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Keywords:

  • ZAP70;
  • CLL;
  • flow cytometry;
  • T/B ratio;
  • isoclonic control;
  • T cells;
  • blocking antibody;
  • SBZAP monoclonal antibody

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

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. © 2012 International Clinical Cytometry Society


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Chronic Lymphocytic Leukemia (CLL) is an indolent B-cell neoplasm related to the accumulation of mature monoclonal small lymphocytes coexpressing CD19, CD5, and CD23, in the absence or low expression of surface CD22, CD79b, and FMC7. Surface immunoglobulins, usually IgM, associated or not with IgD, are clonal but weakly expressed or undetectable. Patients with CLL may have a long survival without therapy, while others die rapidly despite aggressive treatment (1). Since 1999, breakthroughs have been made in the identification of molecular and cellular markers that may predict disease progression, including the mutational status of the immunoglobulin heavy chain variable region (IGHV) genes (2, 3), cytogenetic abnormalities such as del(11q22.3), del(17p13), trisomy 12 or del 13q and expression of Zeta-chain-associated protein kinase 70 (ZAP70) (4). ZAP70 is a tyrosine kinase initially described in the transduction complex of the T cell receptor (TCR) and is believed to increase the sensitivity of CLL B-cell BCRs to antigenic stimulation (5). Flow cytometry (FCM) can specifically assess ZAP70 levels in B-CLL cells (4, 6–8). Compared with molecular genetics and cytogenetics, FCM is usually considered as a simple and rapid technique. ZAP70 is thus thought to be one of the most promising phenotypic markers because of its correlation with the IGHV mutational status as well as other cytogenetic prognostic markers (1, 3, 9, 10). Different techniques for ZAP70 FCM detection have been published since the first publication in 2003 and different consensus protocols have been established. 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 (11–13). 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, 15–19). 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. ZAP70 T/B ratio in CLL B-cells does not take into account the fact that ZAP70 levels in T-cells can vary between patients. For example, it has been reported that ZAP70 expression is variable in T-cells from patients with CLL (20) and can be increased when compared with T-cells from normal subjects, a feature that seems to be associated with a poorer prognosis (21).

Negative controls are very often a key point to validate FCM results with confidence. Isoclonic negative controls, also called blocking antibodies, are often considered as the true negative controls since they give the true nonspecific background of the tested mAbs themselves. For example, the International Society of Hematotherapy and Graft Engineering (ISHAGE) recommends using an isoclonic control for CD34 labeling to count CD34 positive stem cells in peripheral blood by FCM (22).

Following the ISHAGE CD34 principle and to circumvent difficulties of expressing results for ZAP70 expression in CLL B-cells as a ratio between ZAP70 MFIs in CLL B-cells and normal B-cells or as a ZAP70 T/B ratio, we adapted the published SBZAP labeling procedure (13, 23), including an isoclonic control for the PE-conjugated SBZAP mAb. Results were expressed as ratios between MFIs of PE-SBZAP and isoclonic controls in CLL B-cells (ZAP70 isoclonic ratio). Comparison between ZAP70 isoclonic and T/B ratio demonstrated that the latter ratio did not reflect levels of ZAP70 expression in tumor cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Biological Samples

A total of 42 subjects, including 10 controls, were included in this study after informed verbal consent (absence of written opposition) and approval of the ethical committee of the University Campus Hospital of the Limousin Region. All patients were either untreated or off therapy for at least 6 months before beginning the study (Table 1). Residual blood samples were obtained from patients during the normal course of staging without any additional samples after completion of all workups needed for their pathology. All samples were collected using EDTA as anticoagulant.

Table 1. Patients Characteristics
CLL #SexAgeBinet StageIGHV mutation rate (%)% CD38 positive cellsB-cell ZAP70 isoclonic ratioZAP70 T/B ratioT-cell ZAP70 isoclonic ratio
  1. N.D., not determined.

1M59A013.384.3910.16
2F69B0152.0812.6512.65
3M79C004.486.6423.91
4M63B2.0312.598.5818.28
5F87A0385.545.4618.28
6M80B0696.234.6722.40
7M90N.D.071.5616.7017.16
8F64B4.813.488.4823.17
9M68A9.1721.9915.8819.82
10M67A8.8511.8916.2521.46
11M85A7.05121.5019.5917.94
12F74A9.61ND1.4318.6518.55
13M70A5.2621.2819.2317.43
14M83A2181.538.458.61
15M65A4.3311.0914.6711.11
16F46A2.19391.515.027.68
17M77A13.9502.2216.3938.86
18F62A3.904.707.6626.50
19F87A8.0411.3430.8629.00
20M61N.D.2.5413.959.1630.87
21F59N.D.0294.584.2515.60
22M80AN.D.01.3611.0910.72
23M87A6.6401.0515.1112.57
24F60B0623.807.5025.89
25M89A9.6511.3514.2916.71
26F80A10.9201.7611.1912.71
27M70A0282.8610.1626.94
28M82A6.9501.6528.7734.31
29M66A2.64595.178.6640.70
30M53B026.045.8636.01
31M55A2119.275.9546.15
32M72A8.2301.7036.1046.11

Analysis of IGHV Gene Sequences

After DNA extraction, IGHV genes were amplified by polymerase chain reaction (PCR) according to the BIOMED-2 protocol and purified PCR products were directly sequenced (24). B-CLL IGHV sequences were aligned to germinal sequences on the IMGT database using V-QUEST and junction analysis software (http://imgt.cines.fr; initiator and coordinator: Marie-Paule Lefranc, Montpellier, France) and the GenBank database using the IgBLAST software (http://www.ncbi.nlm.nih.gov/igblast/). IGHV genes with less than 98% sequence homology to the closest germ line counterpart were considered mutated as described elsewhere (25, 26).

Flow Cytometry

Antibodies used in this study were: fluorescein isothiocyanate (FITC) conjugated anti-CD5 mAb (clone BL1a, Beckman-Coulter, Miami, Florida), phycoerythrin cyanin-5 (PC5) conjugated anti-CD19 mAb (clone J3-119, Beckman-Coulter) and phycoerythrin cyanin-7 (PC7) conjugated anti-CD45 mAb (clone J33, Beckman-Coulter). Monoclonal antibodies against ZAP70 (SBZAP, Beckman Coulter) were either unconjugated (SBZAP-NC) or phycoerythrin (PE) conjugated (SBZAP-PE). Each mAb was added as recommended by the supplier. One hundred microliters of whole EDTA blood samples were first labeled with anti-CD19, anti-CD5, and anti-CD45 mAbs for 15 min in the dark at room temperature. Red blood cells were then lysed and fixed for 10 min with 1 ml of “fix and lyse” Versalyse solution (Beckman-Coulter) containing 25 µl Iotest-3 fixative reagent (Beckman-Coulter). White blood cells were then washed in PBS, 0.5% paraformaldehyde (PFA). Dried cells were permeabilized in 100 µl permeabilizing agent (Perm and Stab reagent, Beckman-Coulter) for 5 min. Fifty microliters of a stabilizing reagent (Beckman Coulter) were added before intracytoplasmic staining. Cell suspensions were divided into 2 aliquots of 75 µl each. The first aliquot was incubated for 5 min with 20 µl unconjugated SBZAP mAb (isotypic control). The second aliquot was incubated for 5 min with 20 µl PBS. Then, 20 µl of PE-conjugated anti-ZAP70 mAb was added to each aliquot for 30 min. Cells from both aliquots were washed and resuspended in 0.5 ml PBS/0.5% PFA immediately prior to flow cytometry analysis. Acquisitions were performed on a FC500 flow cytometer (Beckman Coulter). FITC, PE, PC5, and PC7 were excited with a 488 nm argon laser. FITC, PE, PC5, and PC7 fluorescences were collected with a 530 ± 30 nm band pass filter, a 585 ± 42 nm band pass filter, and a 670 nm long pass filter respectively. Hardware fluorescence compensations were applied during acquisitions according to recommendations of the French Association for Cytometry (27). Fluidic stability and intervariation of MFIs between ZAP70 labeling and isoclonic control are shown in Supporting Information Figure 1. Original data files can be obtained by contacting the website of the CIM (Cytometry Imagery Mathematics) platform (http://www.unilim.fr/CIMLimoges). Analysis was performed with CXP software (Beckman Coulter). Lymphocytes were first gated on a CD45/Side Scatter (SS) histogram (28). Normal T-cells (CD5+, CD19-) and clonal CLL B-cells (CD5+, CD19+) were then gated on the CD5/CD19 biparametric histogram (Fig. 1), in order to specifically assess MFIs for ZAP70 in the absence of (SBZAP-PE total signal for CLL B-cell or T-cells) or presence of the SBZAP-NC isoclonic control (SBZAP-PE background, Fig. 1C). The isoclonic ratio for ZAP70 was calculated for both lymphocyte subpopulations as the ratio between the SBZAP-PE total signal and the SBZAP-PE background (in presence of the isoclonic control). The T/B ZAP70 ratio was calculated as the ratio between SBZAP-PE total signals of T-cells and CLL B-cells.

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Figure 1. ZAP70 expression in different lymphocyte subsets. A: Staining of different lymphocyte subsets. Lymphocytes were first gated on both FSC/SSC and then CD45/SSC biparametric histograms (not shown). On the CD5-FITC/CD19-PC5 biparametric histogram, normal B-cells, CLL B-cells, T-cells and NK cells were stained in red, blue, purple, and cyanine, respectively. B: Monoparametric histogram of ZAP70 labeling with SBZAP mAb for normal B-cells, CLL B-cells, T-cells, and NK cells. C: Monoparametric histogram for ZAP70 labeling with PE-conjugated SBZAP mAb in presence of excess unconjugated SBZAP mAb (isoclonic control) for the same cell subset. For panel A, each cell subset is given in the frame of the biparametric histogram. For panels B and C, the color code of each cell subset is indicated on the top right of the panel.

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Statistical Analysis

Statistical significance of the results was determined with the Mann-Whitney non parametrical test, χ2 test with Yates correction, Fisher's exact test or Pearson correlation test when applicable and according to standard uses of statistical analyses. We used Excel software (Microsoft) and R software (http://www.R-project.org.) for their assessment.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

The gating strategy used to assess levels of ZAP70 expression in lymphocyte subsets is shown in Figure 1. Lymphocytes subsets were gated based on both FSC/SSC and CD45/SSC biparametric histograms (not shown). Specific gatings of nonclonal B lymphocytes, CLL tumor cells, T and NK lymphocytes were performed on the CD5/CD19 biparametric histogram, in order to assess fluorescence levels of each lymphocyte subpopulation after labeling with PE-conjugated ZAP70 mAb alone or in presence of saturating concentrations of unlabeled ZAP70 mAb, so called isoclonic controls (Fig. 1). Isoclonic ratios and T/B ratios were calculated as described (12, 15–19, 29 and see Materials and Methods).

To set-up the decisional threshold for ZAP70 isoclonic ratios, we compared results of ZAP70 labeling with the IGHV mutation status of patients. Figure 2 shows that low ZAP70 isoclonic ratios were associated with CLL cases having a mutated IGHV gene (<98% homology, M-CLL) whereas increased ratios were found for CLL patients with unmutated IGHV genes (≥98% homology, UM-CLL; Mann-Whitney test, P = 0.004). The best threshold for the ZAP70 isoclonic ratios between M-CLL and UM-CLL was 2.6 (χ2 maximization test (13), Supporting Information Fig. 2A), which was used to predict the unmutated and mutated IGHV status with 75% sensitivity and 73.7% specificity. The best threshold value for ZAP70 T/B ratios was 8 (Supporting Information Fig. 2B), which predicted the unmutated and mutated IGHV status with a 66.7% sensitivity and 89.5% specificity.

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Figure 2. Distribution of ZAP70 MFIs (left panel), isoclonic ratios (middle panel), and T/B ratios (right panel) in B-cells from CLL patients with a mutated (M-CLL) or unmutated (UM-CLL) IGHV gene rearrangement and in controls. For each series, mean values and SD are indicated by solid and dashed lines respectively. Nonparametric Mann-Whitney P-values between UM-CLL and controls are given at the top of each panel.

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We next compared both ZAP70 isoclonic and T/B ratios to evaluate their respective relevance. Means of ZAP70 isoclonic ratios were 1.15 ± 0.22 for normal B cells, 2.19 ± 1.23 for mutated CLL and 4.28 ± 2.60 for unmutated CLL (Fig. 2). Means for ZAP70 T/B ratios were 6.81 ± 2.68, 16.03 ± 8.33, and 7.72 ± 3.78, with no significant difference between control B-cells (no ZAP70 expression) and UM-CLL B-cells (increased ZAP70 expression). As stated above, patients were declared ZAP70 negative or positive for a threshold value of 2.6 for ZAP70 isoclonic ratios and 8 for the ZAP70 T/B ratios. ZAP70 expression of CLL patients was concordant according these two methods for 26/31 patients (concordance = 84%, Yates corrected χ2 P-value = 8.10−4, Fig. 3). Five cases were dissimilar. One case (CLL#8) was a M-CLL with a mutated VH3-21 IGHV gene rearrangement but with the common HCDR3 subset (id est HCDR3 amino-acid sequence ARDANA/GMDV) known to be of poor prognosis (30,31), and with an increased ZAP70 isoclonic ratio of 3.48 and a normal ZAP70 T/B ratio at 8.48 (slightly above the threshold, Fig. 3). For the four remaining cases, three (CLL#27, CLL#20, and CLL#29) had high ZAP70 isoclonic ratios (interpreted as ZAP70 positive) and ZAP70 T/B ratios slightly above the threshold (interpreted as ZAP70 negative). For the last case (CLL#16), the difference was due to a low ZAP70 isoclonic ratio (interpreted as ZAP70 negative) and a decreased ZAP70 T/B ratio (interpreted as ZAP70 positive). Altogether, 4/5 divergent cases had increased ZAP70 isoclonic ratios and ZAP70 T/B ratios slightly above the T/B ratio threshold. Flow cytometry of ZAP70 labeling showed that the ZAP70 isoclonic ratio almost perfectly followed ZAP70 MFI values in CLL B-cells for these cases (not shown). These differences were all due to variations in ZAP70 MFI values in T cells. ZAP70 MFI values were decreased for CLL#16 and increased for the 4 other cases. With the exception of CLL#8, for the four remaining patients, ZAP70 isocolonic and T/B ratios were in agreement with the IGHV mutation status for two cases each: one UM-CLL and one M-CLL for ZAP70 isoclonic ratios, and two M-CLLs for ZAP70 T/B ratios. Thus, it cannot be concluded that the isoclonic or the T/B ZAP70 ratio was more relevant for prediction of IGHV mutation status. We looked at other parameters that predict prognosis such as the Binet stage, CD38 expression and cytogenetic abnormalities, and again, it could not be determined whether the ZAP70 isoclonic ratio was better than the ZAP70 T/B ratio.

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Figure 3. Concordance between ZAP70 isoclonic (x axis) and T/B (y axis) ratios. Concordant and discordant cases are represented by • and ○ respectively. The CLL case number is indicated for each discordant case. Thresholds for ZAP70 isoclonic and T/B ratios are represented by vertical and horizontal dashed black lines respectively, and corresponding values are given by numbers in bold type. The number of cases is given in each quadrant.

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Figure 4. ZAP70 expression in T-cells. A) Increased ZAP70 expression in T-cells from CLL patients. Panels A and C: CD5-FITC/CD19-PC5 biparametric histograms for a healthy control (panel A) and a CLL patient (panel C). Panels B and D: Overlay between monoparametric histograms for ZAP70 (purple) and isoclonic (grey) labeling for the same healthy control (panel B) and CLL patient (panel D) as for panels A and C, respectively. For panels A and C, the color code is the same as in Figure 1 and each lymphocyte subset is given in the frame of the biparametric histogram. B) Distribution of MFIs for isoclonic controls (left panel) and ZAP70 (middle panel) as well as distribution of ZAP70 isoclonic ratios (right panel) in T-cells from healthy controls and CLL patients. For each series, mean values and SD are indicated by solid and dashed lines, respectively. Non parametric Mann-Whitney P-values between controls and patients are given at the top of each panel.

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As shown above, differences between ZAP70 isoclonic ratios and T/B ratios were likely to be due to variations in ZAP70 levels in T-cells. To compare the significance of both ZAP70 isoclonic and T/B ratios we examined ZAP70 expression in T-cells of CLL patients and controls. The intensity of ZAP70 labeling in T-cells was increased for patients with CLL when compared with controls (Fig. 4A). Distribution of ZAP70 MFIs and isoclonic ratios clearly showed that T-cells from most CLL patients had higher ZAP70 expression levels than controls (Fig. 4B). ZAP70 MFIs means were respectively 22.2 ± 6.5 for CLL T-cells and 9.7 ± 4.7 for normal T-cells (P < 0.001). ZAP70 isoclonic ratios in T-cells paralleled those of MFI values with means of 22.5 ± 10.7 and 7.3 ± 3.4 for CLL T-cells from patients and controls respectively (P < 0.001). No correlation was found between ZAP70 expression in T cells and the IGHV mutation status of CLL B-cells (P = 0.761), or CD38 surface expression (P = 0.645). A weak correlation was found between ZAP70 levels in B and T cells from patients with CLL (coefficient correlation r = 0.495, P = 0.004, Supporting Information Fig. 3). In fact, ZAP70 expression was highly variable in T-cells from CLL patients regardless of ZAP70 expression levels in B-cells. This demonstrated that T/B ZAP70 ratios depend on two rather unrelated biological processes, modulation of ZAP70 expression in T-cells and dysregulation of ZAP70 expression in CLL cells. This certainly explains why the sensitivity of the ZAP70 T/B ratio was lower than the ZAP70 isoclonic ratio whereas ZAP70 T/B ratio specificity was better.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

In this study, we compared a new method for assessing ZAP70 expression in CLL cells, a ZAP70 isoclonic ratio, and the ZAP70 T/B ratio, considered as the reference in terms of expressing results of ZAP70 expression.

Increased ZAP70 expression in CLL cells with unmutated IGHV gene rearrangements is a well-established feature (1, 3–5). It is also accepted that dysregulated ZAP70 expression in CLL is associated with a poor prognosis (6, 12). Flow cytometry is the reference technique for detection and quantification of ZAP70 in cells. This technology is highly quantitative and provides a differential analysis of lymphocyte subsets without the necessity of cell purification. However, the manner of expressing ZAP70 levels in CLL cells has been matter of debate in the literature. 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, 15–18). 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).

Determining the ZAP70 isoclonic ratio corresponds to a different approach. With this method, the ZAP70 MFI values in CLL cells were not compared to a supposed invariant internal control. Rather, MFI ZAP70 values are normalized to those of isoclonic controls (ZAP70 labeling with excess unlabeled ZAP70 mAb). The negative control set-up for labeling has been shown to be more robust and reliable for quantification of CD34 positive cells by flow cytometry (22). Our comparison between the ZAP70 iscolonic and T/B ratios showed 84% concordance. Only 5/32 cases were discrepant, 4 of them due to abnormal ZAP70 levels in T-cells. Even if we could not demonstrate the superiority of one or the other ratios in terms of biological and clinical correlations, it was striking that the ZAP70 T/B ratio was less sensitive but more specific than the ZAP70 isoclonic ratio in predicting the IGHV mutation status of CLL cells. However, evaluating ZAP70 expression with the isoclonic ratio enabled comparison of ZAP70 levels between cases, which was not possible if the ZAP70 T/B ratio was used. The ZAP70 isoclonic ratio method demonstrated that ZAP70 expression was increased in T-cells from CLL patients. Almost all CLL cases with increased ZAP70 expression in CLL B-cells had also increased expression in T-cells, in agreement with results published by Herishanu et al. in 2005 (21). In contrast, ZAP70 levels were highly variable in T-cells from patients with ZAP70 negative CLL B-cells. Altogether, our results show that ZAP70 MFIs from T-cells are most likely not correct internal controls for normalising ZAP70 MFIs from B-cells. Of note, it has been recently reported that ZAP70 is modulated by TLR7 and TLR9 activation of normal B-cells (33). CLL cells have been reported to secrete various cytokines such as interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and interleukin 8 (34). It would be of interest to examine the effect of these agents on ZAP70 expression in T-cells from both healthy controls and CLL patients.

Altogether the data presented here show that the ZAP70 isoclonic ratio is a reliable method for analysis of ZAP70 expression in CLL cells. In addition, ZAP70 levels were increased in T-cells from CLL patients and highly variable. Whereas ZAP70 isoclonic ratios in B-cells depend only on their ZAP70 expression, the ZAP70 T/B ratio depends on ZAP70 expression by both T and B cells. This demonstrates that ZAP70 MFI values from T-cells of CLL patients cannot be taken as internal controls and would explain some difficulties encountered in interpreting the ZAP70 T/B ratio in routine practice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

The authors thank Dr Jenny Cook-Moreau for scientific English editing and Jean-Luc Faucher for the excellent technical assistance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information
  • 1
    Dighiero G. CLL biology and prognosis. Hematology Am Soc Hematol Educ Program 2005: 278284.
  • 2
    Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N. IgHV gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999; 94: 18401847.
  • 3
    Montserrat E. New prognostic markers in CLL. Hematology Am Soc Hematol Educ Program 2006: 279284.
  • 4
    Rosenwald A, Alizadeh AA, Widhopf G, Simon R, Davis RE, Yu X, Yang L, Pickeral OK, Rassenti LZ, Powell J, Botstein D, Byrd JC, Grever MR, Cheson BD, Chiorazzi N, Wilson WH, Kipps TJ, Brown PO, Staudt LM. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. J Exp Med 2001; 194: 16391647.
  • 5
    Orchard JA, Ibbotson RE, Davis Z, Wiestner A, Rosenwald A, Thomas PW, Hamblin TJ, Staudt LM, Oscier DG. ZAP- 70 expression and prognosis in chronic lymphocytic leukaemia. Lancet 2004; 363: 105111.
  • 6
    Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG, Neuberg DS, Flinn IW, Rai KR, Byrd JC, Kay NE, Greaves A, Weiss A, Kipps TJ. ZAP- 70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 2004; 351: 893901.
  • 7
    Montillo M, Hamblin T, Hallek M, Montserrat E, Morra E. Chronic lymphocytic leukemia: novel prognostic factors and their relevance for risk-adapted therapeutic strategies. Haematologica 2005; 90: 391399.
  • 8
    Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, Marcé S, López-Guillermo A, Campo E, Montserrat E. ZAP- 70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003; 348: 17641775.
  • 9
    Shanafelt TD, Geyer SM, Kay NE. Prognosis at diagnosis: integrating molecular biologic insights into clinical practice for patients with CLL. Blood 2004; 103: 12021210.
  • 10
    Oscier DG, Gardiner AC, Mould SJ, Glide S, Davis ZA, Ibbotson RE, Corcoran MM, Chapman RM, Thomas PW, Copplestone JA, Orchard JA, Hamblin TJ. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 2002; 100: 11771184.
  • 11
    Shankey TV, Forman M, Scibelli P, Cobb J, Smith CM, Mills R, Holdaway K, Bernal-Hoyos E, Van Der Heiden M, Popma J, Keeney M. An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia. Cytometry B Clin Cytom B 2006; 70B: 259269.
  • 12
    Kern W, Dicker F, Schnittger S, Haferlach C, Haferlach T. Correlation of flow cytometrically determined expression of ZAP-70 using the SBZAP antibody with IgVH mutation status and cytogenetics in 1,229 patients with chronic lymphocytic leukemia. Cytometry B Clin Cytom B 2009; 76B: 385393.
  • 13
    Gachard N, Salviat A, Boutet C, Arnoulet C, Durrieu F, Lenormand B, Leprêtre S, Olschwang S, Jardin F, Lafage-Pochitaloff M, Penther D, Sainty D, Reminieras L, Feuillard J, Béné MC. Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method. Haematologica 2008; 93: 215223.
  • 14
    Degheidy HA, Venzon DJ, Farooqui MZH, Abbasi F, Arthur DC, Wilson WH, Wiestner A, Stetler-Stevenson MA, Marti GE. Combined normal donor and CLL: Single tube ZAP-70 analysis. Cytometry B Clin Cytom B 2012; 82B: 6777.
  • 15
    Smolej L, Vroblova V, Motyckova M, Jankovicova K, Schmitzova D, Krejsek J, Maly J. Quantification of ZAP-70 expression in chronic lymphocytic leukemia: T/B-cell ratio of mean fluorescence intensity provides stronger prognostic value than percentage of positive cells. Neoplasma 2011; 58: 140145.
  • 16
    Rossi FM, Del Principe MI, Rossi D, Irno Consalvo M, Luciano F, Zucchetto A, Bulian P, Bomben R, Dal Bo M, Fangazio M, Benedetti D, Degan M, Gaidano G, Del Poeta G, Gattei V. Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: Mean fluorescence intensity T/B ratio versus percentage of positive cells. J Transl Med 2010; 8: 23.
  • 17
    Marquez M-E, Deglesne P-A, Suarez G, Romano E. MFI ratio estimation of ZAP-70 in B-CLL by flow cytometry can be improved by considering the isotype-matched antibody signal. Int J Lab Hematol 2011; 33: 194200.
  • 18
    Letestu R, Rawstron A, Ghia P, Villamor N, Boeckx N, Boettcher S, Buhl AM, Duerig J, Ibbotson R, Kroeber A, Langerak A, Le Garff-Tavernier M, Mockridge I, Morilla A, Padmore R, Rassenti L, Ritgen M, Shehata M, Smolewski P, Staib P, Ticchioni M, Walker C, Ajchenbaum-Cymbalista F. Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process. Cytometry B Clin Cytom B 2006; 70B: 309314.
  • 19
    Bakke AC, Purtzer Z, Leis J, Huang J. A robust ratio metric method for analysis of Zap-70 expression in chronic lymphocytic leukemia (CLL). Cytometry B Clin Cytom B 2006; 70B: 227234.
  • 20
    Zucchetto A, Bomben R, Bo MD, Nanni P, Bulian P, Rossi FM, Del Principe MI, Santini S, Del Poeta G, Degan M, Gattei V. ZAP- 70 expression in B-cell chronic lymphocytic leukemia: Evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgV(H) mutations. Cytometry B Clin Cytom B 2006; 70B: 284292.
  • 21
    Herishanu Y, Kay S, Rogowski O, Pick M, Naparstek E, Deutsch VR, Polliack A. T-cell ZAP-70 overexpression in chronic lymphocytic leukemia (CLL) correlates with CLL cell ZAP-70 levels, clinical stage and disease progression. Leukemia 2005; 19: 12891291.
  • 22
    Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. Int Soc Hematotherapy Graft Eng Cytometry 1998; 34: 6170.
  • 23
    Shankey TV, Forman M, Scibelli P. Optimized whole-blood assay for measurement of ZAP-70 protein expression. Curr Protoc Cytom 2007;Chapter 9:Unit9.22.
  • 24
    van Dongen JJM, Langerak AW, Brüggemann M, Evans PAS, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, García-Sanz R, van Krieken JHJM, Droese J, González D, Bastard C, White HE, Spaargaren M, González M, Parreira A, Smith JL, Morgan GJ, Kneba M, Macintyre EA. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17: 22572317.
  • 25
    Peková S, Baran-Marszak F, Schwarz J, Matoska V. Mutated or non-mutated? Which database to choose when determining the IgVH hypermutation status in chronic lymphocytic leukemia? Haematologica 2006;91:ELT01.
  • 26
    Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 18481854.
  • 27
    Bayer J, Grunwald D, Lambert C, Mayol JF, Maynadié M. Thematic workshop on fluorescence compensation settings in multicolor flow cytometry. Cytometry B Clin Cytom B 2007; 72B: 813.
  • 28
    Béné MC, Nebe T, Bettelheim P, Buldini B, Bumbea H, Kern W, Lacombe F, Lemez P, Marinov I, Matutes E, Maynadié M, Oelschlagel U, Orfao A, Schabath R, Solenthaler M, Tschurtschenthaler G, Vladareanu AM, Zini G, Faure GC, Porwit A. Immunophenotyping of acute leukemia and lymphoproliferative disorders: A consensus proposal of the European LeukemiaNet Work Package 10. Leukemia 2011; 25: 567574.
  • 29
    Shenkin M, Maiese R. Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLL. Cytometry B Clin Cytom B 2006; 70B: 251258.
  • 30
    Tobin G, Thunberg U, Johnson A, Thörn I, Söderberg O, Hultdin M, Botling J, Enblad G, Sällström J, Sundström C, Roos G, Rosenquist R. Somatically mutated Ig V(H)3-21 genes characterize a new subset of chronic lymphocytic leukemia. Blood 2002; 99: 22622264.
  • 31
    Ghia P, Stamatopoulos K, Belessi C, Moreno C, Stella S, Guida G, Michel A, Crespo M, Laoutaris N, Montserrat E, Anagnostopoulos A, Dighiero G, Fassas A, Caligaris-Cappio F, Davi F. Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocytic leukemia: The lesson of the IGHV3-21 gene. Blood 2005; 105: 16781685.
  • 32
    Rawstron AC, Villamor N, Ritgen M, Böttcher S, Ghia P, Zehnder JL, Lozanski G, Colomer D, Moreno C, Geuna M, Evans PAS, Natkunam Y, Coutre SE, Avery ED, Rassenti LZ, Kipps TJ, Caligaris-Cappio F, Kneba M, Byrd JC, Hallek MJ, Montserrat E, Hillmen P. International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia 2007; 21: 956964.
  • 33
    Bekeredjian-Ding I, Doster A, Schiller M, Heyder P, Lorenz H-M, Schraven B, Bommhardt U, Heeg K. TLR9-activating DNA up-regulates ZAP70 via sustained PKB induction in IgM+ B cells. J Immunol 2008; 181: 82678277.
  • 34
    di Celle PF, Carbone A, Marchis D, Zhou D, Sozzani S, Zupo S, Pini M, Mantovani A, Foa R. Cytokine gene expression in B-cell chronic lymphocytic leukemia: evidence of constitutive interleukin- 8 (IL-8) mRNA expression and secretion of biologically active IL-8 protein. Blood 1994; 84: 220228.

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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