Florence Ajchenbaum-Cymbalista

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

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

Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process

The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patients prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold‐standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta‐associated protein (ZAP‐70) expression was associated with unmutated IgVH genes and ZAP‐70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP‐70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP‐70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti‐ZAP‐70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti‐ZAP‐70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP‐70 levels were expressed as percentage positive (POS) relative to isotype control or normal B‐cells or normal T‐cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T‐cells. The reported level of ZAP‐70 expression varied greatly depending on the antibody and the method used to express the results. The CLL/T‐cell ZAP‐70 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP‐70 expression. We assessed the variation of ZAP‐70 expression levels in fresh cells according to storage time, which demonstrated that ZAP‐70 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures.

Down-regulation of CXCR4 and CD62L in Chronic Lymphocytic Leukemia Cells Is Triggered by B-Cell Receptor Ligation and Associated with Progressive Disease

Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases.

Constitutive and B-cell receptor-induced activation of STAT3 are important signaling pathways targeted by bortezomib in leukemic mantle cell lymphoma

Background The deregulation of several transcription factors contribute to the aggressive course of mantle cell lymphoma. This study focuses on survival signals emanating from the tumor environment and involving the signal transducer and activator of transcription (STAT) 3 through cytokines or antigen recognition. Design and Methods Primary mantle cell lymphoma cells were isolated from 20 leukemic patients. The phosphorylation status of STAT3 was evaluated by immunoblottting and immunofluorescence, the levels of cytokine secretion by enzyme-linked immunosorbent assay and the cell survival signals by apoptosis and cell viability assays. Results STAT3 was constitutively phosphorylated in the Jeko-1 mantle cell lymphoma cell line and in 14 out of 20 (70%) cases of leukemic mantle cell lymphoma as the result of an autocrine secretion of interleukin-6 and/or interleukin-10. In addition, B-cell receptor engagement resulted in an increase of both in vitro cell survival and STAT3 phosphorylation in primary mantle cell lymphoma cells. Inhibition of the Janus-activated kinase/STAT3 pathway increased spontaneous apoptosis and suppressed B-cell receptor-induced cell survival in all cases analyzed. The impact of in vitro exposure to the proteasome inhibitor bortezomib was next evaluated in primary mantle cell lymphoma cells. Bortezomib induced apoptosis and a decrease of both interleukin-6/interleukin-10 secretion and STAT3 phosphorylation. In addition, bortezomib inhibited B-cell receptor-triggered STAT3 phosphorylation and cell survival. Conclusions We demonstrated that STAT3 was activated in primary mantle cell lymphoma cells either constitutively through a cytokine autocrine loop or in response to B-cell receptor engagement, both processes leading to a survival signal inhibited by bortezomib. STAT3 appears, therefore, to play a pivotal role in mantle cell lymphoma and represents a promising therapeutic target.

Over-expression of cyclin D1 in chronic B-cell malignancies with abnormality of chromosome 11q13

Accurate identification of B‐cell chronic malignancies is sometimes uncertain, despite careful cytologic and immunophenotypic evaluation. Cytogenetics and molecular biology studies may therefore prove useful, because some of these disorders are associated with nonrandom abnormalities, such as the t(11;14)(q13;q32) translocation and bcl‐1 rearrangement mainly observed in mantle‐cell lymphoma (MCL). We studied the expression of cyclin D1 in malignant lymphoid cells from the peripheral blood of 32 patients with various B‐cell chronic lympho‐proliferative disorders, using Northern blot (NB) and RNA in situ hybridization (ISH). Cytogenetic analysis was informative in 18 cases, and most of the missing karyotype data were from typical B‐CLL cases where a t(11;14) is unlikely to be found. Over‐expression of cyclin D1 mRNA was observed by both NB and ISH in four samples (MCL: two cases; lymphoplasmacytic lymphoma: one case, unclassified B‐cell chronic disorder: one case). In each of these cases there was an abnormality of chromosome 11q13, either a t(11;14)(q13;q32) translocation (three cases) or a del(11)(q13) without evidence of chromosome 14 involvement (one case). Cytogenetic and gene rearrangement studies are not available in all institutions and have some technical pitfalls. Because of its close association with bcl‐1 rearrangement and/or t(11;14), the demonstration of cyclin D1 mRNA over‐expression either by NB, or, more conveniently, by ISH, may represent additional information which could be of help for the identification of B‐cell malignancies.

National Standardization of ZAP-70 Determination by Flow Cytometry: The French Experience

ZAP‐70, after being considered as a potential surrogate for VH mutational status, has seen its own prognostic value emerge. We aimed at standardizing a simple, fast, and reproducible flow cytometry method.

Use of hematopoietic progenitor cell count on the Sysmex XE-2100 for peripheral blood stem cell harvest monitoring

Successful peripheral blood stem cell (PBSC) collection depends on the timing of apheresis based on CD34+ cell enumeration. Because this analysis is expensive and induces organization difficulties, we evaluated hematopoietic progenitor cell (HPC) quantification on the Sysmex XE-2100 as a surrogate analysis. We tested 157 blood samples for CD34+ cells and HPC counts. We found a good linear correlation between HPC and CD34+ and determined simple rules allowing to use HPC count in daily practice. We set a positive cut-off >30 HPC/mm3 for allowing PBSC harvest and a negative cut-off at 0 HPC/mm3 for which collection should be delayed. These two situations accounted for 62% of cases and CD34+ cell count by flow cytometry confirmed HPC result in 95% of cases. Between 0 and 30 HPC/mm3, CD34+ enumeration is required for decision-making. We conclude that HPC count may be a useful surrogate for CD34+ enumeration in PBSC harvest monitoring.

Deciphering leukemic B-cell chronic lymphoproliferative disorders

Diagnosis of leukemic B-cell chronic lymphoproliferative disorders (B-CLPD) is a frequent challenge in hematology. In this multicentric study, we prospectively studied 165 new consecutive leukemic patients with B-CLPD selected on the basis of Royal Marsden Hospital scoring system ≤3. The primary aim of the study was to try to decipher the atypical cases and identify homogenous subgroups. Overall, morphological examination contributed to diagnosis in only 20% cases, all of them CD5 negative. Thirty additional cases were CD5 negative suggestive of leukemic marginal zone lymphoma in most cases. The significantly poorer survival of the 26 cyclin D1 positive cases justifies recommending its systematic determination among atypical B-CLPD. CD20 expression segregated clearly two subgroups among CD5 positive cyclin D1 negative B-CLPD. The 17 patients with the CD20 dim profile represent a homogeneous subgroup very close to typical B-cell chronic lymphocytic leukemia (B-CLL) on morphological, phenotypical and cytogenetical criteria. In contrast, the subgroup of 51 patients with a CD20 bright profile is heterogeneous. Their significantly lower p27 expression level suggest the presence of a proliferative component, underlying a more aggressive disease. Further genomic studies are warranted to establish their precise nature. These cases should not be included in the same therapeutic trials as B-CLL.

Protein kinase D-dependent CXCR4 down-regulation upon BCR triggering is linked to lymphadenopathy in chronic lymphocytic leukaemia

In Chronic Lymphocytic Leukemia (CLL), infiltration of lymph nodes by leukemic cells is observed in patients with progressive disease and adverse outcome. We have previously demonstrated that B-cell receptor (BCR) engagement resulted in CXCR4 down-regulation in CLL cells, correlating with a shorter progression-free survival in patients. In this study, we show a simultaneous down-regulation of CXCR4, CXCR5 and CD62L upon BCR triggering. While concomitant CXCR4 and CXCR5 down-regulation involves PKDs, CD62L release relies on PKC activation. BCR engagement induces PI3K-δ-dependent phosphorylation of PKD2 and 3, which in turn phosphorylate CXCR4 Ser324/325. Moreover, upon BCR triggering, PKD phosphorylation levels correlate with the extent of membrane CXCR4 decrease. Inhibition of PKD activity restores membrane expression of CXCR4 and migration towards CXCL12 in BCR-responsive cells in vitro. In terms of pathophysiology, BCR-dependent CXCR4 down-regulation is observed in leukemic cells from patients with enlarged lymph nodes, irrespective of their IGHV mutational status. Taken together, our results demonstrate that PKD-mediated CXCR4 internalization induced by BCR engagement in B-CLL is associated with lymph node enlargement and suggest PKD as a potential druggable target for CLL therapeutics.

Unexpectedly high frequency of European parentage in Venezuelan patients with chronic lymphocytic leukemia

Abstract There is insufficient information on the characteristics of chronic lymphocytic leukemia (CLL) in Latin American patients. Immunoglobulin variable-region heavy-chain (IGVH) gene usage and mutation status and prognostic factors were investigated in patients resident in Venezuela. The most frequently used IGVH family genes were: VH3 > VH1 > VH4 > VH5, with a high incidence of IGVH1.69 and IGVH3.21 genes, and 55.2% of IGVH genes were mutated. Analysis of HCDR3 (third complementarity-determining region of the heavy chain) revealed that 24% of Venezuelan HCDR3s belonged to a CLL stereotyped HCDR3. Results for prognostic factors were similar to those reported previously for Caucasian populations. Interestingly, we found an over-representation of people of European extraction among Venezuelan patients with CLL, suggesting the possibility of a higher frequency of susceptibility genes for CLL in Europeans in comparison with Latin American mestizos.