RNA sequencing profiles comparable to blood-derived NK cells. Importantly, iNK cells are fully functional with the ability to produce key pro-inflammatory cytokines and to form lytic immunological synapses leading to efficient killing of cancer cell lines. In addition to the killing of established tumor cell lines, we also evaluated the ability of iNK cells to kill primary leukemia cells ex vivo by using blood samples from B-cell chronic lymphocytic leukemia patients. In combination with a standard of care anti-CD20 antibody, iNK cells demonstrated the ability to kill primary leukemia cells via ADCC. Taken together, these promising results obtained with iNK cells show the potential of the EVOcells Oncology platform to deliver the next generation of cell therapies in oncology paving the way to develop a portfolio of iPSC-derived immune effector cells. Citation Format: Michael Esquerré, Audrey Holtzinger, Mélanie Pichery, Stefanie Pfaender, Saniye Yumlu, Mandy Richter, Delphine Betous, Oriane Bombarde, Mylène Gador, Nadja Sailer, Michael Paillasse, Loïc Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, Markus Dangl. iPSC-derived natural killer cells as the front-runner program of the EVOcells Oncology platform: From inception to translational validation using patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2749.

Circulating Endocannabinoids in Canine Multicentric Lymphoma Patients.

The endocannabinoid system is increasingly being implicated in the pathogenesis and progression of various human cancers. Specifically, increased levels of 2-arachidonoylglycerol (2-AG) and oleoythanolamide (OEA) have been demonstrated in human diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) patients, respectively. The objectives of this paper were to compare 2-AG, OEA, N-arachidonoylethanolamine (AEA), and palmitoylethanolamide (PEA) levels between dogs with multicentric lymphoma and healthy control dogs. In addition, evaluate 2-AG, OEA, AEA, and PEA levels as biomarkers for progression free interval (PFI) and overall survival time (OST) in the dogs with lymphoma. The study consisted of 26 dogs with multicentric B cell lymphoma, 14 dogs with multicentric T cell lymphoma, and 12 healthy control dogs. Serum 2-AG, OEA, AEA, and PEA levels were measured using liquid chromatography combined with tandem mass spectrometry (LC—MS/MS) in dogs with lymphoma and in healthy dogs. OEA, AEA, and PEA levels were significantly elevated in dogs with lymphoma compared to healthy controls (p < 0.05). Total AG was significantly higher in healthy control dogs (p = 0.049). There was no significant difference between dogs with B cell and T cell lymphoma for any of the measured endocannabinoids. Elevated PEA was significantly associated with decreased PFI (p = 0.04) in dogs with lymphoma with a hazards ratio of 1.816 [95% Confidence Interval (CI): 1.020–3.232]. Overall, dogs with lymphoma have elevated levels of OEA, AEA, and PEA. PEA levels have the potential to be a prognostic biomarker.

CYTOGENETIC STUDY OF MANIFESTATIONS OF THE UNIVERSAL PHENOMENON OF THE BYSTANDER RESPONSE.

to establish the level of chromosomal instability in peripheral blood lymphocytes (PBL) from healthyindividuals and cells from patients with B-cell chronic lymphocytic leukemia (CLL) under co-cultivation conditions and to determine the possibility of inducing manifestations of the universal phenomenon of bystander response in them. Cytogenetic analysis of uniformly stained chromosomes from human PBL, which differed by cytogenetic markers of sex; the presence of oncological transformation and in vitro irradiation of 137Cs in a dose of 0.50 Gy under the conditions of their joint cultivation was performed. The frequency of chromosome aberrations in PBL from healthy individuals when co-cultured with the blood from CLL patients was 3.35 per 100 cells, exceeded the control (1.48 per 100 cells, р < 0.01), did not significantly differ from the rate in non-irradiated blood T-lymphocytes from patients with B-cell CLL (3.18 per 100 cells, р > 0.05) and was lower than when co-cultivated with irradiated blood from CLL patients (5.00 per 100 cells, р < 0.01). In irradiated in vitro blood lymphocytes from CLL patients, the mean group level of chromosome aberrations under separate cultivation was 12.36 per 100 cells and exceeded the indicator during their co-cultivation with lymphocytes from healthy individuals (8.35 per 100 cells, р < 0.05). A tumor-induced bystander effect (TIBE) develops in PBL from healthy individuals when co-cultured with the blood from CLL patients and in T lymphocytes of B-cell CLL patients, the cytogenetic manifestation of which is an increase in the frequency of single chromatid fragments. The interaction of irradiated blood cells from CLL patients with lymphocytes from healthy individuals causes an increase in chromosomal instability in the latter due to the development of a radiation-induced bystander effect (RIBE) and leads to a decrease in the level of chromosomal instability in irradiated lymphocytes from CLL patients (rescue effect). An increase in the level of chromatid-type aberrations in T-lymphocytes of CLL patients during in vitro irradiation is a consequence of the development of RIBE against the background of TIBE.

Intronic SNP - rs9288953 Of Suppressor Molecule Btla Confers Susceptibility To B-Cell Chronic Lymphocytic Leukemia

The contribution of the immune system to the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) have been receiving growing attention. The increase of cancer risk in immunodeficient individuals has been documented in population and experimental studies (Gurlich et al 2007; Swann et al 2007, Smyth et al 2006). A new member of co-inhibitory molecules B - and T-lymphocyte attenuator (BTLA) that inhibits lymphocyte activation could influence cancer susceptibility (Watanabe et al 2003). A high expression of BTLA was observed on malignant B cells from chronic lymphocytic leukemia/small lymphocytic leukemia (M'Hidi et al. 2009). As BTLA synthesis depends on the rate of gene transcription and/or translation, polymorphism in the corresponding gene might result in abnormal expression and function.The aim of this studywas to evaluate possible associations between selected on the basis of literature (Fu et al 2009) single nucleotide polymorphisms (SNPs) existing within exons and introns of BTLA gene: rs9288952A>G, rs1844089G>A, rs2705535C>T and 9288953C>T and susceptibility to B-CLL.Genotyping was done using allelic discrimination method with the TaqMan SNP Genotyping Assays in 300 B-CLL patients (pts.) (203 pts. from Department of Hematology, Medical University Lodz, Poland and 97 from Department of Clinical Immunology and Immunotherapy, Medical University of Lublin, Poland (138 female/162 male) and in 417 healthy individuals (168 female/243 male) originating from the same region of Poland.The significant differences in distribution of alleles and genotypes was observed for intronic SNP -rs9288953. The frequency of T allele and T alleles carriers (CT+TT genotypes) were higher in patients than in healthy controls as compare to C allele and CC genotypes (0.458 vs. 0.375, p=0.0016; OR:1.41, 95%CI - 1.13-1.75 and 0.677 vs. 0.606, p=0.0022; OR:1.67, 95%CI- 1.16-1.2.40, respectively). None of other investigated SNPs have shown different frequency of alleles or genotypes in group of patients and controls.The haplotype analysis of rs9288952A>G, rs1844089G>A, rs2705535C>T and 9288953C>T showed that the most common haplotypes rs9288952A>G[A]/rs1844089G>A[G]/rs2705535C>T[C]/ 9288953C>T[C] (named as AGCC) and rs9288952A>G[A]/rs1844089G>A[G]/rs2705535C>T[C]/ 9288953C>T[T] (named as AGCT) which were differentiate only by variants of 9288953C>T SNP were completely contrary associated with disease susceptibility. Haplotype AGCC is associated with decreased risk of B-CLL (OR: 0.72, 95%CI-0.59-0.90 p with Bonferroni correction=0.01), while AGCT with increased risk of disease (OR: 1.42, 95%CI-1.14-1.76 p with Bonferroni correction=0.005)Our data indicated for the first time the possible role of intronic polymorphism 9288953C>T in gene encoding suppressor molecule BTLA in susceptibility to B-CLL. ReferencesFu Z, Li D, Jiang W, Wang L, Zhang J, Xu F, Pang D, Li D. Association of BTLA gene polymorphisms with the risk of malignant breast cancer in Chinese women of Heilongjiang Province. Breast Cancer Res Treat. 2010 Feb;120(1):195-202.Grulich AE, van Leeuwen MT, Falster MO, Vajdic CM. Incidence of cancers in people with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis. Lancet. 2007 Jul 7;370(9581):59-67.M'Hidi H, Thibult ML, Chetaille B, Rey F, Bouadallah R, Nicollas R, Olive D, Xerri L. High expression of the inhibitory receptor BTLA in T-follicular helper cells and in B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia.Am J Clin Pathol. 2009 Oct;132(4):589-96.Smyth MJ, Dunn GP, Schreiber RD. Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol. 2006;90:1-50.Swann JB, Smyth MJ. Immune surveillance of tumors. J Clin Invest. 2007 May;117(5):1137-46.Watanabe N, Gavrieli M, Sedy JR, Yang J, Fallarino F, Loftin SK, Hurchla MA, Zimmerman N, Sim J, Zang X, Murphy TL, Russell JH, Allison JP, Murphy KM. BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1.Nat Immunol. 2003 Jul;4(7):670-9. Disclosures:No relevant conflicts of interest to declare.

In vivo potential of recombinant granulysin against human melanoma

9-kDa granulysin is a protein expressed into the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It has been shown to exert cytolysis on microbes and tumors. We showed previously that 9-kDa granulysin exerted cell death by apoptosis in vitro on hematological tumor cell lines and also on cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. In addition, we have shown the anti-tumor efficiency of granulysin as a single agent in two in vivo models of human tumor development in athymic mice, the MDA-MB-231 mammary adenocarcinoma and the NCI-H929 multiple myeloma, without signs of overt secondary effects by itself. In this work, we have tested recombinant 9-kDa granulysin in an in vivo and especially aggressive model of melanoma development, xenografted UACC62 cells in athymic mice. Recombinant granulysin was administered once UACC62-derived tumors were detectable and it substantially retarded the in vivo development of this aggressive tumor. We could also detect apoptosis induction and increased NK cell infiltration inside granulysin-treated tumor tissues. These observations are especially interesting given the possibility of treating melanoma by intra-tumor injection.

Role of cytogenetic abnormalities detected by fluorescence in situ hybridization as a prognostic marker: Pathogenesis & clinical course in patients with B-chronic lymphocytic leukaemia.

Background & objectives:B-cell chronic lymphocytic leukaemia (B-CLL) is one of the most common forms of adult leukaemia, with a highly variable clinical course. Specific chromosomal and genetic aberrations are used clinically to predict prognosis, independent from conventional clinical markers. Molecular cytogenetic methods such as fluorescence in situ hybridization (FISH) detect aberrations in up to 80 per cent B-CLL patients. This study was conducted to score the frequencies of recurrent aberrations, i.e., del(13q14), trisomy 12, del(11q22), del(17p13), del(6q21) and IgH (immunoglobulin heavy chain) translocations and to understand their role in prognostication and risk stratification.Methods:FISH studies were performed on bone marrow aspirate or peripheral blood of 280 patients using commercially available disease-specific probe set. The data were correlated with clinical and haematological parameters such as low haemoglobin, splenomegaly and lymphadenopathy.Results:Chromosomal aberrations were detected in 79 per cent of patients, with del(13q14) (57%) as the most common cytogenetic aberration, followed by trisomy 12 (27%), del(11q22) (22%), t(14q32) (19%), del(17p13) (18%) and del(6q21) (9%). Single or in coexistence with other aberration del(13q14) had a favourable outcome in comparison to del(11q22), t(14q32), del(17p13) and del(6q21) which were associated with advanced stages of the disease. Trisomy 12 had a variable clinical course.Interpretation & conclusions:FISH was found to be a sensitive and efficient technique in detecting the prevalence of recurrent cytogenetic abnormalities. Each of these aberrations is an important independent predictor of disease progression and survival which aids in designing risk-adapted treatment strategies for better disease management.

Flow Cytometric Analysis of ZAP-70 Protein Expression for B-Cell Chronic Lymphocytic Leukemia Prognostication: Usefulness and Limitations.

Objectives: The heterogenous clinical course in B-cell chronic lymphocytic leukemia (B-CLL) can be linked to several genetic and phenotypic characteristics of malignant B-cells. Prognostic analysis in B-CLL is routinely carried out to assist patient management; particularly to predict the time to initiate treatment. Increased ZAP-70 expression is a surrogate marker for unmutated immunoglobulin genes and inferior clinical outcomes which can be quantified to predict future outcomes in B-CLL patients. The study determined the ZAP-70 expression pattern using Z-index in Pakistani patients with B-CLL.Methods: A retrospective analysis of B-CLL cases diagnosed and confirmed on flow cytometry at Aga Khan University Hospital for the last six years which had also undergone ZAP-70 analysis were included. In all these cases, ZAP-70 expression was quantified by measuring mean fluorescence intensities (MFIs) of normal B-cells, T-cells, and CLL-cells (CD19 and CD5 double-positive population). ZAP-70 expression was divided into high, low, and negative categories based on Z-index calculation. Mann-Whitney U test was utilized to determine the significance of ZAP-70 variations in different age groups and genders. P-value <0.05 was considered significant.Results: A total of 120 patients of B-CLL had ZAP-70 analysis during the study period. The median age was 62 with an interquartile range of 35-87 and male to female ratio of 2:1. ZAP-70 expression was high in 18 (15%), low in 52 (43.3%) and negative in 50 (41.7%) cases. No significant difference in ZAP-70 expression with respect to the age or gender of the study population was identified using appropriate statistical calculations.Conclusions: This study showed only 15% of B-CLL cases showing high ZAP-70 expression, a surrogate biomarker for possible aggressive behavior which may necessitate therapeutic intervention and close surveillance.

EFFECT OF HLA-DR ANTIBODY CONCENTRATION ON ESTIMATION OF THE NUMBER OF CD14+HLA-DRLOW /–-CELLS IN THE BLOOD OF PATIENTS WITH B-CELL chronic lymphocytic leukemia

Introduction. Immunoregulatory functions of myeloid-derived suppressor cells have been extensively studied over the recent decades. Additionally, myeloid-derived suppressor cells have been investigated as a prognostic factor.Objective. To optimize the measurement of monocytic myeloid-derived suppressor cells in the peripheral blood of patients with B-cell chronic lymphocytic leukemia.Materials and methods. The number of myeloid-derived suppressor cells with CD14+HLA-DRlow/–-phenotype was determined in the peripheral blood of patients with B-cell chronic lymphocytic leukemia and healthy donors by flow cytometry. The measurement was carried out at two points, which differed in the concentration of anti-HLA-DR antibodies − 15 and 4 μl.Results. The median amount of myeloid-derived suppressor cells in the peripheral blood of B-cell chronic lymphocytic leukemia patients with 15 μl of anti-HLA-DR antibody was 1.9 %, and with 4 μl of antibody concentration – 7 %. Healthy donors had that median of 0.15 % with 15 μl of antibody and 0.3 % with 4 μl concentration.Conclusion. The number of CD14+HLA-DRlow/–-cells in the blood of patients with B-cell chronic lymphocytic leukemia is sensitive to the concentration of the HLA-DR antibody.Compliance with patient rights and principles of bioethics. The study protocol No 3 of 13.02.2020 was approved by the biomedical ethics committee of Research Institute of Experimental Diagnostics and Therapy of Tumors N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation. All patients gave written informed consent to participate in the study.

Clinical Relevance of +936 C>T VEGFA and c.233C>T bFGF Polymorphisms in Chronic Lymphocytic Leukemia.

Angiogenesis process contributes to the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) being the levels of VEGFA and bFGF higher in patients than in healthy controls. Our aim was to evaluate the implication of angiogenesis factors genetic variants in the predisposition to B-CLL and their association with clinical factors and survival. We performed a population-based case-control study in 224 Spanish B-CLL patients and 476 healthy randomly selected controls to evaluate susceptibility to developing B-CLL. Six polymorphisms were evaluated: rs1109324, rs1547651, rs3025039 (+936 C>T), rs833052 of the VEGFA gene, rs1449683 (c.233C>T) of the bFGF gene and (−710 C>T) of the VEGFR1 gene. The association between clinical parameters and patient outcome was analyzed. Carriers of the CT/TT variants of rs3025039 showed a significant protective effect against developing B-CLL. The CT/TT variants of rs1449683 show a tendency towards the development of the disease and the same variants associated significantly with higher genetic risk and with reduced disease free survival. Moreover, the association persisted in the early-stage disease subgroup. Our study provides evidence of the protective effect of the T/- rs3025039 VEGFA variant against B-CLL development and the association of CT/TT variants of the rs1449683 bFGF gene with genetic risk and an adverse survival.

Cytokine Release Ensuing Interaction Between Human Peripheral Blood Mononuclears and Epstein-Barr Virus Transformed B-CLL Cell Line.

B-cell chronic lymphocytic leukemia (B-CLL) is a common form of leukemia affecting mostly elderly individuals. The course of the disease is usually unremarkable, but because it may proceed with impaired immune defense, B-CLL might be complicated with infections and even death. The leukemic microenvironment containing a number of immune cells, mainly lymphocytes and macrophages capable to produce various molecules including inflammatory cytokines, plays an important role in the development and outcome of the disease. We studied the capacity of Epstein-Barr virus (EBV)-transformed B-cell chronic lymphocytic leukemia (B-CLL) cell line (EHEB) cells, an EBV-transformed line established from a B-CLL patient, to affect the production of inflammatory cytokines by human peripheral blood mononuclear cells (PBMC). PBMC isolated from peripheral blood of healthy donors were incubated either with EHEB cells or with their supernatants and the production of the following cytokines: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, IL-1ra, and IL-10 were detected using the enzyme-linked immunosorbent assay method. Direct contact of PBMC incubated with EHEB cells induced a marked increase of TNFα, IL-1β, IL-6, IFNγ, and IL-10 release by the immune cells. Yet, incubation of PBMC with EHEB cells' supernatant resulted in a mild production of the same cytokines. The noticeable increased production of inflammatory cytokines by PBMC following direct contact with EHEB cells and to a lesser degree with their supernatants implies the existence of an immune dialogue between these two types of cells. The results support the concept that not only leukemic cells, but also peripheral blood mononuclears could serve as a therapeutic target for B-CLL.

Clinico-Biological Implications of Modified Levels of Cytokines in Chronic Lymphocytic Leukemia: A Possible Therapeutic Role.

B-cell chronic lymphocytic leukemia (B-CLL) is the main cause of mortality among hematologic diseases in Western nations. B-CLL is correlated with an intense alteration of the immune system. The altered functions of innate immune elements and adaptive immune factors are interconnected in B-CLL and are decisive for its onset, evolution, and therapeutic response. Modifications in the cytokine balance could support the growth of the leukemic clone via a modulation of cellular proliferation and apoptosis, as some cytokines have been reported to be able to affect the life of B-CLL cells in vivo. In this review, we will examine the role played by cytokines in the cellular dynamics of B-CLL patients, interpret the contradictions sometimes present in the literature regarding their action, and evaluate the possibility of manipulating their production in order to intervene in the natural history of the disease.

Mechanism for IL-15-Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins.

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.

CD14 as a potential prognostic factor and Bcl-2 as a therapeutic target in egyptian B-Cell chronic lymphocytic leukemia patients

INTRODUCTION: B-cell chronic lymphocytic leukemia (B-CLL) is a unique lymphoproliferative disorder that scarcely occurs under the age of 40. B-CLL represents a neoplastic disorder caused primarily by defective programmed cell death and accompanied by a myriad of cellular and humoral immune defects. AIM: This study aimed to assess Bcl-2 and CD14 expression in B-CLL patients and their study as probable prognostic and therapy targeting factors. PATIENTS AND METHODS: In this study, we assessed Bcl-2 and cluster of differentiation (CD14) expression in a group of Egyptian patients with B-CLL. Forty B-CLL patients and 20 apparently healthy individuals served as the control group were included in this study. STATISTICAL ANALYSIS: SPSS statistical software (IBM SPSS Inc., version 20, Chicago, Illinois, USA) was used for statistical analysis. RESULTS: Aberrant expression of Bcl-2 protein appeared in all B-CLL patients (100%). Bcl-2 expression showed a highly positive correlation with total lymphocyte count and lymphocyte count (P =0.000 for both) and a positive correlation with lactate dehydrogenase (P = 0.044). The expression of myelomonocytic antigen “CD14” above the cutoff value 5 × 109/L was reported in 70% (28/40) of B-CLL patients, 55.6% (10/18) of the intermediate-risk group, and 81.8% (18/22) of high-risk group. CONCLUSION: There was a significant increase in Bcl-2 protein and CD14 in B-CLL patients. Bcl-2 was highly increased in all patients and CD14 more observed in the high-risk group than that of the intermediate risk group.

CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated Bruton's tyrosine kinase.

Bruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment.

Identification of new phosphorylation sites of CD23 in B-cells of patients with chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) is the most common lymphoproliferative disorder in adults. Patients with B-CLL strongly express the CD23 – C type of lectin (low affinity IgE receptor, Fc epsilon RII), which is linked to B cell activation and proliferation. Phosphorylation in lymphocytes is tightly associated with regulation of protein activities, functional regulation and cell signaling, and may thus affect initiation and/or progression of the disease. Here we report changes in the phosphorylation of CD23 on threonine (pThr314) and two serine residues (pSer254, pSer265) in B lymphocytes of B-CLL patients, using a flow cytometry approach. The majority of tested patients with active forms of B-CLL presented a notable overexpression of CD23 along with pThr314, pSer254, and pSer265 CD23 phosphorylation positivity. Moreover, we have experimentally stimulated the CD23 phosphorylations in a subset of peripheral blood lymphocytes of healthy controls by phorbol-12-myristate-13-acetate treatment. This affects the activation of competent phosphorylation mediating kinases, resulting in the enhanced phosphorylation pattern. Together, these data confirm that CD23 protein is phosphorylated in B cells of B-CLL patients, report the identification of new CD23 phosphorylation sites, and suggest a possible role(s) of such phosphorylations in the activation of CD23 during the process of lymphocytic activation in B-CLL.

EBV Positive Diffuse Large B Cell Lymphoma and Chronic Lymphocytic Leukemia Patients Exhibit Increased Anti-dUTPase Antibodies.

The Epstein-Barr virus (EBV), which is a ubiquitous γ-herpesvirus, establishes a latent infection in more than 90% of the global adult population. EBV-associated malignancies have increased by 14.6% over the last 20 years, and account for approximately 1.5% of all cancers worldwide and 1.8% of all cancer deaths. However, the potential involvement/contribution of lytic proteins to the pathophysiology of EBV-associated cancers is not well understood. We have previously demonstrated that the EBV-deoxyuridine triphosphate nucleotidohydrolase (dUTPase) modulates innate and adaptive immune responses by engaging the Toll-Like Receptor 2 (TLR2), which leads to the modulation of downstream genes involved in oncogenesis, chronic inflammation, and in effector T-cell function. Furthermore, examination of serum samples from diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia patients revealed the presence of increased levels of anti-dUTPase antibodies in both cohorts compared to controls with the highest levels (3.67-fold increase) observed in DLBCL female cases and the lowest (2.12-fold increase) in DLBCL males. Using computer-generated algorithms, dUTPase amino acid sequence alignments, and functional studies of BLLF3 mutants, we identified a putative amino acid motif involved with TLR2 interaction. These findings suggest that the EBV-dUTPase: TLR2 interaction is a potential molecular target that could be used for developing novel therapeutics (small molecules/vaccines).

Proteomics and metabolomics identify molecular mechanisms of aging potentially predisposing for chronic lymphocytic leukemia

B cell chronic lymphocytic leukemia (B-CLL), the most common type of leukemia in adults, is still essentially incurable despite the development of novel therapeutic strategies. This reflects the incomplete understanding of the pathophysiology of this disease. A comprehensive proteome analysis of primary human B-CLL cells and B cells from younger as well as elderly healthy donors was performed. For comparison, the chronic B cell leukemia cell line JVM-13 was also included. A principal component analysis comprising 6,945 proteins separated these four groups, placing B cells of aged-matched controls between those of young donors and B-CLL patients, while identifying JVM-13 as poorly related cells. Mass spectrometric proteomics data have been made fully accessible via ProteomeXchange with identifier PXD006570-PXD006572, PXD006576, PXD006578, and PXD006589-PXD006591. Remarkably, B cells from aged controls displayed significant regulation of proteins related to stress management in mitochondria and ROS stress such as DLAT, FIS1, and NDUFAB1, and DNA repair, including RAD9A, MGMT, and XPA. ROS levels were indeed found significantly increased in B cells but not in T cells or monocytes from aged individuals. These alterations may be relevant for tumorigenesis and were observed similarly in B-CLL cells. In B-CLL cells, some remarkable unique features like the loss of tumor suppressor molecules PNN and JARID2, the stress-related serotonin transporter SLC6A4, and high expression of ZNF207, CCDC88A, PIGR and ID3, otherwise associated with stem cell phenotype, were determined. Alterations of metabolic enzymes were another outstanding feature in comparison to normal B cells, indicating increased beta-oxidation of fatty acids and increased consumption of glutamine. Targeted metabolomics assays corroborated these results. The present findings identify a potential proteome signature for immune senescence in addition to previously unrecognized features of B-CLL cells and suggest that aging may be accompanied by cellular reprogramming functionally relevant for predisposing B cells to transform to B-CLL cells.