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

Letestu, Rémi; Rawstron, Andy C.; Villamor, Neus; Leuven, Nancy Boeckx; Böettcher, Sebastian; Buhl, Anne Mette; Duerig, Jan; Ibbotson, Rachel; Kroeber, Alexander; Langerak, Anton W.; Garff‐Tavernier, Magali Le; Mockridge, Ian; Morilla, Alison; Padmore, Ruth; Rassenti, Laura Z.; Ritgen, Matthias; Shehata, Medhat; Smolewski, Piotr; Staib, Peter; Ticchioni, Michel; Walker, Clare; Ajchenbaum‐Cymbalista, Florence

doi:10.1002/cyto.b.20132

Cited by 71 publications

(87 citation statements)

References 16 publications

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“…1). Isoclonic ratios and T/B ratios were calculated as described (12,(15)(16)(17)(18)(19)29 and see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“…The main problem was to find the correct internal control. Among proposed methods, calculating the ZAP70 T/B ratio is one of the earliest and most common approaches (12,(15)(16)(17)(18). Other methods based on ratios calculated on normal B-cell MFIs have drawbacks such as the very low percentage of residual B-cells or the difficulty of separating them from CLL cells by flow cytometry in the case of weak CD5 expression (11,13,32).…”

Section: T/b Ratio Does Not Reflect Zap70 Expression Levelsmentioning

confidence: 99%

“…Regarding the antibody and fluorochrome, various authors agree that the phycoerythrin-conjugated (PE-conjugated) SBZAP monoclonal antibody (mAb) is one of the best reagents for this method (11)(12)(13). Currently most groups express FCM results of ZAP70 expression in CLL B-cells as a ratio between mean fluorescence intensities (MFI) of ZAP70 in CLL B-cells and in normal residual or externally added B-cells (11,13,14) or, more frequently, as a ratio between ZAP70 MFIs in residual T-cells and CLL B-cells (ZAP70 T/ B ratio) (12,(15)(16)(17)(18)(19). The main disadvantage of the former method is that residual normal B-cells may be virtually absent in peripheral blood of patients with CLL and external addition of normal B-cells to the blood sample prior to staining supposes that ZAP70 levels in normal subjects are biologically stable.…”

Section: Introductionmentioning

confidence: 99%

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T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

Rizzo

Bouvier

Youlyouz-Marfak

et al. 2012

Cytometry Part B Clinical

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Flow cytometry is the reference technique for assessing ZAP70 expression, a marker of poor prognosis in CLL. One of the most common methods is to assess ZAP70 levels in CLL cells by calculating the ratio between ZAP70 mean fluorescence intensities (MFIs) in residual T-cells and CLL B-cells (ZAP70 T/B ratio). In this study, we developed a new method for ZAP70 labeling. Cells were labeled with a combination of anti ZAP70 phycoerythrin-conjugated SBZAP monoclonal antibody (mAb) and mAbs against CD45, CD19, and CD5. The latter three were used to specifically gate on different lymphocyte subsets. Staining was performed in absence (test) or in presence of excess unconjugated SBZAP mAb (isoclonic control). A so-called ZAP70 isoclonic ratio between SBZAP MFIs in the test and isoclonic control was calculated. A series of 32 patients with CLL and 10 normal controls were studied. Prediction of IGHV mutation status by ZAP70 isoclonic and T/B ratios was similar. By using the ZAP70 isoclonic ratio, we showed that ZAP70 expression was increased in T-cells from CLL patients. Nearly all cases with increased ZAP70 expression in CLL cells were associated with high ZAP70 expression in cognate T-cells. Therefore, the ZAP70 isoclonic ratio was more likely to closely reflect the biology of ZAP70 dysregulation rather than the T/B ratio. These results also explained why ZAP70 T/B ratios were artefactually close to normal in cells from CLL patients with high levels of ZAP70. V C 2012 International Clinical Cytometry Society

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“…1). Isoclonic ratios and T/B ratios were calculated as described (12,(15)(16)(17)(18)(19)29 and see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

Section: T/b Ratio Does Not Reflect Zap70 Expression Levelsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

Rizzo

Bouvier

Youlyouz-Marfak

et al. 2012

Cytometry Part B Clinical

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“…63 Markers like IgVH mutational status and minimal residual disease detection by PCR are harmonized, but cannot easily used in daily routine. 48,83 The least developed group, including lipoprotein lipase, activationinduced cytidine deaminase, or micro RNAs, have not reached either one of these stages, but are potentially interesting prognostic markers.…”

Section: Discussionmentioning

confidence: 99%

“…While convincing results were obtained by several groups using different protein detection methods, efforts by the European Initiative in CLL Research (ERIC) to standardize their study approach have proved problematic. 63 Furthermore, investigations into the association with IgVH mutational status have yielded differing results, particularly 17p-samples cluster in the ZAP-70 negative group. 64 Assessment of ZAP-70 mRNA expression by real time PCR requires positive (CD19) or negative selection of B-cells.…”

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confidence: 99%

Impact of cytogenetic and molecular prognostic markers on the clinical management of chronic lymphocytic leukemia

Hauswirth¹,

Jäger²

2008

Haematologica

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© F e r r a t a S t o r t i F o u n d a t i o nsubgroups for further analysis in microarray studies. Gene expression profiling revealed the association of a number of genes previously thought to be unrelated to CLL. Depending on the use of CD19-selected/T-cell depleted or unpurified B-CLL samples, a variety of markers have been found since the initial experiments of Klein and Rosenwald in 2001. 19,52-54 A comprehensive analysis of more than 500 patients suggests a complex expression pattern with many CLL subgroups and overlaps.55 Furthermore, Fernàndez et al. have shown considerable intra-individual modulation of gene expression patterns during the course of disease. 56The first established marker emerging from microarray analysis was the receptor kinase ZAP-70. The prognostic value of this genetic marker has now been well established by FACS-analysis or PCR.33,34,57-62 ZAP-70 protein expression predicts for treatment-free survival (TFS) and overall survival independently of mutation status. While convincing results were obtained by several groups using different protein detection methods, efforts by the European Initiative in CLL Research (ERIC) to standardize their study approach have proved problematic.63 Furthermore, investigations into the association with IgVH mutational status have yielded differing results, particularly 17p-samples cluster in the ZAP-70 negative group.64 Assessment of ZAP-70 mRNA expression by real time PCR requires positive (CD19) or negative selection of B-cells. 33,34 Despite these drawbacks, ZAP-70 is a useful clinical marker and may also serve as a future target for specific signal transduction inhibitors.Among the markers with an even stronger correlation to IgVH mutational status, 52,53 lipoprotein lipase has been extensively studied. [35][36][37][38][39][40] Its association with other markers (cytogenetic risk groups, molecular markers) as well as patient outcome (time to treatment, overall survival) is also strong. Lipoprotein lipase is a stable marker which has been studied by real-time PCR in several large series using purified or unpurified CLL cells or even whole blood. No difference between purified and unpurified samples was observed in several studies, indicating its potential for easy and general use. Its specificity regarding IgVH mutational status is 89%, with a sensitivity of 68%, a positive predictive value of 83% and a negative predictive value of 78%.39 Discordant results are also observed.36 Lipoprotein lipase can be combined with a downregulated marker (ADAM29) to increase specificity.35 While lipoprotein lipase protein can also be detected on normal B-cells, its cytoplasmatic expression correlates well with RNA levels. 36 We have used the level of lipoprotein lipase-expression as a discriminator for microarray analysis.19 Several markers emerging from this experiment have been validated by real time PCR. Among those, septin 10 and dystrophin (DMD) were strongly associated with time to treatment. The prognostic significance of some of these factors have also be...

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ZAP‐70 expression is associated with increased risk of autoimmune cytopenias in CLL patients

et al. 2010

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Autoimmune cytopenias (AIC) are frequent in chronic lymphocytic leukemia (CLL) patients, but risk factors and prognostic relevance of these events are controversial. Data about the influence on AIC of biological prognostic markers, as ZAP-70, are scanty. We retrospectively evaluated AIC in 290 CLL patients tested for ZAP-70 expression by immunohistochemistry on bone marrow biopsy at presentation. They were 185 men, median age 63 years, 77.9% Binet stage A, 17.6% B and 4.5% C. AIC occurred in 46 patients (16%): 31 autoimmune hemolytic anemias, 10 autoimmune thrombocytopenias, four Evans syndromes, and one pure red cell aplasia. Of the 46 cases of AIC, 37 (80%) occurred in ZAP-70 positive patients and nine (20%) in ZAP-70 negatives. ZAP-70 expression [Hazard Ratio (HR) = 7.42; 95% confidence interval (CI): 2.49-22.05] and age >65 years (HR = 5.41; 95% CI: 1.67-17.49) resulted independent risk factors for AIC. Among the 136 patients evaluated both for ZAP-70 expression and IGHV status, the occurrence of AIC was higher in ZAP-70 positive/IGHV unmutated cases (35%) than in patients ZAP-70 negative/IGHV mutated (6%) or discordant for the two parameters (4%; P < 0.0001). In ZAP-70 positive patients, occurrence of AIC negatively influenced survival (HR = 1.75; 95% CI: 1.06-2.86). The high risk of developing AIC in ZAP-70 positive CLL, particularly when IGHV unmutated, should be considered in the clinical management.

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

Cited by 71 publications

References 16 publications

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

Impact of cytogenetic and molecular prognostic markers on the clinical management of chronic lymphocytic leukemia

ZAP‐70 expression is associated with increased risk of autoimmune cytopenias in CLL patients

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