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Florence Ajchenbaum-Cymbalista

Publications and source records attributed to Florence Ajchenbaum-Cymbalista.

9 recordsLinked to original sources
PubMed2007

National standardization of ZAP-70 determination by flow cytometry: the French experience.

BACKGROUND: 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. METHODS: AntiZAP-70 antibody 2F3.2 was used with indirect labeling and secondary anti-IgG2a antibody. The reference values for the expression of the results were determined on 45 normal blood samples. ZAP-70 protein expression was investigated in 192 CLL samples. The indirect technique was compared with FITC-conjugated 2F3.2 clone, and with clone 1E7.2-FITC, -PE or -AlexaFluor 488. RESULTS: Using FITC or PE-conjugated antibodies, 2F3.2 and 1E7.2 clones allowed a much less adequate discrimination between positive and negative cells and discordant cases were most likely true negative cases. Using the AlexaFluor 488 conjugated 1E7.2 clone, the discordant cases were mostly negative with the conjugated antibody and positive with the 2F3.2 clone but Western blotting or RNA microarray confirmed discordant cases were false negative with the conjugated antibody. Subsequently, recommendations were used by 13 centers participating in an interlaboratory quality control protocol. The use of MFI ratio appeared to be more reliable. CONCLUSIONS: Results suggested that slight differences in the procedure had little impact on the interpretation in characteristic cases; however, careful interpretation was required for values close to threshold.

Antibodies↗
PubMed2006

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 patient's 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.

Antibodies, Monoclonal↗
PubMed2006

Survival response to B-cell receptor ligation is restricted to progressive chronic lymphocytic leukemia cells irrespective of Zap70 expression.

Despite very similar gene expression profiles, the clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is heterogeneous. Immunoglobulin VH (IgVH) mutational status and expression of B-cell receptor (BCR) signaling mediators have been associated with disease progression. However, the consequences of BCR engagement on cell survival and evolution of the disease remain unclear. We show here that B-CLL cell survival is dependent on the threshold of BCR stimulation induced by immobilized antibody, in contrast to soluble anti-mu F(ab)'2 antibody, which leads to apoptosis. Measurement of metabolic activity and apoptotic response discriminated two subgroups. "Nonresponders" showed low metabolic activity and unmodified apoptotic response upon BCR stimulation. In contrast, "responders" exhibited increased metabolic activity and inhibition of spontaneous apoptosis. This survival advantage was associated to a BCR-dependent activation profile leading to induction of cyclin D2/cyclin-dependent kinase 4 (cdk4) expression and G1 cell cycle progression. The ability to respond to BCR ligation correlated with an unfavorable clinical course and allowed to define an additional group of patients among IgVH-mutated cases exhibiting a risk of progression. Remarkably, we show that Zap70 expression was neither mandatory nor sufficient to generate downstream survival signals and cyclin D2/cdk4 up-regulation. In conclusion, BCR engagement has a significant effect on B-CLL cell survival, activation, and G1 progression. Furthermore, our results provide new insights in the physiopathology of progressive IgVH-mutated cases.

Antibodies, Anti-Idiotypic↗
PubMed2006

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 < or =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.

Aged↗
PubMed2006

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.

B-Cell Activating Factor↗
PubMed2003

Binet's staging system and VH genes are independent but complementary prognostic indicators in chronic lymphocytic leukemia.

PURPOSE: Rai's and Binet's staging systems have contributed significantly to the identification of major prognostic groups in chronic lymphocytic leukemia (CLL), though they fail to accurately predict disease progression at the individual level. Biologic factors, such as the mutational status of the immunoglobulin heavy-chain variable genes (VH, cytogenetics, CD38 expression, and some serum markers, have recently improved prognostic assessment in CLL. In this study, we analyzed the prognostic value of VH mutational status within the different stages of Binet's classification in 145 patients. PATIENTS AND METHODS: Our series consisted of 83 VH mutated (MT) and 62 VH unmutated (UM) patients. MT cases predominated within Binet's stage A (70%), whereas UM cases predominated among stages B and C (62%). RESULTS: Median overall survival (OS) was 84 months for UM patients and was not achieved for the MT group (70% 12-year survival, P <.0001). Concerning Binet's stage A, both median OS and progression-free survival were significantly shorter for UM patients when compared with those of MT patients (97 months v not achieved, P =.0017; and 42 v 156 months, P <.0001), which compared favorably with the classical A' and A" substaging. The VH mutational profile could also segregate stage B and C patients into two groups with different survival patterns (median OS, 78 v 120 months for UM and MT patients, respectively; P =.002). CONCLUSION: The significant survival differences observed between the VH mutational groups, among stage A and stage B and C patients, indicate that Binet's classification and VH genes are independent prognostic variables and are most likely complementary.

Brazil↗
PubMed2003

Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway.

Tumor necrosis factor (TNF) superfamily members BAFF, or B-cell activation factor of the TNF family, and APRIL, a proliferation-inducing ligand, are involved in normal B-cell survival and differentiation. They interact with 3 receptors: BAFF-R, specific to BAFF; and TACI and BCMA, which are shared by BAFF and APRIL. We tested the potential role of these proteins in B-cell chronic lymphocytic leukemia (B-CLL) resistance to apoptosis. TACI and BAFF-R mRNAs were found in leukemic B cells. BAFF and APRIL mRNAs and proteins were detected in B-CLL leukemic cells and normal blood or tonsil-derived B lymphocytes. Yet, in contrast to normal B lymphocytes, BAFF and APRIL were expressed at the membranes of leukemic cells. Adding soluble BAFF or APRIL protected B-CLL cells against spontaneous and drug-induced apoptosis and stimulated NF-kappaB activation. Conversely, adding soluble BCMA-Fc or anti-BAFF and anti-APRIL antibodies enhanced B-CLL apoptosis. Moreover, a soluble form of BAFF was detected using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF MS) in the sera of B-CLL patients but not of healthy donors. Taken together, our results indicate that B-CLL cells can be rescued from apoptosis through an autocrine process involving BAFF, APRIL, and their receptors. Inhibiting BAFF and APRIL pathways may be of therapeutic value for B-CLL treatment.

Aged↗