scholarly journals Autoantibody-associated kappa light chain variable region gene expressed in chronic lymphocytic leukemia with little or no somatic mutation. Implications for etiology and immunotherapy.

1988 ◽  
Vol 167 (3) ◽  
pp. 840-852 ◽  
Author(s):  
T J Kipps ◽  
E Tomhave ◽  
P P Chen ◽  
D A Carson
Keyword(s):  
B Cells ◽  
Nucleic Acid ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Light Chain ◽  
Variable Region ◽  
Lymphocytic Leukemia ◽  
Kappa Light Chain ◽  
V Genes

Recently the minor B cell subpopulation that expresses the CD5 (Leu-1) antigen has been implicated as a source of IgM autoantibodies. Chronic lymphocytic leukemia (CLL), the most common leukemia in humans, represents a malignancy of small B lymphocytes that also express the CD5 antigen. However, little is known concerning the antibody variable region genes (V genes) that are used by these malignant CD5 B cells. We have found that a relatively high frequency of CLL patients have leukemic B cells with surface immunoglobulin (sIg) recognized by 17.109, a murine mAb specific for a kappa light chain associated crossreactive idiotype (CRI) associated with rheumatoid factor and other IgM autoantibodies. Flow cytometric analyses revealed that the relative expression of the 17.109-CRI by circulating leukemic B cells was directly proportional to the levels of sIg kappa light chain, indicating that there exists stable idiotype expression in the leukemic population. To examine this at the molecular level, the nucleic acid sequences encoding the Ig kappa light chains of two unrelated patients with CLL bearing sIg with the 17.109-CRI were determined. Analyses of multiple independent kappa light chain cDNA clones did not reveal any evidence for sequence heterogeneity in the CLL cell population. Furthermore, the nucleic acid sequences expressed by the leukemic cells of these two patients were identical or very homologous to a germline V kappa gene isolated from placental DNA, designated Humkv 325, or "V kappa RF" because of its association with IgM autoantibodies. This study suggests; (a) that the malignant CD5+ B lymphocytes in CLL use the same V kappa gene that has been highly associated with IgM autoantibodies and (b) that the expression of V genes is stable in CLL, in contrast to other B cell malignancies examined to date. We propose that many CLL cases represent malignancies of autoreactive CD5 B cells that use a restricted set of conserved V genes. This property may render CLL particularly amenable to immunotherapy with antiidiotypic antibodies.

scholarly journals Autoantibody-associated cross-reactive idiotypes expressed at high frequency in chronic lymphocytic leukemia relative to B-cell lymphomas of follicular center cell origin

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 422-428 ◽  
Author(s):  
TJ Kipps ◽  
BA Robbins ◽  
P Kuster ◽  
DA Carson
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Light Chain ◽  
Variable Region ◽  
Lymphocytic Leukemia ◽  
Kappa Light Chain ◽  
Region Gene ◽  
Variable Regions ◽  
Hodgkin's Lymphomas ◽  
Center Cell

Using murine monoclonal antibodies (MoAbs) specific for immunoglobulin (Ig) cross-reactive idiotypes (CRI), we performed immunohistochemical analyses on frozen tissue sections and cytocentrifuge preparations of Ig-expressing malignant cells from patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphomas (NHL) of follicular center cell origin. Twenty percent (4/20) of the Ig kappa light chain- expressing CLL cells reacted with 17.109, a MoAb against a major CRI on human IgM autoantibodies that is encoded by a conserved Ig variable- region gene (V gene) of the V kappa IIIb sub-subgroup. Another MoAb specific for V kappa IIIb framework determinant(s) reacted exclusively with all the 17.109-reactive CLL cells. Only one of 20 kappa light- chain-expressing CLL cells reacted with 6B6.6, a monoclonal antibody specific for a CRI commonly found on rheumatoid factor (RF) paraproteins with light-chain variable regions of the V kappa IIIa sub- subgroup. Finally, greater than 20% (8/34) of all CLL reacted with G6, a MoAb specific for an Ig heavy chain-associated CRI present on several RF paraproteins. In contrast, these CRIs were expressed at significantly lower frequencies in NHL of follicular center cell origin. Only one of 30 NHL expressing kappa light chains reacted with the 17.109 MoAb. Also, in contrast to the concordance between the 17.109-CRI and V kappa IIIb framework determinant(s) in CLL, two lymphomas in addition to the 17.109-reactive lymphoma were recognized by the anti-V kappa IIIb framework MoAb. None of the NHL reacted with either the 6B6.6 or the G6 MoAbs. These results are the first to demonstrate that CLL and NHL differ with respect to the expression of autoantibody-associated CRIs. The data support the notion that NHL of follicular center cell origin differs from CLL in its utilization and/or somatic mutation of Ig variable-region genes. The physiological and immunotherapeutic implications of these findings are discussed.

Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4609-4614 ◽  
Author(s):  
Liguang Chen ◽  
George Widhopf ◽  
Lang Huynh ◽  
Laura Rassenti ◽  
Kanti R. Rai ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Variable Region ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cytosolic Proteins ◽  
V Genes

We examined isolated leukemia B cells of patients with chronic lymphocytic leukemia (CLL) for expression of zeta-associated protein 70 (ZAP-70). CLL B cells that have nonmutated immunoglobulin variable region genes (V genes) expressed levels of ZAP-70 protein that were comparable to those expressed by normal blood T cells. In contrast, CLL B cells that had mutated immunoglobulin variable V genes, or that had low-level expression of CD38, generally did not express detectable amounts of ZAP-70 protein. Leukemia cells from identical twins with CLL were found discordant for expression of ZAP-70, suggesting that B-cell expression of ZAP-70 is not genetically predetermined. Ligation of the B-cell receptor (BCR) complex on CLL cells that expressed ZAP-70 induced significantly greater tyrosine phosphorylation of cytosolic proteins, including p72Syk, than did similar stimulation of CLL cells that did not express ZAP-70. Also, exceptional cases of CLL cells that expressed mutated immunoglobulin V genes and ZAP-70 also experienced higher levels tyrosine phosphorylation of such cytosolic proteins following BCR ligation. Following BCR ligation, ZAP-70 underwent tyrosine phosphorylation and became associated with surface immunoglobulin and CD79b, arguing for the involvement of ZAP-70 in BCR signaling. These data indicate that expression of ZAP-70 is associated with enhanced signal transduction via the BCR complex, which may contribute to the more aggressive clinical course associated with CLL cells that express nonmutated immunoglobulin receptors.

scholarly journals Autoantibody-associated cross-reactive idiotypes expressed at high frequency in chronic lymphocytic leukemia relative to B-cell lymphomas of follicular center cell origin

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 422-428 ◽  
Author(s):  
TJ Kipps ◽  
BA Robbins ◽  
P Kuster ◽  
DA Carson
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Light Chain ◽  
Variable Region ◽  
Lymphocytic Leukemia ◽  
Kappa Light Chain ◽  
Region Gene ◽  
Variable Regions ◽  
Hodgkin's Lymphomas ◽  
Center Cell

Abstract Using murine monoclonal antibodies (MoAbs) specific for immunoglobulin (Ig) cross-reactive idiotypes (CRI), we performed immunohistochemical analyses on frozen tissue sections and cytocentrifuge preparations of Ig-expressing malignant cells from patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphomas (NHL) of follicular center cell origin. Twenty percent (4/20) of the Ig kappa light chain- expressing CLL cells reacted with 17.109, a MoAb against a major CRI on human IgM autoantibodies that is encoded by a conserved Ig variable- region gene (V gene) of the V kappa IIIb sub-subgroup. Another MoAb specific for V kappa IIIb framework determinant(s) reacted exclusively with all the 17.109-reactive CLL cells. Only one of 20 kappa light- chain-expressing CLL cells reacted with 6B6.6, a monoclonal antibody specific for a CRI commonly found on rheumatoid factor (RF) paraproteins with light-chain variable regions of the V kappa IIIa sub- subgroup. Finally, greater than 20% (8/34) of all CLL reacted with G6, a MoAb specific for an Ig heavy chain-associated CRI present on several RF paraproteins. In contrast, these CRIs were expressed at significantly lower frequencies in NHL of follicular center cell origin. Only one of 30 NHL expressing kappa light chains reacted with the 17.109 MoAb. Also, in contrast to the concordance between the 17.109-CRI and V kappa IIIb framework determinant(s) in CLL, two lymphomas in addition to the 17.109-reactive lymphoma were recognized by the anti-V kappa IIIb framework MoAb. None of the NHL reacted with either the 6B6.6 or the G6 MoAbs. These results are the first to demonstrate that CLL and NHL differ with respect to the expression of autoantibody-associated CRIs. The data support the notion that NHL of follicular center cell origin differs from CLL in its utilization and/or somatic mutation of Ig variable-region genes. The physiological and immunotherapeutic implications of these findings are discussed.

Different Expression of FcgammaRIIb in Chronic Lymphocytic Leukemia and Human Normal B Lymphocytes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3134-3134
Author(s):  
Carol Moreno ◽  
Rajendra Damle ◽  
Sonia Jansa ◽  
Gerardo Ferrer ◽  
Pau Abrisqueta ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Surface Membrane ◽  
Memory B Cells ◽  
Lymphocytic Leukemia ◽  
Cd38 Expression ◽  
B Cell Subsets

Abstract The Fcgamma receptors (FcγRs) are a family of molecules that modulate immune responses. FcγRIIb is an inhibitory FcγR that bears immunoreceptor tyrosine-based inhibitory motifs which transduce inhibitory signals on coligation with the surface membrane Ig of the B-cell antigen receptor (BCR). The role of FcγRIIb in controlling B cell activation through inhibition of BCR signaling has been extensively studied in animal models. Nevertheless, data on FcγRIIb are scant in human normal and neoplastic B cells, this being due to the lack of a specific antibody for human FcγRIIb. Consequently, there is little information on this receptor in chronic lymphocytic leukemia (CLL). Considering the activated nature of CLL cells and the central role of the BCR in the biology of the disease, studies of FcγRs are warranted. We used a novel specific mAb directly conjugated with Alexa 488 fluorophore that solely reacts with the human FcγRIIb (MacroGenics, Inc.) to investigate the receptors expression on CLL and normal human B cells. The study population included 84 patients with CLL and 24 age- and sex-matched controls. FcγRIIb expression was assessed as the mean fluorescence intensity (MFI) of surface membrane staining. In CLL cells, FcγRIIb was measured on CD19+CD5+ cells in combination with CD38, CD49d or CD69. Normal B cells were immunostained for CD19, CD5, IgD and CD38 expression and B cell subsets: naïve (IgD+CD38−), activated (IgD+CD38+) and memory B cells (IgD−CD38−) were studied for their relative expression of FcγRIIb. FcγRIIb expression was found significantly higher in naïve B cells compared to activated and memory B cells [median MFI: 17420 (11960–21180) vs. 11.140 (7899–16970) and 11.830 (6984–17100); p<0.001]. Significant differences were also observed between CD5− and CD5+ normal B cells. In contrast, FcγRIIb expression was lower in CLL cells than in CD5+ and CD5− normal B lymphocytes [median MFI: 6901(1034–42600), 10180 (5856–14820) and 12120 (7776–16040); p<0.05)]. Interestingly, FcγRIIb expression was variable within individual CLL clones, this being higher in CD38+ and CD49d+ cells than in CD38− and CD49d− cells (p<0.05). Furthermore, the highest density of FcγRIIb was observed on those cells which coexpressed CD38 and CD49d. In contrast, no significant differences were observed between FcγRIIb and the expression of the activation antigen CD69. Although CD69 and CD38 expression was significantly higher on unmutated IGHV cases, no correlation was found between FcγRIIb levels and IGHV mutational status. Similarly, there was no correlation between FcγRIIb and other poor prognostic variables such as ZAP-70 (≥20%), CD38 (≥ 30%) or high risk cytogenetics. Nevertheless, cases with ≥ 30% CD49d+ cells had higher FcγRIIb expression than those with <30% CD49d+ cells (p=0.006). The findings presented in this study suggest a hierarchy of FcγRIIb expression in normal B-cells, CLL cells and their subpopulations: circulating normal CD5− B cells > circulating normal CD5+ B cells > circulating CD5+ CLL B cells. In addition, although FcγRIIb is present on all normal B cell subsets its expression is higher in naïve B cells. Furthermore, in CLL FcγRIIb density is greater in CD38+ and CD49d+ cells within the clone. Although CD49d and FcγRIIb on CLL clones is linked in a direct manner, there is no relationship with FcγRIIb density and IGHV mutations, ZAP-70, CD38 and unfavorable cytogenetic markers. Finally, the relationship between FcγRIIb expression on CLL cells and functional responses to BCR and other receptor-mediated signals deserve further investigation.

scholarly journals Immunoglobulin secretory function of B cells from untreated patients with chronic lymphocytic leukemia and hypogammaglobulinemia: role of T cells

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 767-774 ◽  
Author(s):  
LA Fernandez ◽  
JM MacSween ◽  
GR Langley
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Cell Function ◽  
Lymphocytic Leukemia ◽  
Suppressor Activity ◽  
Normal Peripheral Blood

Abstract The mechanism of the hypogammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) was studied by determining the generation of specific immunoglobulin-secreting cells in response to mitogen and antigen stimulation in culture. Normal peripheral blood B lymphocytes from 18 normal subjects cocultured with equal numbers of autologous T cells generated cells secreting 2,542 +/- 695 IgG, 2,153 +/- 615 IgA, and 2,918 +/- 945 IgM. Normal B lymphocytes cocultured with normal allogeneic T cells generated similar numbers. However, B lymphocytes from patients with chronic lymphocytic leukemia cocultured with T cells from the same patient generated only 0.5% as many cells secreting IgG and 11% and 23% as many secreting IgA and IgM, respectively. The reason for this markedly defective generation of immunoglobulin-secreting cells was investigated by evaluating T-helper, T-suppressor, and B-cell function using B cells from tonsil and T and B cells from peripheral blood of normal and leukemic individuals. T cells from patients with chronic lymphocytic leukemia provided somewhat greater help than did normal T cells to normal peripheral blood B cells and normal help to tonsil B cells, whether stimulated with mitogen or antigen. T cells from patients with chronic lymphocytic leukemia did not demonstrate increased suppressor function compared to normals with B cells from normal peripheral blood. The hypogammaglobulinemia in these patients therefore was associated with a markedly defective generation of immunoglobulin secreting cells, and as there was normal or increased T- cell helper activity without excessive suppressor activity, it seems likely that this was due to an intrinsic B-cell defect.

scholarly journals Analysis of signal transduction in B chronic lymphocytic leukemia cells

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1461-1469
Author(s):  
HG Drexler ◽  
MK Brenner ◽  
E Coustan-Smith ◽  
SM Gignac ◽  
AV Hoffbrand
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Kinase C

We report here experiments on the analysis of cellular signal transduction in a series of patients with chronic B cell disorders (B cell chronic lymphocytic leukemia [B-CLL] and prolymphocytic leukemia). We compared the response of the leukemic cells with primary external signals (interleukin 2 [IL-2] or B cell differentiation factors [BCDF or IL-6]) with their response to secondary inducers (the phorbol ester (12–O-tetradecanoylphorbol-13-acetate [TPA] or the calcium ionophore A23187) that circumvent the first part of the signal transduction pathway by directly activating the key enzyme protein kinase C. One BCDF was synthesized by mitogen-activated peripheral blood B lymphocytes; a second BCDF was constitutively produced by the human bladder carcinoma cell line T24. Changes in morphology, Tac (IL-2 receptor) expression, RNA synthesis measured by 3H-uridine uptake, and immunoglobulin production tested by enzyme-linked immunosorbent assay were used as parameters of successful signal transduction. TPA alone and TPA plus A23187 (synergistically) effectively initiated differentiation in all the leukemia cases. Neither IL-2 nor BCDF (singly or in combinations) caused equivalent responses. On the other hand, IL-2 and BCDF produced a substantial differentiation effect on normal B lymphocytes. Our data suggest that (a) B-CLL cells are able to respond to direct stimulation of the second messenger pathway (through protein kinase C) but not to the physiological stimuli IL-2 or BCDF; (b) the defect in signal transduction appears to be located upstream of protein kinase C (a possible candidate is a G protein); (c) malignant B cells may spontaneously or after treatment with inducers express the IL- 2 receptor (Tac antigen) in the absence of a functional differentiating response to IL-2; and (d) signs of proliferation/differentiation in B- CLL samples after incubation with IL-2 or BCDF might be due to contamination of the cell populations with residual normal B cells.

scholarly journals Expression of FAK and Its Involvement in the Progression of B-Cell Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3309-3309
Author(s):  
Cristina Gattazzo ◽  
Andrea Visentin ◽  
Alberto Pavan ◽  
Veronica Martini ◽  
Federica Frezzato ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Focal Adhesion ◽  
Poor Prognosis ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
Cell Chronic Lymphocytic Leukemia

Abstract INTRODUCTION B-cell chronic lymphocytic leukemia (B-CLL) is a disorder characterized by the accumulation of clonal CD5+ B lymphocytes, due to uncontrolled growth and resistance to apoptosis. Although the prognosis and clinical outcome has dramatically improved by recent innovative therapies, B-CLL still remains an incurable disease. Since signaling events downstream the BCR engagement are important for the progression of B cells, BCR signaling has been investigated in B-CLL in order to design new agents to specifically treat this disease. We demonstrated that Lyn, one of the first kinases involved in BCR signaling pathway, is overexpressed, constitutively active and anomalously distributed in malignant B cells, as compared to normal B lymphocytes. The Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, is the primary enzyme involved in the engagement of integrins and assembly of Focal Adhesion. FAK is regulated primarily through tyrosine phosphorylation by Lyn after BCR engagement and was found to be overexpressed in many kinds of human cancers. However, a downmodulation of FAK expression and its association to poor prognosis have also been reported. The aim of this study was to investigate the role of FAK in CLL patients. METHODS Blood samples were collected from 5 controls and 50 B-CLL patients. Informed consent was obtained according to the Declaration of Helsinki. Untouched peripheral blood B cells were purified using the RosetteSep for human B cells isolation kit. The samples that were used had at least 95% of normal CD19+ or neoplastic CD5+/CD19+ cells, as assessed by flow-cytometry. Level of FAK protein was evaluated by Western blotting (Wb) and Flow Cytometry assay (FC). Levels of FAK were correlated to clinical parameters of patients. RESULTS We observed that FAK was downmodulated in 56% of analyzed patients with respect to healthy subjects (respectively, Wb: 0.28±0.25 vs 0.85±0.32, p<0.001; FC: 35%±29 vs 60%±16, p<0.05). We also identified that lower levels of FAK expression were related to the prognostic markers of poor outcome (the expression of ZAP70, CD38 and an unmutated-IGHV genes status, p<0.05) and to a shorter Treatment Free Survival (p<0.05). Moreover, patients (n=6) who had an indolent course and were responsive to the standard treatment, showed normal expression of this kinase already at diagnosis. In contrast, patients (n=6) with a more aggressive disease, had a lower expression of FAK, that was further downmodulated during the progression of disease, irrespective of how the patients were treated. CONCLUSIONS From the data presented in this report we propose that FAK downmodulation could be considered as a new marker of poor prognosis and as a putative predictor for high-risk subgroups of CLL, even in early-stage disease. Disclosures No relevant conflicts of interest to declare.

Demonstration of Biclonal Chronic Lymphocytic Leukemia with Mutated and Unmutated Clones and Concurrent but Clonally Unrelated Myeloma in the Same Patient by Single Cell Immunoglobulin Heavy and Light Chain Gene Analysis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4772-4772
Author(s):  
Selim A. Yavuz ◽  
Dan-Paul Hartmann ◽  
Said Baidas ◽  
Peter E. Lipsky ◽  
Metin Ozdemirli
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Single Cell ◽  
Light Chain ◽  
Lymphocytic Leukemia ◽  
Chain Gene ◽  
Gene Rearrangements ◽  
Kappa Light Chain ◽  
B Cell Malignancies ◽  
Myeloma Cells

Abstract Patients with chronic lymphocytic leukemia (CLL) may develop other B cell malignancies in their clinical course including aggressive diffuse large B-cell lymphomas and rarely myelomas. In a large proportion of cases, the secondary B cell malignancies reflected the emergence of immunophenotypically and genetically different clones. An immature type plasma cell myeloma developed in a 73-year-old female patient in whom CLL was diagnosed four years previously. The CLL expressed CD5, CD19, CD23, CD38 and surface kappa light chain, but were negative for ZAP-70. Trisomy 12 was detected by FISH analysis. PCR analysis of the peripheral blood for immunoglobulin heavy chain genes demonstrated two sharp bands that were initially interpreted as biallelic heavy chain gene rearrangements. Myeloma cells were CD38 and CD138 positive, CD19 negative and expressed cytoplasmic kappa light chain, but not heavy chains. In order to investigate the clonal relationship between these B cell malignancies, a detailed analysis of VHDJH and VκJκ gene rearrangements in individually sorted CD5 and CD19 double-positive CLL cells and also in CD38-positive and CD19-negative myeloma cells by single cell PCR of genomic DNA and direct sequencing was carried out. This technique permitted identification and pairing of both the heavy and light chain immunoglobulin genes from the same individually sorted cell. A total of 17 individual CLL and 23 myeloma cells were successfully analyzed. Our analysis demonstrated (a) the presence of two discrete clones of CLL, one with usage of [VH1-2*04/D3-3*01/J3*02]-[Vκ2-28*01/J1*01] without VH and Vκ hypermutation and the other with usage of [VH1-2*04/D4-11*01/J6*02]-[Vκ1-5*03/J1*01] with VH and Vκhypermutation; (b) no clonal relationship between the CLL and myeloma cells that utilized different VHDJH and VκJκ rearrangements [VH3-66*02/3-10*01/J4*03]-[Vκ1-33*01/J2*02] with VH and Vκ hypermutation. To our knowledge, this is the first demonstration of a biclonal CLL with mutated and unmutated clones in the same patient along with a third clonally unrelated B cell malignancy. This result suggests that single cell analysis may be necessary to detect subtle biclonality of CLL that might be associated with a more aggressive phenotype.

Inhibition of JAK2/STAT3 Pathway Leads to Apoptosis in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2023-2023 ◽  
Author(s):  
Filippo Severin ◽  
Federica Frezzato ◽  
Veronica Martini ◽  
Flavia Raggi ◽  
Valentina Trimarco ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
B Lymphocytes ◽  
Sh2 Domain ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Stat3 Phosphorylation ◽  
Jak2 Inhibition

Abstract INTRODUCTION Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus kinases)-STAT (Signal Transducers and Activators of Transcription) pathways. We particularly focused on the JAK2/STAT3 axis since Interleukin-6 (IL-6), one of the most abundant cytokines released in the CLL microenvironment, is the key ligand of the receptor triggering this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. METHODS B cells were collected from 12 controls and 46 CLL patients. Purified cells (2x106cells/ml) were cultured, and treated with AG490 (10, 50 and 100μM), AZD1480 (1, 4 and 10μM), Fedratinib (1, 5 and 10μM), and Ruxolitinib (0.313, 2.5 and 10μM) (which are JAK2 inhibitors), and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs. STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test. RESULTS We demonstrated that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. As far as STAT3 phosphorylation at Tyr705, that is an essential step for STAT3 activation, we demonstrated a constitutive phosphorylation in CLL cells by FC and WB analyses, although in some patients STAT3 Tyr705 phosphorylation is barely detected. We also pointed out that the in vitroincubation of leukemic B cells with AG490 and Stattic and Fedratinib, induces a dose-dependent apoptosis of CLL B cells. However, the tested doses of Ruxolitinib and AZD1480 did not seem to CLL B cell viability but only STAT3 phosphorylation. Both AG490 and Stattic were able to bypass the microenvironmental protection when neoplastic B cells were co-cultured with MSCs. STAT3 Tyr705 localization was analyzed in normal and leukemic B cells by a subcellular protein fractionation. We separated nuclei from cytosol, detecting STAT3 Tyr705 both in the cytosolic and in the nuclear fractions of CLL B cells. We showed that AG490 and Fedratinib treatment on CLL cells can mediate other effects: i) SHP-1 activity is turned on by JAK2 inhibition, decreasing its phosphorylation at Ser591; ii) AG490 administration inactivates protein Lyn, reducing the phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SH2-domain containing Tyrosine Phosphatase (SHP-1), a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells. To confirm the link between JAK2 inhibition by AG490 and Lyn dephosphorylation, we added sodium orthovanadate (Na3VO4, 100 μM), a phosphatase inhibitor, to cell culture to restore Lyn activation (resulting from the inactivation of SHP-1 phosphatase); as a result, Lyn Tyr396 phosphorylation was restored. On the contrary, the treatment of CLL cells with Stattic did not induce any change in SHP-1 status with respect to untreated cells since Stattic is effective on STAT3, that is a downstream protein with respect to JAK2. Since Stattic did not affect SHP-1 activation, it does not impact on Lyn activation/phosphorylation. CONCLUSIONS The ability of AG490 and Stattic to induce apoptosis in leukemic B cells bypassing the pro-survival stimuli provided by the tumor microenvironment and the Fedratinib effectiveness at low doses, represents a starting point for the development of new therapeutic strategies in CLL. This study also provides new insights for the investigation of the pathogenesis of CLL focusing the attention on the cross-talk between JAK/STAT and BCR/Lyn axes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lack of extensive mutations in the VH5 genes used in common B cell chronic lymphocytic leukemia.

1993 ◽  
Vol 177 (4) ◽  
pp. 1039-1046 ◽  
Author(s):  
L Z Rassenti ◽  
T J Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Somatic Mutation ◽  
Sequence Homology ◽  
Common Property ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
V Genes ◽  
Cell Chronic Lymphocytic Leukemia

B cell chronic lymphocytic leukemia (CLL) is a malignancy of the CD5+ B cells. Prior studies indicated that CLL B cells generally express immunoglobulin (Ig) VH and VL genes with little or no somatic mutations. However, a recent report indicated that VH251, one of three VH genes belonging to the VH5 subgroup (e.g., VH251, VH32, and VH15), not only is frequently rearranged in this disease, but also has extensive and selective mutations when expressed by CLL B cells. The extent and nature of these mutations contrasts markedly from the low level of mutations noted in VH5 genes used by normal B cells or other Ig V genes found expressed in CLL. To determine whether this difference reflects a unique property of VH251 or a previously unrecognized subgroup of CLL, we examined for VH5 Ig gene rearrangements in leukemia cells from 68 patients that satisfied clinical and diagnostic criteria for CD5+ B cell CLL. Southern blot hybridization studies with probes for VH251 and the JH locus revealed that only 7 (10%) of the 68 monoclonal CLL cell populations had undergone Ig gene rearrangement involving VH5 genes. Two (3%) were found to have functionally rearranged VH5 genes that shared &gt; or = 98% sequence homology with 5-2R1, a VH251 gene isolated from a pre-B cell acute lymphocytic leukemia. The other five CLL (7%) had functionally rearranged VH5 genes that each shared &gt; or = 99% nucleic acid sequence homology with a germline VH32 isolated from human sperm DNA. These data indicate that VH251 or VH32 also may be expressed by CD5+ CLL B cells with little or no somatic mutation. These findings contrast with a recently published study on VH5 gene expression in B CLL and contest the hypothesis that extensive somatic mutation is a common property of the VH5 genes used in this disease. Further work to define the clinical and/or phenotypic characteristics of patients with leukemia cells that express mutated versus nonmutated Ig V genes may reveal subsets of CLL that possibly differ in their cytogenesis, etiopathogenesis, and/or clinical behavior.

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