Raimundo Fernando Bezares

Chronic lymphocytic leukemia cells bind and present the erythrocyte protein band 3: possible role as initiators of autoimmune hemolytic anemia.

The mechanisms underlying the frequent association between chronic lymphocytic leukemia (CLL) and autoimmune hemolytic anemia are currently unclear. The erythrocyte protein band 3 (B3) is one of the most frequently targeted Ags in autoimmune hemolytic anemia. In this study, we show that CLL cells specifically recognize B3 through a still unidentified receptor. B3 interaction with CLL cells involves the recognition of its N-terminal domain and leads to its internalization. Interestingly, when binding of erythrocyte-derived vesicles as found physiologically in blood was assessed, we observed that CLL cells could only interact with inside-out vesicles, being this interaction strongly dependent on the recognition of the N-terminal portion of B3. We then examined T cell responses to B3 using circulating CLL cells as APCs. Resting B3-pulsed CLL cells were unable to induce T cell proliferation. However, when deficient costimulation was overcome by CD40 engagement, B3-pulsed CLL cells were capable of activating CD4+ T cells in a HLA-DR-dependent fashion. Therefore, our work shows that CLL cells can specifically bind, capture, and present B3 to T cells when in an activated state, an ability that could allow the neoplastic clone to trigger the autoaggressive process against erythrocytes.

Biallelic deletion 13q14.3 in patients with chronic lymphocytic leukemia: cytogenetic, FISH and clinical studies

Background and objective:  Monoallelic deletion of 13q14.3 (13q14x1) is the most common abnormality in chronic lymphocytic leukemia (CLL). As a sole alteration, it predicts a favorable outcome. Biallelic 13q14.3 (13q14x2) deletion or concomitant 13q14x1/13q14x2 has been scarcely evaluated in the literature. We present the clinical, cytogenetic and fluorescence in situ hybridization (FISH) analysis of six CLL patients with normal karyotypes and 13q14x2 and their comparison to cases with 13q14x1 as a single abnormality.

Ibrutinib impairs the phagocytosis of rituximab-coated leukemic cells from chronic lymphocytic leukemia patients by human macrophages

Ibrutinib is an oral irreversible inhibitor of the bruton tyrosine kinase (Btk) which has shown promising efficacy and excellent tolerability in patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies.[1][1] Ibrutinib inhibits BCR signaling, impairs chemokine-mediated adhesion

The cytotoxic activity of Aplidin in chronic lymphocytic leukemia (CLL) is mediated by a direct effect on leukemic cells and an indirect effect on monocyte-derived cells

SummaryAplidin is a novel cyclic depsipeptide, currently in Phase II/III clinical trials for solid and hematologic malignancies. The aim of this study was to evaluate the effect of Aplidin in chronic lymphocytic leukemia (CLL), the most common leukemia in the adult. Although there have been considerable advances in the treatment of CLL over the last decade, drug resistance and immunosuppression limit the use of current therapy and warrant the development of novel agents. Here we report that Aplidin induced a dose- and time-dependent cytotoxicity on peripheral blood mononuclear cells (PBMC) from CLL patients. Interestingly, Aplidin effect was markedly higher on monocytes compared to T lymphocytes, NK cells or the malignant B-cell clone. Hence, we next evaluated Aplidin activity on nurse-like cells (NLC) which represent a cell subset differentiated from monocytes that favors leukemic cell progression through pro-survival signals. NLC were highly sensitive to Aplidin and, more importantly, their death indirectly decreased neoplasic clone viability. The mechanisms of Aplidin-induced cell death in monocytic cells involved activation of caspase-3 and subsequent PARP fragmentation, indicative of death via apoptosis. Aplidin also showed synergistic activity when combined with fludarabine or cyclophosphamide. Taken together, our results show that Aplidin affects the viability of leukemic cells in two different ways: inducing a direct effect on the malignant B-CLL clone; and indirectly, by modifying the microenvironment that allows tumor growth.

CXCL12-induced chemotaxis is impaired in T cells from patients with ZAP-70-negative chronic lymphocytic leukemia

Background T cells from patients with chronic lymphocytic leukemia may play an important role in contributing to the onset, sustenance, and exacerbation of the disease by providing survival and proliferative signals to the leukemic clone within lymph nodes and bone marrow. Design and Methods By performing chemotaxis assays towards CXCL12, CCL21 and CCL19, we sought to evaluate the migratory potential of T cells from chronic lymphocytic leukemia patients. We next analyzed the chemokine-induced migration of T cells, dividing the chronic lymphocytic leukemia samples according to their expression of the poor prognostic factors CD38 and ZAP-70 in leukemic cells determined by flow cytometry. Results We found that T cells from patients with chronic lymphocytic leukemia are less responsive to CXCL12, CCL21 and CCL19 than T cells from healthy adults despite similar CXCR4 and CCR7 expression. Following separation of the patients into two groups according to ZAP-70 expression, we found that T cells from ZAP-70-negative samples showed significantly less migration towards CXCL12 compared to T cells from ZAP-70-positive samples and that this was not due to defective CXCR4 down-regulation, F-actin polymerization or to a lesser expression of ZAP-70, CD3, CD45, CD38 or CXCR7 on these cells. Interestingly, we found that leukemic cells from ZAP-70-negative samples seem to be responsible for the defective CXCR4 migratory response observed in their T cells. Conclusions Impaired migration towards CXCL12 may reduce the access of T cells from ZAP-70-negative patients to lymphoid organs, creating a less favorable microenvironment for leukemic cell survival and proliferation.

Structural alterations in chronic lymphocytic leukaemia. Cytogenetic and FISH analysis

In this study, we described cytogenetics and fluorescence in situ hybridization (FISH) analysis performed in chronic lymphocytic leukaemia (CLL) patients with structural alterations. Results were correlated with clinical characteristics. A total of 38 CLL patients: 16 cases with complex and 22 with simple karyotypes were studied. For comparison of clinical parameters, a control group of 78 CLL patients with normal karyotype and without FISH genomic alterations were also evaluated. We found 38 structural abnormalities not previously described in the literature, 28 (74%) of them were translocations. In cases with complex karyotypes, chromosomes 6, 8 and 13 were the most frequently involved in new alterations (nine each), followed by chromosomes 12, 14 and 15 (six each). Chromosome 8p was particularly involved in losses, being 8p21‐pter the commonest region of overlap. Cases with simple karyotypes, showed del(6q) as the most frequent alteration (39%). Del(9)(q11) was recurrent in our series. Analysis of clinical parameters showed significant differences in white blood count (p = 0.005) and platelet count (p = 0.015) between patients with structural alterations and the control group. In addition, patients with structural alterations had a significantly shorter time to first treatment (TFT) (29 months) than the control group (69 months) (p = 0.037). Cases with complex karyotypes had a lower proportion of patients in Rai 0 clinical stage (15.4% vs 75%) (p = 0.005) and higher β2 microglobulin levels (3.3 vs 2.5 µg/mL) (p = 0.037) than those with simple karyotypes. Furthermore, a shorter TFT (13 months) and overall survival (56 months) in the complex karyotypes group compared with controls (69 and 144 months, respectively) (p = 0.015 and p = 0.005, respectively) were also found. Our results support the importance of cytogenetic analysis for clinical outcome in CLL and suggest that the diversity of genomic alterations is much greater than previously appreciated. Copyright

CXCL12 is a costimulator for CD4+ T cell activation and proliferation in chronic lymphocytic leukemia patients

Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4+ T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4+ T cells from CLL patients, similarly in T cells from ZAP-70+ to ZAP-70− patients. Autologous nurse-like cells establish a close contact with CD4+ T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.

The Expression of Sphingosine-1 Phosphate Receptor-1 in Chronic Lymphocytic Leukemia Cells Is Impaired by Tumor Microenvironmental Signals and Enhanced by Piceatannol and R406

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L+, BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow–resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38low counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues.

SHIP‐1 protein level and phosphorylation status differs between CLL cells segregated by ZAP‐70 expression

The clinical course of B-cell chronic lymphocytic leukaemia (CLL) is very heterogeneous, with some patients that present an indolent disease and others that succumb rapidly despite therapy. Leukaemic cells from aggressive CLL patients typically display B-cell receptors (BCR) encoded by unmutated immunoglobulin variable heavy-chain genes (IGHV) and express the protein tyrosine kinase ZAP-70 (Crespo et al, 2003). In contrast, mutated IGHV genes and the absence of ZAP-70 are mostly found in patients with indolent disease (Crespo et al, 2003). It has been proposed that survival differences between these two subgroups are related to the differential ability of the BCR to respond to stimulation (Chen et al, 2002; Stevenson & Caligaris-Cappio, 2004). Thus, ZAP-70 patients display a more effective BCR signal transduction that could contribute to their relatively aggressive clinical behaviour (Chen et al, 2002). Inhibitory phosphatases SHP-1 (SH2 domain-containing tyrosine phosphatase-1), SHIP-1 (SH2 domain-containing phosphatidylinositol 5-phosphatase-1) and SHIP-2 are involved in the complex and organized machinery aimed at counterbalancing B-cell activation upon BCR engagement by restricting the duration and/or intensity of the signalling (Zhang et al, 2000; March & Ravichandran, 2002). As ZAP-70 patients present an impaired BCR signalling capacity, we have explored the possibility that these phosphatases were preferentially expressed in CLL cells from this subgroup. Following informed consent and ethical approval, Western blotting analysis was performed on 49 purified CLL samples. SHP-1 and SHIP-1 proteins were found to be expressed in B cells from ZAP-70 and ZAP-70 CLL patients, while SHIP-2 could not be detected in any of the samples analysed (Fig 1A), even when the amount of protein loaded in each lane was duplicated (data not shown). Normal tonsillar B lymphocytes (TBL), used as controls, showed specific bands at 68, 145 and 150 kDa corresponding to SHP-1, SHIP-1 and SHIP-2 respectively (Fig 1A). There were no significant differences in SHP-1 expression between CLL subgroups (data not shown). By contrast, ZAP-70 CLL cells not only displayed a higher SHIP-1 protein expression compared with ZAP-70 subgroup (Fig 1B), but also it was constitutively tyrosine phosphorylated to a greater extent (Fig 1C and D). As SHIP-1 phosphorylation may correlate with its inhibitory capacity by enhancing the interaction with other adapter proteins (March & Ravichandran, 2002), ZAP-70 CLL cells might be more prone to inhibition by SHIP-1 than ZAP-70 ones. Finally, we evaluated SHIP-1 phosphorylation status upon BCR cross-linking with goat anti-human IgM (anti-l) and rabbit anti-goat IgG (RAG). We found that, in ZAP-70 patients, who present an impaired IgM signalling capacity (Chen et al, 2002), BCR cross-linking led to a time-dependent increase in reactivity of the anti-P-SHIP-1 antibody, which was maximal at 15 min and then decreased back towards baseline by 30 min (Fig 1E and F). By contrast, ZAP-70 samples did not modify SHIP-1 phosphorylation status upon BCR engagement (Fig 1E and F) although they expressed similar levels of surface IgM compared with ZAP-70 samples (data not shown). In conclusion, we found that the inhibitory phosphatase SHIP-1 exclusively participated in BCR signal transduction in ZAP-70 CLL cells, wherein it is expressed and constitutively tyrosine phosphorylated to a greater extent compared with ZAP-70 samples. Taken together, our data suggest that SHIP1 might be involved in the impaired BCR signalling commonly found in the former subgroup of patients. Moreover, given that SHIP-1 can negatively modulate not only BCR but also cytokine and chemokine receptor signalling (March & Ravichandran, 2002), the possibility exist that ZAP-70 CLL cells, by expressing higher SHIP-1 levels, hold higher signalling thresholds to different microenvironment stimuli. Experiments are in progress to determine whether SHIP-1 can regulate CLL cell responsiveness.

Immunoglobulin Gene Rearrangements and Mutational Status in Argentinian Patients With Chronic Lymphocytic Leukemia

BACKGROUND Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. The mutational status of the immunoglobulin heavy chain variable (IGHV) region represents one of the best prognostic markers and defines 2 disease subgroups: mutated (M-CLL) and unmutated (UM-CLL), with different clinical course. MATERIALS AND METHODS IGHV-D-J gene rearrangements and mutational status were analyzed in 73 Argentinian patients with CLL, 22 previously treated, by reverse transcriptase-polymerase chain reaction and bidirectional sequencing. The results were compared with those reported in other geographic regions. Fluorescence in situ hybridization analysis was also performed. RESULTS A total of 43 (58.9%) cases were of patients with M-CLL, and 30 (41.1%) were patients with UM-CLL. Deletion of chromosome 13q14 as a single alteration was more frequently observed in the M-CLL group (48%) than in the UM-CLL group (24%). In the M-CLL group, the proportion of cases with deletion of chromosome 13q14 was significantly higher than those with +12 and those with deletions of chromosomes 17p and 11q (P = .003). The most frequently used IGHV families were IGHV3 > IGHV1 > IGHV4, which are different from those observed in Asian, Brazilian, and Uruguayan series. The IGHV3-23 gene (10.8%) was the most commonly used, followed by IGHV1-69 (9.5%), IGHV4-59 and IGHV2-5 (6.8% each), and IGHV3-21 and IGHV3-30 (5.4% each). IGHV4-34 showed the lowest frequency (2.7%) in our cohort compared with published data, whereas IGHV4-59, IGHV3-72, and IGHV2-5 were overexpressed in our series. Stereotyped HCDR3 (heavy chain complementary determining region 3) was found in 9.5% of patients. CONCLUSIONS Our results showed that Argentinian patients with CLL display an IGHV gene usage that resembles that observed in Western countries and exhibited interesting similarities and differences with respect to published series from other Latin American populations, which reflect variations in the genetic background.