Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods: CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results: IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions: CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures: No relevant conflicts of interest to declare.

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Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,

Blood ◽

10.1182/blood.v118.21.3895.3895 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3895-3895

Author(s):

Yair Herishanu ◽

Inbal Hazan-Hallevi ◽

Sigi Kay ◽

Varda Deutsch ◽

Aaron Polliack ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Anti Inflammatory

Abstract Abstract 3895 Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL. Disclosures: No relevant conflicts of interest to declare.

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T cell chronic lymphocytic leukemia: presence in bone marrow and peripheral blood of cells that suppress erythropoiesis in vitro

Blood ◽

10.1182/blood.v52.1.255.255 ◽

1978 ◽

Vol 52 (1) ◽

pp. 255-260 ◽

Cited By ~ 86

Author(s):

R Hoffman ◽

S Kopel ◽

SD Hsu ◽

N Dainiak ◽

ED Zanjani

Keyword(s):

Bone Marrow ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Peripheral Blood ◽

Lymphocytic Leukemia ◽

Transfusion Therapy ◽

Cell Chronic Lymphocytic Leukemia ◽

Marrow Cells

Abstract The pathogenesis of the anemia associated with malignancy was investigated in a patient with T cell chronic lymphocytic leukemia. The plasma clot culture system was used as a measure in vitro of erythropoiesis. The patient's peripheral blood and marrow T lymphocytes obtained both before and after transfusion therapy suppressed erythroid colony formation by normal human bone marrow cells. Pretreatment of the patient's bone marrow T cells by antithymocyte globulin (ATG) and complement reversed this suppression. In addition, pretreatment of the patient's marrow cells with ATG and complement markedly augmented erythropoiesis in vitro. The expression of erythroid activity caused by the selective destruction of the suppressor T lymphocytes in the patient's bone marrow with ATG and the suppression of normal erythropoiesis by the patient's bone marrow and peripheral blood lymphocytes suggest that interaction between the malignant T cell and the erythropoietin-responsive stem cell is important in production of anemia in this patient.

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CD200 and Chronic Lymphocytic Leukemia: Biological and Clinical Relevance

Frontiers in Oncology ◽

10.3389/fonc.2020.584427 ◽

2020 ◽

Vol 10 ◽

Author(s):

Giovanni D’Arena ◽

Vincenzo De Feo ◽

Giuseppe Pietrantuono ◽

Elisa Seneca ◽

Giovanna Mansueto ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

B Cell ◽

Cell Lymphoma ◽

Prognostic Significance ◽

Cell Types ◽

Lymphocytic Leukemia ◽

Mantle Cell ◽

Type Ia

CD200, a transmembrane type Ia glycoprotein belonging to the immunoglobulin protein superfamily, is broadly expressed on a wide variety of cell types, such as B lymphocytes, a subset of T lymphocytes, dendritic cells, endothelial and neuronal cells. It delivers immunosuppressive signals through its receptor CD200R, which is expressed on monocytes/myeloid cells and T lymphocytes. Moreover, interaction of CD200 with CD200R has also been reported to play a role in the regulation of tumor immunity. Overexpression of CD200 has been reported in chronic lymphocytic leukemia (CLL) and hairy cell leukemia but not in mantle cell lymphoma, thus helping to better discriminate between these different B cell malignancies with different prognosis. In this review, we focus on the role of CD200 expression in the differential diagnosis of mature B-cell neoplasms and on the prognostic significance of CD200 expression in CLL, where conflicting results have been published so far. Of interest, increasing evidences indicate that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing malignancies, such as CLL.

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Effect of a thymic factor on T lymphocytes in B cell chronic lymphocytic leukemia: in vitro and in vivo studies

Blood ◽

10.1182/blood.v64.3.667.667 ◽

1984 ◽

Vol 64 (3) ◽

pp. 667-671 ◽

Cited By ~ 7

Author(s):

F Lauria ◽

D Raspadori ◽

S Tura

Keyword(s):

T Cell ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

B Cell ◽

Proliferative Response ◽

Lymphocytic Leukemia ◽

Before And After ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abnormalities of T lymphocytes in B cell chronic lymphocytic leukemia (B-CLL) have been extensively documented by several immunologic investigations. Following recent studies pointing to the favorable effect of TP-1, a partially purified extract of calf thymus, on the T cell-mediated immunity of several diseases, including Hodgkin's disease, we have used monoclonal antibodies and the enriched T lymphocytes of 16 untreated B-CLL patients to evaluate the proportion of T cell subsets before and after the administration of TP-1. In addition, the proliferative response to phytohemagglutinin (PHA) and the helper function in a pokeweed mitogen (PWM) system were assessed. In ten cases, the effect of TP-1 was also studied in vitro by evaluating the same parameters before and after incubation of B-CLL T cells with the drug. The study demonstrated that in vivo administration of TP-1 increases significantly (P less than .001) the proportion of the defective helper/inducer T cell population (OKT4-positive cells) in B-CLL, leading to a near normal OKT4/OKT8 ratio. Furthermore, the improved phenotypic profile was accompanied by an increased proliferative response to PHA and, in particular, by a significant increase (P less than .01) of T helper capacity; this increase was, however, insufficient to enable the normalization of the serum immunoglobulin levels. The in vitro incubation of B-CLL T lymphocytes did not succeed in producing significant modifications in distribution and function.

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Comparative Efficacy of First-Line Chemotherapy-Free Combinations in Chronic Lymphocytic Leukemia (CLL): A Network Meta-Analysis

Blood ◽

10.1182/blood-2020-136411 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 25-26

Author(s):

Philip A Haddad ◽

Nowell Ganey ◽

Kevin M. Gallagher

Keyword(s):

Chronic Lymphocytic Leukemia ◽

English Language ◽

Conflicts Of Interest ◽

Residual Disease ◽

Meta Analysis ◽

The Elderly ◽

Lymphocytic Leukemia ◽

First Line ◽

Significant Difference ◽

Anti Cd20

Introduction: Chronic Lymphocytic Leukemia (CLL) is an incurable B-cell malignancy which disproportionately affects the elderly. Although first-line chemoimmunotherapy (CIT) improved CLL clinical outcomes, recent randomized trials revealed superior outcomes with novel chemotherapy-free combinations (CFC) incorporating anti-CD20 monoclonal antibodies and inhibitors of BTK or Bcl-2. So far, these CFC have not been compared head-to-head. We conducted this network meta-analysis to evaluate their relative efficacy to each other. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language; diagnosis of CLL; trials that explored the efficacy of first-line CFC with Obinutuzumab (O), Rituximab (R), Ibrutinib (IB), Acalabrutinib (ACAL), Venetoclax (VEN) compared to standard CIT that included Chlorambucil (CHLOR) with either R or O, Bendamustine+Rituximab, or Fludarabine+ Cyclophosphamide+R; and phase 3 randomized studies reporting responses, progression, death, and adverse (AE) events. A frequentists network meta-analysis was conducted using netmeta package and random-effects model. Results: Five studies comprising a total of 2,272 participants were included. When O-based CFC data was analyzed, only ACAL-O had a significant lower relative risk (RR) of progression and death (P&D). There were no significant differences with respect to overall response rates (ORR), complete remission (CR), minimal residual disease (MRD), or grade >3 adverse events (Grd3+) among O-based CFC. When R-based CFC data was analyzed, IB and IB-R were not different with respect to RR of P&D, ORR, CR, MRD, or Grd3+. When the data was analyzed as CFC versus combined CIT, only ACAL-O was found to be significantly superior to other O- and R-based CFC with respect to RR of P&D. ORR and Grad3+ rates of O- and R-based CFC were not significantly different. While ACAL-O, IB-O, and VEN-O had superior CR and MRD rates compared to other CFC, there were no significant differences among each other. Conclusions: This network meta-analysis is the first to compare and rank first-line CFC therapies in CLL. It indicates that ACAL-O has a superior profile having the lowest RR of P&D without significant difference in Grd3+ among CFC. Disclosures No relevant conflicts of interest to declare.

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Silvestrol, a Rocaglate Derivative from the Indonesian Plant Aglaia foveolata, Has Significant Bcl-2- and p53-Independent Anti-Tumor Activity against Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v108.11.2600.2600 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2600-2600 ◽

Cited By ~ 1

Author(s):

Ryan B. Edwards ◽

David M. Lucas ◽

Gerard Lozanski ◽

Amy J. Johnson ◽

Bao-Ning Su ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Mtt Assay ◽

Whole Blood ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Equal Distribution ◽

Significant Difference ◽

Anti Tumor Activity

Abstract Chronic Lymphocytic Leukemia (CLL) is an incurable disease with limited therapeutic options. The development of drug resistance through multiple pathways, especially in advanced disease, further restricts these options. Thus, new agents with unique mechanisms of action are crucial to make an impact on patient survival. Silvestrol, a rocaglate derivative with an unusual dioxanyloxy unit, was isolated from Aglaia species using bioassay-guided fractionation. Silvestrol exhibited potent in vitro cytotoxic activity against several tumor cell lines. Silvestrol was further evaluated in vivo in the hollow fiber test and in the murine P-388 leukemia model, in which it demonstrated promising anti-tumor activity with no significant weight loss up to 2.5 mg/kg (3.7 μM, assuming equal distribution) (1). Based on these results, we tested silvestrol against tumor cells obtained from CLL patients. Silvestrol exhibited significant antitumor activity with an estimated LC50 (concentration lethal to 50% of cells relative to untreated control) of 10 nM at 72 hours by MTT assay. In contrast, at this same timepoint using normal human peripheral blood mononuclear cells, an LC50 for silvestrol could not be defined even up to 4.0 μM. Under identical conditions, silvestrol was 50 to 100 fold more potent than the active metabolite of fludarabine, commonly used in the treatment of CLL. To determine the minimum exposure time required for silvestrol to have an effect, cells were incubated for various times in 80 nM silvestrol, then washed and resuspended in media with or without drug and incubated for a total of 72 hours. With only a four hour exposure, an average of 56% cytotoxicity was observed relative to untreated cells, and with a 24 hour exposure, results between samples in which the drug was removed and those incubated continuously were indistinguishable. T cell depletion and concomitant immunodeficiency is a serious risk with therapies currently available for CLL. We therefore tested the relative effects of silvestrol on B and T cells. By MTT assay with selected cells and in whole blood incubations followed by flow cytometry, using blood from both CLL patients and healthy volunteers, silvestrol demonstrated significantly more cytotoxicity toward B cells than T cells. Although some variability was observed between patient samples, silvestrol had activity against all samples tested and there was no detectable difference in average potency against cells from patients with a 17p13 deletion (chromosomal site of p53) relative to those without this risk factor. Furthermore, there was no significant difference in silvestrol-mediated cytotoxicity between lymphoblastic cells with a ten-fold overexpression of Bcl-2 relative to control cells. Together, these data demonstrate that silvestrol has efficacy against CLL cells in vitro and in whole blood, has highly unusual B-cell specificity, and is independent of key CLL resistance mechanisms. Our data strongly support further investigation of silvestrol as an antitumor agent in CLL. Studies are underway to determine the precise mechanism of action of this compound in CLL cells.

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CCL3 and CCL4, the Major Chemokines Produced by CD38+ Chronic Lymphocytic Leukemia Cells, Facilitate Microenvironmental Interactions of Neoplastic Cells Via the CD49d/VCAM Pair.

Blood ◽

10.1182/blood.v112.11.1055.1055 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1055-1055

Author(s):

Antonella Zucchetto ◽

Dania Benedetti ◽

Riccardo Bomben ◽

Claudio Tripodo ◽

Fleur Bossi ◽

...

Keyword(s):

Endothelial Cells ◽

Tumor Microenvironment ◽

Tumor Cells ◽

Umbilical Vein ◽

Fold Increase ◽

Peripheral Blood Cell ◽

Lymphocytic Leukemia ◽

Bone Marrow Biopsies ◽

L Cells

Abstract CD38, a negative prognostic marker for patients with CLL, has been demonstrated to be a key molecule in the interactions occurring in the context of tumor microenvironment, mediating both survival and migratory signals for CLL cells. By taking advantage of gene expression profiling studies (GEP) comparing 11 CD38pos (CD38>30%) and 15 CD38neg (CD38<10%) CLLs, we identified as over-expressed in CD38pos CLL cells: i) genes for the two C-C chemokines CCL3 and CCL4 (median-log difference, MLD-CCL3= 3.5; MLD-CCL4=4.4); real-time quantitative PCR (RTQ-PCR) of selected cases confirmed GEP results; ii) the gene for CD49d (MLD=4.4); a high correlation between CD38 and CD49d protein expression, also characterizing the CLL series of the present study, has been reported previously. In vitro experiments, performed on purified tumor cells from additional 11 CD38pos CLL cases cultured for 14 (t14) and 24 (t24) hours in the presence of either the agonist anti-CD38 monoclonal antibody (mAb) IB4 or the non-agonistic anti-CD38 mAb IB6 as control, demonstrated upregulation of CCL3/CCL4 transcripts at t14 (CCL3: mean fold increase=18, p=0.041; CCL4: mean fold increase=13.8, p=0.005), as assessed by RTQ-PCR, and an increased release of CCL3/CCL4 proteins at t24 (CCL3: mean =0.9 ng/mL, mean fold increase=14, p=0.003; CCL4: mean =1.7 ng/mL, mean fold increase=49, p=0.01), as assessed by ELISA. Consistently, immunohistochemistry (IHC) analysis performed in bone marrow biopsies (BMB) from 20 CLL patients (10 CD38pos and 10 CD38neg cases) showed detectable levels of CCL3 in 8 cases, all but one belonging to the CD38pos group (p=0.02). Expression of the CCL3/CCL4 specific receptors CCR1 and CCR5 was examined by flow cytometry in peripheral blood cell subpopulations from 30 CLL (12 CD38pos and 18 CD38neg). Irrespectively of CD38 expression by CLL cells, monocytes showed the highest expression levels for CCR1 and, although at a lesser extent, CCR5. Consistently, CCL3 was able to attract CLL-derived monocytes by in-vitro chemotaxis experiments, and a higher number of infiltrating CD68pos macrophages were found in BMB of CD38pos compared to CD38neg CLLs (p=0.016). In parallel experiments, conditioned media (CM) from CCL3-stimulated macrophages were collected; these CM were able to induce expression of the CD49d-ligand VCAM in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (ADMEC). As shown by ELISA, TNFalpha was among the cytokines contained in macrophage-CM. This citokine was likely responsible for VCAM up-regulation by HUVEC and ADMEC, as suggested by TNFalpha neutralization experiments leading to a suppression of VCAM-1 induction in endothelial cell models. Again, IHC analysis of CLL BMB showed a meshwork of VCAM-1-positive cells more prominent in the context of lymphoid infiltrates of CD38pos, as compared to CD38neg cases (p=0.002). To verify whether CD49d engagement through VCAM-1 could enhance the protection against spontaneous apoptosis of CLL cells in vitro, we cultured purified CD38pos/CD49dpos CLL cells from 5 cases onto VCAM-1-transfected L cells or mock-transfected L cells. Results demonstrated a substantial improvement in cell viability after CD49d engagement: as high as 70%±25 cells were viable after 10 days of culture on L-VCAM cells compared to 50%±25 in control conditions (p=0.009). Altogether, these results identify molecules involved in a functional cross-talk between CD38/CD49d-expressing CLL and cells of the tumor microenvironment. This interplay may eventually affect survival and recirculation of tumor cells via the CD49d/VCAM pair.

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Mapping the Targets of Dasatinib in Chronic Lymphocytic Leukemia Reveals Distinct Roles for Abl and Btk in Drug Resistance and Adhesion, and Explains Clinical Effects On Lymph Node Reduction

Blood ◽

10.1182/blood.v120.21.3900.3900 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3900-3900

Author(s):

Eric Eldering ◽

Christian R Geest ◽

Martin FM de Rooij ◽

Nora Liu ◽

Bogdan I Florea ◽

...

Keyword(s):

Drug Resistance ◽

Lymph Node ◽

Chronic Lymphocytic Leukemia ◽

Broad Spectrum ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Clinical Effects ◽

Open Label

Abstract Abstract 3900 In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche. This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL. Disclosures: No relevant conflicts of interest to declare.

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Four ricin chain A-based immunotoxins directed against the common chronic lymphocytic leukemia antigen: in vitro characterization

Blood ◽

10.1182/blood.v82.2.536.bloodjournal822536 ◽

1993 ◽

Vol 82 (2) ◽

pp. 536-543

Author(s):

GB Faguet ◽

JF Agee

Keyword(s):

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Ex Vivo ◽

Lymphocytic Leukemia ◽

Fab Fragment ◽

In Vitro Characterization ◽

The Common ◽

Dose Dependent

The common B-chronic lymphocytic leukemia (B-CLL) antigen (cCLLa) appears to be ideal for targeted immunotherapy in that it is the most prevalent and disease-restricted marker in B-CLL. To assess this potential, we developed four immunotoxins (ITs) of anti-cCLLa monoclonal antibody CLL2m (an IgG2a kappa), using ricin chain A (RTA) or its deglycosylated derivative (dgA), each conjugated to either the whole IgG molecule or its Fab' fragment. Each IT was tested in vitro for specificity and cytotoxic activity (assessed by protein synthesis inhibition [PSI] and by cell kill [CK] in the clonogenic assay) against B-CLL cells. RTA-based anti-CD5 ITs and enriched normal B and T lymphocytes were used as controls. Each IT exhibited antigen-specific, dose-dependent activity. Thus, whereas B-CLL cells exhibited dose- dependent PSI and CK (whether the B-CLL clone was CD5+ or CD5-), normal B (cCLLa-/CD5-) and T lymphocytes (cCLLa-/CD5+) remained unaffected. IT potency was independent of toxin glycosylation, but was slightly influenced by antibody valence; divalent ITs were twice as potent as monovalent ITs (IC50, 2.3 v 7.1 x 10(-11) mol/L; CK, 2.6- v 2.0-log reached with 524 v 1,072 IT molecules bound/cell, respectively). In the presence of ammonium chloride or Verapamil, IT-induced CK was enhanced 10- to 80-fold. These data suggest that the cCLLa is a promising target for IT-based immunotherapy of B-CLL in vivo and ex vivo.

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98% IGHV Gene Identity Is Still the Optimal Cutoff to Predict the Prognosis of Chronic Lymphocytic Leukemia Patients in China

Blood ◽

10.1182/blood-2018-99-118141 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5545-5545

Author(s):

Yi Xia ◽

Ke Shi ◽

Qian Sun ◽

Chun Qiao ◽

Huayuan Zhu ◽

...

Keyword(s):

Prognostic Factor ◽

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Somatic Hypermutation ◽

Lymphocytic Leukemia ◽

Study Cohort ◽

Optimal Cutoff ◽

Entire Cohort ◽

Mutational Status ◽

Significant Difference

Abstract Background: Immunoglobulin heavy chain variable region (IGHV) has been an important prognostic factor for chronic lymphocytic leukemia (CLL) for decades. 98% being a cut-off value for IGHV is a mathematical choice and researches on the best cut-off value have never been stopped. Chinese CLL patients are known to differ from Caucasian CLL patients on both clinical and genetical features. However, the optimal cutoff for IGHV mutational status has not yet been studied in this particular ethnic group. Method: We carried out a study on 595 Chinese CLL patients in order to find out whether 98% is the best cut-off value for IGHV in Chinese CLL patients. Genomic DNA from peripheral blood or bone marrow was subjected to PCR amplification following the IGH Somatic Hypermutation Assay v2.0 protocol (InVivoScribe). Sequences were aligned to ImMunoGeneTics/V-QUEry and Standardization (IMGT/-VQUEST) database. Result: 600 sequences were received after IGHV rearrangement sequencing. IGHV3-23, IGHV4-34, IGHV3-7, IGHV4-39 and IGHV1-69 were the most frequently used IGHV genes. 352 (58.7%) cases were IGHV-mutated while 248 (41.3%) cases were IGHV-unmutated if the classical 98% classification by ERIC was used. In order to determine the optimal cut-off value, we used 1% as the interval to divide the entire cohort into 7 groups according to the mutational rate, which were <95%, 95%-95.99%, 96%-96.99%, 97%-97.99%, 98%-98.99%, 99%-99.99% and 100% respectively. Binet A patients had a relatively indolent course of disease and cases with different IGHV mutational rates had no significant differences in time to first treatment (TTFT) apart from truly unmutated (100%) cases. For the whole study cohort, significant difference appeared at 98% interval (P<0.001 and P=0.005 for TTFT and OS respectively) while intervals less than 98% had no significant difference compared with the <95% group. Similarly, there was no clear dissimilarities among 98%, 99% and 100% intervals (Table 1a and b). All the other prognostic factors including del(17p), del(11q), TP53 mutation, MYD88 mutation, NOTCH1 mutation, SF3B1 mutation, CD38, ZAP-70, Binet staging, gender, β2-microglobulin and EBV-DNA were differently distributed between group <98% and group ³98%, but not among subgroups in ³98%. In multivariate analysis, the 98% IGHV was also an independent prognostic factor for TTFT and OS. Conclusion: 98% is the optimal cutoff value for IGHV mutational status to predict the prognosis of CLL patients in China. Table 1. Table 1. Disclosures No relevant conflicts of interest to declare.

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