Abstract
Introduction
ZAP-70 has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment.
Methods
Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP /PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ ND B-cell ratio; and modified Z-index.
Result
Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and IGHV mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (p<0.0001).
Conclusion
The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.
Keywords: Chronic Lymphocytic Leukemia, ZAP-70, Flow cytometry, One tube assay, IGHV, Cytogenetics
Introduction
The presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IGHV) of chronic lymphocytic leukemia (CLL) cells provides important prognostic information. Patients whose leukemic cells express un-mutated IGHV regions (U-IGHV) often have progressive disease, whereas patients whose leukemic cells express mutated IGHV regions (M-IGHV) more often have indolent disease (1, 2).
Additionally, cytogenetic abnormalities such as deletions of 13q, 11q, and 17p, and trisomy 12 have been reported to be of significant prognostic value in CLL (3, 4). A correlation exists between U-IGHV genes and high- risk cytogenetic aberrations (4, 5). Although there is a general agreement that the mutational status of IGHV genes and cytogenetic abnormalities constitute strong and reliable prognostic factors for patients with CLL (6, 7), routine analysis, especially that of mutational status, is labor intensive and inaccessible in most clinical diagnostic laboratories.
Among the available prognostic immunophenotypic markers in CLL, zeta-chain-associated protein kinase 70 (ZAP-70) is one of the most promising markers because of its strong correlation with IGHV mutational status (8–11). Flow cytometric analysis of ZAP -70 gives an advantage of the simultaneous assessment of its levels in the clonal B-cell as well as the residual T- & NK- cells (internal positive control), and normal remaining (NR) B-cell (internal negative control). However, the detection of this intracellular protein needs to be robust and reproducible in order to reduce intra-laboratory variations. This means that the intrinsic variability of blind replicate must be strictly controlled. An early step in this direction was the introduction of the use of a normal donor sample as an external control for ZAP-70 assessment. This was previously described by Rassenti et al. using normal donor T-cells as a reference for “percent of positive cells” (11). More recently, a normalization step of adding B-cells from a pool of normal donor peripheral blood mononuclear cells constitutes a second step toward standardization (12). We previously reported the advantage of using two clones for ZAP-70 expression analysis and utilizing normal donor blood as a reference control. In this report, combined ND and patient sample in one tube is proposed as an optimized assay for determination of ZAP-70 expression using two anti ZAP-70 clones (13,14). This step allows simultaneous assessment of ZAP-70 expression by five methods of analysis for each anti-ZAP-70 reagent in one tube. A correlation analysis between IGHV mutational status, cytogenetic features, and ZAP-70 results obtained by both the combined and the non-mixed single tube assay was undertaken.
Material and Method
Patients
Forty-eight untreated CLL patients were included for evaluation at the time of diagnosis or during the subsequent years before treatment. There were 22 males and 26 females with 1.18:1 male: female ratio. The age of these patients ranged from 47–82 years (median 62.8). The majority of the patients had early or intermediate stage disease Binet A+B (45 cases), or Rai 0+I+II (44 cases) with the median and average absolute B-cell count 14.8 cells/ μL and 36.3 cells/ μL respectively (range 5.100–176.000 cells/μL). These patients were enrolled in an NHLBI IRS approved clinical study registered with clinicaltrials.gov under identifier (NCT00923507), and under NCI study 97-C-0178 (clinicaltrial.gov identifier: NCT00019370). The diagnosis of CLL was made on the basis of clinical examination, as well as morphological and immunological criteria (15) as outlines by iwCLL revision of NCI guideline. Anonymous normal donor blood samples were obtained from the NIH Department of Transfusion Medicine and used as controls.
Modified One-Step Mixed Tube Method for ZAP-70 Assessment
The ZAP-70 assay was modified to obtain an optimized one-tube method. This method is based on mixing normal donor blood and patient blood in the same tube after surface staining and fixation. In brief, the normal donor and CLL samples are surface stained with two different sets of reagents. Then the combined sample is permeabilized and stained for ZAP-70. First, both normal donor blood and patients’ blood were washed twice using [1X Phosphate Buffered Saline (PBS)]. The procedure was carried out using 100 μL of washed normal donor whole blood (Tube A) stained with CD19-APC and CD3 APC-Cy7. At the same time, 100 μL of washed patient whole blood (Tube B) was stained with CD20 eFluor 450, CD19 PerCP-Cy5.5, and CD5 PE-Cy7. Both tubes were incubated for 30 min in the dark at room temperature. The cells werethen fixed for 10 min in 4% formaldehyde. Both tubes were washed once with 5% fetal bovine serum (FBS)/ PBS) prior to mixing. After this separate staining and fixation, the normal donor blood control tube and the patient sample tube were combined in one tube (tube #1). This combined mixture was permeabilized for 15 min using the saponin-based method (11). The permeabilized cells were stained for 30 minutes at room temperature with both anti-ZAP-70 clones 1.E7.2-AF488 and SBZAP-PE (target different epitopes). The cells were washed twice with ice-cold saponin, re-suspended in the 5% FBS/PBS, and acquired immediately.
Two separate single tubes for each ZAP-70 fluorochrome were prepared as control for the mix tube as reported previously (13, 14). Also to test the reproducibility of blind replicates, two negative and two positive ZAP-70 cryo-preserved samples were repeated and analyzed in triplicate using the combined patient/ND-one tube method for both fluorochromes and for all five methods of analysis.
Both normal donor and patient control samples were separately processed as a non-mixed single sample tube using CD3 APC-Cy7, CD20 eFluor 450, CD19 PerCP-Cy5.5, and CD5 PE-Cy7 before staining with anti-ZAP-70 reagent (tubes # 2–5). These control samples were run at the same time in a side-by-side comparison. See Table 1 for the reagent panel used in this study. The 1E7.2 clone was obtained from Invitrogen (1E7.2 AF488 Camarillo, California). The SBZAP clone conjugated with phycoerythrin (PE) (SBPE, Fullerton CA) was obtained from Beckman Coulter, Inc. Saponin permeabilization reagent was purchased from (Sigma, St.Louis, MO). CD20-eFluor 450 was obtained from eBiosciences (San Diego, CA). CD19-PerCPCy 5.5, CD5 PECy7, CD19 APC, and CD3 APC-Cy7 were obtained from BD Biosciences (San Diego, USA).
Table 1.
Antibody Panel used in the study.
| Tube # | AF488 | PE | PerCp Cy5.5 | PE -Cy7 | APC | APC -Cy7 | eFluor 450 |
|---|---|---|---|---|---|---|---|
| Tube A ND | --------- | --------- | --------- | --------- | CD19 | CD3 | --------- |
| Tube B pt | --------- | --------- | CD19 | CD5 | --------- | --------- | CD20 |
| Tube# 1 Mix (A+B) tube | ZAP-70* | ZAP-70** | CD19 | CD5 | ND CD19 | ND CD3 | CD20 |
| Tube#2 ND | ZAP-70* | --------- | CD19 | CD5 | --------- | CD3 | CD20 |
| Tube#3 ND | --------- | ZAP-70** | CD19 | CD5 | --------- | CD3 | CD20 |
| Tube#4 pt | ZAP-70* | --------- | CD19 | CD5 | --------- | CD3 | CD20 |
| Tube#5 pt | --------- | ZAP-70** | CD19 | CD5 | --------- | CD3 | CD20 |
ND: normal donor sample. ZAP-70*: 1E7.2 AF488 clone; ZAP-70 **: SBZAP PE clone. AF488: Alexa Fluor 488; PE: phycoerythrin; PerCPCy 5.5: peridin chlorophyll protein-cyanine 5.5; PECy7: PE-cyanine 7; APC: allophycocyanin; APC-Cy7: allophycocyanin-Cy7; and CD20-eFluor 450. The unstained control tubes and a single stained tube for each fluorochrome were not included in the table.
Flow Cytometric Acquisition
All analyses were performed on a FACSCanto II (Becton Dickinson, CA, USA) flow cytometer. FACS Diva software was used for acquisition. Cytometry setup and tracking beads (CST, BD) were used to initialize PMT settings. Unstained control cells as well as single stained tubes for AF488, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, and eFluor 450 were prepared and used to set up flow cytometric compensation. In some experiments, rat anti-mouse kappa light chain Comp Beads (BD) were used to set the compensation and were stained according to the manufacturer’s instruction. The number of events collected in these experiments varied from 500,000 for the single tube to 1 million events for the mixed one-tube methods. Flow Jo software (Tree Star, Ashland, OR) was used for data analysis and display.
Gating Strategies for Definition of ZAP-70 Expression Status
For ZAP-70 expression in the modified combined patient/normal donor tube, the first gate was set on lymphocytes defined by FSC-A vs. SSC-A characteristics, as shown in Figure 1. The normal donor and patient B- and T-cells were identified based on their surface staining. Normal donor T-cells (R1) were defined as CD3 APC-Cy7 positive cells and normal donor B-cells (R2) were defined as CD19 APC positive. Using a gate based on the CD3 APC-Cy7 negative and CD19 APC negative cells (R3), the CLL clone, residual patient T-cells, NK cells, and normal remaining polyclonal B-cells were identified. B-CLL cells were defined as CD5 PE-Cy-7 positive, and CD20 eFluor 450 dim population. Patient’s residual T-cells were identified in the R3 gate and defined as CD5 PE-Cy7 positive, and CD20 eFluor 450 negative cell population. Normal remaining patient B-cells were identified as CD20 eFluor 450 bright, and CD5 PE-Cy7 negative cell population. Normal patient natural killer cells (NK) were identified in the R3 gate as CD20 eFluor 450 negative, and CD5 PE-Cy7 negative cell population (See Fig.1 for gating strategy in combined mixed tube). The gating strategy for ZAP-70 expression in the single standardized tube was performed as previously reported (13, 14).
Figure 1. Gating Strategy for ZAP-70 Expression in the combined mixed tube.
A. FSC-A vs. SSC-A: lymphocyte gate in red. B. CD3 APC Cy7 vs. CD19 APC: R1 (CD3 + ND T-cells), R2 (CD19+ ND B-cells), and R3 (CD3-/CD19- cells), which includes all patient’s cells, not stained by these two ND labeling gates shown. C. CD20 eFluor450 vs. CD5 PE Cy7, cells gated on R3: Patient’s Clone (CD20 dim/CD5+ CLL cells), Pt T-cell (CD20-/CD5+ patient T-cells), NR (CD20 bright/CD5- normal residual patient polyclonal B cells), and NK (patient’s CD20-/CD5- NK cells) gates are shown. D & E panels showed ZAP-70 negative case using both 1E7.2 AF488 and SBZAP PE clones. F&G panels showed ZAP-70 positive case using both 1E7.2 AF488 and SBZAP PE clones. D. Single color histogram for ZAP-70 expression shows the overlay of the clone (in blue), ND B-cells (in green), ND T-cells, (in red) and NK cells (in orange) for ZAP-70 negative case using the 1E7.2 AF488 clone. E. Single color histogram for ZAP-70 expression shows the overlay of the clone (in blue), ND B-cells (in green), ND T-cells (in red), and NK cells (in orange) for the same ZAP-70 negative case using the SBZAP PE clone. F. Single color histogram for ZAP-70 expression shows the overlay of the clone (in blue), ND B-cells (in green), ND T-cells, (in red) and NK cells (in orange) for ZAP-70 positive case using 1E7.2 AF488 clone. G. Single color histogram for ZAP-70 expression shows the overlay of the clone (in blue), ND B-cells (in green), ND T-cells, (in red) and NK cells (in orange) for the same ZAP-70 positive case using SBZAP PE
Flow cytometric Analysis for ZAP-70 expression
Initially, ZAP-70 mean fluorescence intensity (MFI) was evaluated for the CLL clonal cells, residual patient T-cells, normal remaining patient B-cells, residual patient NK cells, normal donor T-cells, and normal donor B-cells. Five different methods were used for ZAP-70 expression analysis (one percentile and four ratio-metric methods). See Table 2 for summary of methods used for ZAP-70 expression analysis. The percentile method (ND- T-cell %) depends on using normal donor T-cells as a reference point for the determination of “percent positive.” First, both CLL-cells and normal donor T-cells were defined as previously described in the modified one-tube method. Quantification of ZAP-70 positive cells was performed with quadrant analysis. The quadrants were set on gated normal donor T-cells where ≥99 % of T-cells were positive for ZAP-70 expression. This gating was used for all subsequent samples in the experiment. The expression of ZAP-70 was measured by calculating the percentage of CD5 positive, CD20 dim (CLL clonal cells) that was beyond this gated threshold. Samples were considered positive if percentage of positive CLL ZAP-70 cells exceeded 20% (11, 16). The patient T-cell/ clone method is a ratio of – geometric mean MFI of ZAP-70 staining by patient T-cells/ geometric mean MFI of CLL cells (17, 18). The normal donor T-cell/clone is a ratio of the geometric mean MFI of ZAP-70 staining by normal donor T-cells/ geometric mean MFI of CLL cells ratio (13, 14) .The Clone/normal donor B-cell is a ratio of the geometric mean MFI of CLL cells/ geometric mean MFI of normal donor B-cells (12). The modified CLL-Z score methods uses the normal donor B-cells and normal donor T-cells (geometric mean MFI of CLL cells geometric mean MFI of normal donor B-cells)/ (geometric mean MFI of normal donor T-cells - geometric mean MFI of normal donor B-cells) x 100 (13, 14). See Figure 2 for ZAP-70 expression analysis in the combined patient/ND one-tube assay vs. the non-mixed single tube method. Based on our previous reports we used a cut-off value of 20% positivity to determine the percentage of positive cells using the percentile method of analysis. A value of < 3.0 was considered positive for patient T-cell/clone and normal donor T-cell/clone ratio; a value of > 1.4 was considered positive for clone/ normal donor B-cell ratio. Also we used an arbitrary cut-off of 20.0 to discriminate between positive and negative ZAP-70 cases for modified Z-CLL index.
Table 2.
Methods Of Analysis ZAP-70 Expression*
| Method | Description |
|---|---|
| Percentile method | Percentage of ZAP-70 positive B-CLL cells using the normal donor T-cell as a reference. |
| Patient T-cell/clone ratio | ZAP-70 MFI of patient T- cells / MFI of B-CLL cells. |
| ND T-cell/clone ratio | ZAP-70 MFI of ND T- cells /MFI of CLL cells. |
| Clone/ND-B-cell ratio | ZAP-70 MFI of B-CLL/ MFI of ND B-cell. |
| Modified CLL.Z-index | ZAP-70 MFI of B-CLL-MFI of ND B-cell/ MFI of ND T-cell- MFI of ND B-cell x100 |
Figure 2. Comparison of ZAP-70 expression in both single non- mixed and combined mix tubes in a ZAP-70 negative case.
A and B panels show ZAP-70 expression in the single non-mixed tube. A. Dot plot shows setting the vertical line for percent positive determination using normal donor T-cell (in red) and CLL cells in blue. The cursor was set were more than 99% of T-cells shows positive ZAP-70 expression, B. Single color histogram for ZAP-70 expression shows the overlay of clone, ND T-cell and NK- cells. C, D, and E. ZAP-70 expression in the combined mixed tube gated on the clone, ND B-cells, ND T-cells, and NK cells as shown in Fig.1. C. Dot plot for percentile determination normal donor (ND) T-cells (in red) as a positive control, and CLL cells in blue. D. A single color histogram showing the overlay of the clone, ND B-cells, ND T-cells, and NK cells. E. A single color histogram showing the overlay of the clone, NR B-cells, patient (pt) T-cells, and NK cells for the same CLL case. The identical process was used for 1E7.2 AF488 gating strategy analysis.
IGHV Gene and Cytogenetic Analyses
The samples were analyzed by these two methodologies as previously described (14, 19, 20). Twenty-nine out of forty-eight of the CLL patients had available mutational analysis, while forty-three patients had cytogenetic data available.
Statistical Analysis
Fisher's exact test was used to compare the positive test frequency of each method against the presence or absence of IGHV mutation and other cytogenetic abnormalities. Correlations between single and mixed tube expression levels were assessed by the Spearman rank correlation method.
Results
Correlated ZAP-70 Expression Analysis in the Combined one-tube Method vs. Single non-mixed tube method
We compared ZAP-70 expression using five different methods of analysis generated from the combined patient/normal donor one-tube method with those generated from the single non-mixed tubes. We found the following correlation coefficients: 0.83, 0.79, 0.84, 0.78, and 0.85 for percentile method (normal donor T-cells used as reference for determining of percent positive cells), patient T-cell/CLL clone ratio, normal donor T-cell/CLL clone ratio, CLL clone/ normal donor B-cell ratio, and modified CLL Z-index respectively using the 1E7.2/AF488 clone. In contrast, the following correlation coefficients were obtained using SBZAP/PE antibody: 0.82, 0.82, 0.73, 0.57, and 0.76 for percentile method, patient T-cell/CLL clone ratio, normal donor T-cell/CLL clone ratio, CLL clone/ normal donor B-cell ratio and modified Z-CLL index respectively.
Statistical analysis showed a coefficient of variation of 8% with SEM, 1% for the triplicates of the combined /mixed tube for two ZAP-70 positive, and two ZAP-70 negative samples with all five methods of analysis.
ZAP-70 Expressions in the Combined, mixed one-tube Method vs. Single non-mixed tube with IGHV mutational status
In the non-mixed single tube method, the p values obtained for the association between ZAP-70 expression and IGHV mutational status were 0.0012, 0.0012, 0.0001, 0.0080 and 0.018 for percentile method using the normal donor T-cells as a references, patient T-cell/CLL clone ratio, normal donor T-cell/CLL clone ratio, CLL clone/ normal donor B-cell ratio, and modified CLL Z-index respectively using 1E7.2 AF488 clone. For the combined patient/normal donor tube, a statistically significant association was found between ZAP-70 expression with all the five methods of analysis, and IGHV mutational status. The p values obtained were < 0.0001, 0.0004, <0.0001, <0.0001, and 0.0002 for percentile method using the normal donor T-cells as a references, patient T-cell/CLL clone, normal donor T-cell/CLL clone ratio, CLL clone/ normal donor B-cell ratio, and modified CLL Z- index respectively using 1E7.2 AF488 clone (see Table 3A).
Table 3A.
Correlation between IGHV Status and ZAP-70 Expression Analysis using 1 E7.2 AF488 clone.
| 1E7.2 AF488
| ||||||
|---|---|---|---|---|---|---|
| ZAP-70 +ve | ZAP-70 −ve | |||||
|
| ||||||
| Mutated | Un-mutated | Mutated | Un-mutated | P value | ||
|
| ||||||
| Percentile Method | single | 7 | 11 | 11 | 0 | 0.0012 |
| mixed | 4 | 11 | 14 | 0 | <0.0001 | |
|
| ||||||
| Pt T-cell/clone | single | 7 | 11 | 11 | 0 | 0.0012 |
| mixed | 6 | 11 | 12 | 0 | 0.0004 | |
|
| ||||||
| ND T/clone | single | 5 | 11 | 13 | 0 | 0.0001 |
| mixed | 3 | 11 | 15 | 0 | <0.0001 | |
|
| ||||||
| Clone/ ND B | single | 7 | 10 | 11 | 1 | 0.0080 |
| mixed | 2 | 11 | 16 | 0 | <0.0001 | |
|
| ||||||
| Modified Z- index | single | 4 | 8 | 14 | 3 | 0.018 |
| mixed | 2 | 9 | 16 | 2 | 0.0002 | |
M: mutated; U: un-mutated; ND: normal donor; Pt: patient; single: single non-mixed tube; mixed: combined patient/ND one-tube.
For the SBZAP clone using the non-mixed single tube, the p values were 0.0001, 0.0027, < 0.0001, 0.0017 and <0.0001 for percentile method using the normal donor T- cells as a references, patient T-cell/CLL clone, normal donor T-cell/CLL clone ratio, CLL clone/ normal donor B-cell ratio and modified CLL Z- index respectively. And for the combined patient/normal donor tube the p values obtained were <0.0001, 0.0078, <0.0001, <0.0001, and 0.0013 for percentile method, patient T-cell/CLL clone, ND T- cell/CLL clone ratio, CLL clone/ ND B-cell ratio, and modified CLL Z- index respectively using SBZAP PE clone (see Table 3B).
Table 3B.
Correlation between IGHV Status and ZAP-70 Expression Analysis using SBZAP PE clone.
| SBZAP PE
| ||||||
|---|---|---|---|---|---|---|
| ZAP-70 +ve | ZAP-70 -ve | |||||
|
| ||||||
| Mutated | Un-mutated | Mutated | Un-mutated | P value | ||
|
| ||||||
| Percentile Method | single | 5 | 11 | 13 | 0 | 0.0001 |
| mixed | 4 | 11 | 14 | 0 | <0.0001 | |
|
| ||||||
| Pt T-cell/clone | single | 4 | 9 | 14 | 2 | 0.0027 |
| mixed | 5 | 9 | 13 | 2 | 0.0078 | |
|
| ||||||
| ND T/clone | single | 4 | 11 | 14 | 0 | <0.0001 |
| mixed | 3 | 11 | 15 | 0 | <0.0001 | |
|
| ||||||
| Clone/ ND B | single | 5 | 10 | 13 | 1 | 0.0017 |
| mixed | 2 | 11 | 16 | 0 | <0.0001 | |
|
| ||||||
| Modified Z- index | single | 0 | 8 | 18 | 3 | <0.0001 |
| mixed | 2 | 8 | 16 | 3 | 0.0013 | |
M: mutated; U: un-mutated; ND: normal donor; Pt: patient; single: single non-mixed tube; mixed: combined patient/ND one-tube.
ZAP-70 Expressions in the Combined, mixed one-tube vs. the Single non-mixed tube with Cytogenetic Abnormality
Regarding the association between the cytogenetic features and ZAP-70 expression, patients with del 13q14 alone have lower ZAP-70 expression compared to patients with other abnormalities. The best association with 13q14 deletion in the non-mixed single tube using anti-ZAP-70 antibody 1E7.2 AF488 (p value= 0.0019) was seen for the percentile method using normal donor T-cell. On the other hand, the best p value for the combined patient/normal donor one-tube method was 0.0040 for normal donor T-cell/CLL clone ratio. See Table 4A for the p values for the other methods used in this study.
Table 4A.
Correlation between Cytogenetic Abnormality and ZAP-70 Expression Analysis using the 1E7.2 AF488 clone.
| 1E7.2 AF488 | |||||||
|---|---|---|---|---|---|---|---|
| 13q14 deletion alone | Trisomy 12 | ||||||
|
| |||||||
| No n= 21 | Yes n=22 | P value | No n=35 | Yes n=8 | P value | ||
|
| |||||||
| Percentile Method | single | 17 | 7 | 0.0019 | 16 | 8 | 0.0056 |
| mixed | 13 | 4 | 0.0051 | 10 | 7 | 0.0037 | |
|
| |||||||
| Pt T-cell/clone | single | 12 | 9 | 0.37 | 14 | 7 | 0.021 |
| mixed | 13 | 7 | 0.069 | 13 | 7 | 0.017 | |
|
| |||||||
| ND T/clone | single | 13 | 5 | 0.014 | 11 | 7 | 0.0058 |
| mixed | 12 | 3 | 0.0040 | 9 | 6 | 0.014 | |
|
| |||||||
| Clone/ ND B | single | 12 | 7 | 0.13 | 14 | 6 | 0.12 |
| mixed | 11 | 4 | 0.027 | 10 | 5 | 0.10 | |
|
| |||||||
| Modified CLL Z- index | single | 10 | 3 | 0.022 | 8 | 5 | 0.042 |
| mixed | 9 | 3 | 0.045 | 7 | 5 | 0.028 | |
ND: normal donor; Pt: patient; single: single non-mixed tube; mixed: combined patient/ND one-tube. Note that 13q14del is associated with the absence or lack of ZAP-70 expression. For instance, of the 22 samples with 13q14 deletion, 7 cases were positive for ZAP-70 expression in the single non-mixed tube method, while only 4 cases were positive in the combined patient/normal donor tube using the percentile method (ND % T-cell as a reference for “percent positive”).
While using anti-ZAP-70 antibody SBZAP PE, the best p value for the association with 13q14 deletion in the non-mixed single tube was 0.014 for the percentile method using normal donor T-cell as a reference. On the other hand, the best p value for the combined patient/normal donor tube method was 0.0042 for the normal donor T-cell / clone ratio. See Table 4B for the p values for the other methods used in this study.
Table 4B.
Correlation between Cytogenetic Abnormality and ZAP-70 Expression Analysis using the SBZAP PE clone.
| SBZAP PE | |||||||
|---|---|---|---|---|---|---|---|
| 13q14 deletion alone | Trisomy 12 | ||||||
|
| |||||||
| No n= 21 | Yes n=22 | P value | No n=35 | Yes n=8 | P value | ||
|
| |||||||
| Percentile Method | single | 15 | 5 | 0.014 | 13 | 7 | 0.017 |
| mixed | 14 | 5 | 0.031 | 12 | 7 | 0.014 | |
|
| |||||||
| Pt T-cell/clone | single | 9 | 4 | 0.20 | 9 | 4 | 0.22 |
| mixed | 10 | 4 | 0.12 | 9 | 5 | 0.089 | |
|
| |||||||
| ND T/clone | single | 12 | 4 | 0.056 | 12 | 4 | 0.44 |
| mixed | 14 | 3 | 0.0042 | 10 | 7 | 0.0037 | |
|
| |||||||
| Clone/ ND B | single | 12 | 6 | 0.22 | 14 | 5 | 0.43 |
| mixed | 10 | 4 | 0.12 | 9 | 5 | 0.089 | |
|
| |||||||
| Modified CLL Z- index | single | 6 | 3 | 0.47 | 6 | 3 | 0.33 |
| mixed | 7 | 3 | 0.29 | 7 | 3 | 0.36 | |
ND: normal donor, Pt: patient, single: single non-mixed tube, mixed: combined patient/ND one-tube.
In the association of the anti-ZAP-70 antibody 1E7.2 AF488 clone with trisomy 12, the combined patient/normal donor tube method essentially offered no advantage over the non-mixed single tube. The same was true for the anti-ZAP-70 antibody SBZAP PE clone and its correlation with trisomy 12. See Table 4A and 4B.
Intra-clonal and Inter-clonal Bimodal ZAP-70 Distribution Profile
Two examples are shown as evidence that the addition of normal donor cells does not interfere with the detection of inter-clonal or intra-clonal bimodal distributions of ZAP-70 (Fig.3 & Fig. 4). The first example is for a case that shows an apparent bi-clonal sub-population based on CD20 and CD5 fluorescent intensity (CD20 bright, CD5 bright sub-population, and CD20 dim, CD5 dim sub-population). The CD20 bright, CD5 bright sub-population showed high ZAP-70 expression, and it was designated as positive. On the other hand, the CD20 dim, CD5 dim sub-population showed low ZAP-70 expression and it was designated as negative. These two sub-populations were lambda restricted with two different fluorescent intensities. This case showed a mixed cytogenetic abnormality (deletion 13q14 and trisomy 12). Sorted cells showed restriction of the deletion 13q14 cells in the CD20 bright, CD5 bright sub-population (data not shown). There was no difference in detection of the two sub-populations by both anti-ZAP-70 antibodies (1E7.2 AF488 and SBZAP PE) in the case of bright and dim CD20 sub-populations (Fig. 3). Both the AF488 and the SBZAP anti-ZAP-70 antibodies were equal in their ability to detect the inter-clonal bimodal expression profile in the non-mixed single standardized tube as well as the combined mixed patient/normal donor tube. However, in the second case (Fig. 4) it can be clearly seen that the PE SBZAP anti-ZAP-70 antibody clone performs better in resolving the two intra-clonal populations that showed different ZAP-70 fluorescent intensity. Approximately half of leukemic cells segregated in each of the ZAP-70 positive and ZAP-70 negative fraction. Although there is no difference in surface expression for the B-cells markers, ZAP-70 expression easily detects the two sub-populations. We would further point out that these two populations could be seen in the SSC-A versus FSC-A. Large cells consistent with transformation were seen on the patient’s blood film at the time of this study. Furthermore, these two populations express the same kappa light chain restriction.
Figure 3. Bimodal ZAP-70 "Inter-clonal "expression.
A. FSC-A vs. SSC-A: Analysis showing lymphocyte gate in red. B. CD3 vs. SSC-A: Gated on FSC-A vs. SSC-A lymphocyte gate. R1 analysis gate: CD3+ T-cells, R2 analysis gate: CD3- cells. C: CD20 vs. CD5: Gated on R2 analysis gate. Plot shows bimodal CD20 bright and CD20 dim population. D. 1E7.2/AF488 ZAP-70 vs. CD3: Gated on dim CD20 population from C (in green) and T cells (in red). Insert shows the distribution of the ZAP-70 negative CLL population to the internal T-cell control. E. 1E7.2/AF488 ZAP-70 vs. CD3: Gated on bright (Br) CD20 population (in blue) and T cells (in red). Insert shows the single parameter histogram of ZAP-70 positive population to the internal T-cell control (overlapping). F. 1E7.2/AF488 ZAP-70 vs. CD3: Gated on overall bright CD20 population (in blue), dim CD20 population (in green) and T cells in (red). Insert shows single parameter histogram overlay of ZAP-70 expression in CD 20 Br, CD20 dim, and pt T-cell. G. Single parameter histogram shows ZAP-70 1E7.2 AF488 expression for overall CLL clone (red), normal residual B-cells (NR B-cell, blue), patient T-cells (Pt T-cell, green), and NK cells (orange). H. Single parameter histogram of SBZAP PE expression for overall CLL clone (red), normal residual B-cells (NR B-cell, in blue), patient T-cells (Pt T-cell, in green), and NK cells (in orange). Note SBZAP PE clone clearly resolves the two populations.
Figure 4. Bimodal ZAP-70 "Intra-clonal" Expression.
The sequential analysis gates are based upon FSC-A vs. SSC-A. A. FSC-A vs. SSC-A lymphocyte gate (R1 and R2 are two lymphocyte gated subsets). B. Dot plot shows CD20 vs. CD5 in overall lymphocyte gate. C. Dot plot shows CD20 vs. CD5 in R1 gate. D. Dot plot shows CD20 vs. CD5 in R2 gate. E. FSC-A vs. SSC-A shows back-gated R1 and R2 based on ZAP-70 positive and ZAP-70 negative populations. F. Single parameter histogram shows the SBZAP/PE ZAP-70 expression on overall CLL clone (insert shows single parameter histogram for SBZAP ZAP-70 expression for R1 and R2 separately). G. Single parameter histogram shows the SBZAP/PE ZAP-70 expression on R1. H. Single parameter histogram shows the SBZAP/PE ZAP-70 expression on R2. I. Is the bimodal ZAP-70 expression on overall CLL clone using the SBZAP PE clone (Insert shows single parameter histogram for bimodal clone and uni-modal distribution of ZAP-70 in the internal T-cell). J. Is the 1 E7.2 AF488 ZAP-70 expression over the same clone (Note the AF488 can’t clearly resolve the two populations). Insert shows single parameter histogram for CLL clone and uni-modal distribution of ZAP-70 in the internal T-cell.
Discussion
ZAP-70 expression is an independent prognostic marker in CLL (18). However, a single standardized method for its expression has not been reported. In the present study, we have examined ZAP-70 expression using our modified combined patient/normal donor tube in 48 un-treated CLL patients. In this method, the normal donor whole blood and the patient whole blood are stained in a separate tube with antibodies that distinguish clonal B-cells, patient residual T- cells, NK- cells, and normal remaining patient polyclonal B-cells from one another (see Table 1 for antibody panel used). After surface staining and fixation, these two tubes were pooled into one combined tube and intra-cellular ZAP-70 staining was performed with two anti-ZAP-70 clones that target two different epitopes. Five different methods for ZAP-70 expression analysis were evaluated for each ZAP-70 reagent clone (see Fig.1 for gating strategy). The results of ZAP-70 expression obtained from the combined patient/normal donor tube were compared with those obtained concurrently using the single non-mixed patient and normal donor tubes for each ZAP-70 reagent and each method of analysis.
The association of ZAP-70 expression with the IGHV mutational status was statistically more significant with all five methods of analysis generated from the combined patient/normal donor tube compared to those generated from the non-mixed tube method. The highest statistically significant association between ZAP-70 expression and IGHV mutational status was obtained in the combined patient/normal donor tube using the percentile method, normal donor T-cell/CLL clone ratio, or CLL clone/ normal donor B-cell ratio (p values were <0.0001) for both the 1E7.2 AF488 and SBZAP PE anti-ZAP-70 antibody clones. This data confirm the previously published advantage reported by Rassenti et al, of using the normal donor T-cells as a reference for “percent positive cells.” (16). Also it supports the finding of the GEIL group about the advantage of adding unstained B-cells from a pool of normal donor peripheral blood mononuclear cells as a normalization step (12). In our combined patient/normal donor tube, the stained ND control cells and the stained CLL blood sample are combined prior to ZAP-70 labeling. This procedure gives the advantage of using the normal donor T-and B-cells as well as the internal residual T- and NK-cells, and residual patient normal B-cells as internal reference controls in samples that are exposed to the same condition of permeabilization and ZAP-70 antibody staining. This should provide improved intra-and inter-laboratory standardization.
Again, there is an excellent association between ZAP-70 expression and IGHV mutational status as shown in our previous report using the non-mixed single tube method (14). In this present report, there is a further increase in the statistical significance of this correlation as measured by p value using the combined patient/normal donor tube method. This improvement was seen in all the five methods of ZAP-70 expression analysis using the 1 E7.2 AF488.
Although the normal donor T-cell/CLL clone ratio and the patient T-cell/CLL clone ratio in the combined patient/normal donor tube for the 1E7.2 AF 488 show nearly the same statistically significant correlation with IGHV mutational status (p<0.0001, and 0.0004 respectively), a difference was observed using the SBZAP anti-ZAP-70 antibody clone between these two ratio-metric methods (p<0.0001 and 0.0078 respectively). We suspect that this may be related to the nonspecific binding for the PE conjugate.
Due to insufficient number of normal remaining B-cells in many CLL cases, the CLL Z-index and the clone/NR B-cell index could not be determined. The addition of normal donor cells made these determinations possible using the external normal donor B- & T-cells as an internal control instead of patient residual B- & T-cells.
The CLL modified Z-index (an extension of the CLL Z-index previously reported by Shankey et al., (21) uses normal donor T- and B-cells instead of patient T- and B-cells. The p values obtained in our study regarding the association with IGHV mutational status were 0.0002 and 0.0013 for 1E7.2 AF488 and SBZAP PE respectively. This is an improvement from our earlier reported observations using the non-mixed tube method (14). Even though the association between IGHV mutational status and ZAP-70 is well known we were impressed by the tight association observed previously (14) and in the present study.
The association of ZAP-70 expression with cytogenetic abnormalities appears to be more variable depending upon the fluorochrome used, anti-ZAP-70 antibody clone and the method of analysis. Nonetheless, in the majority of methods used in this study, low ZAP-70 expression does associates with 13q14 deletion and high ZAP-70 expression associates with trisomy 12. The percentile method showed the best p value as associated with del 13q14 using the single non-mixed tube method for both fluorochromes. For the combined mixed tube, the best p values for this association were seen using the normal donor T-cell/ clone ratio for both fluorochromes.
For trisomy 12, the significant association was also dependant upon the fluorochrome, antibody clone used, and the method of analysis. It would seem that for the trisomy 12 using the 1E7.2 AF488 that either the percentile method or the normal donor T-cell/clone ratio in the single non-mixed tube is preferable. The opposite was seen for the Anti-ZAP-70 SBZAP clone, where the combined patient/normal donor tube using normal donor T-cell /clone ratio showed the best association.
Our observation of intra- and inter-clonal, bimodal ZAP-70 expression suggests a greater underlying complexity in some CLL cases. This complexity was seen in both the non-mixed single and the combined patient/normal donor tube method. Although the cases are too few, it would seem that both antibody clones are able to detect inter-clonal variation, while the SBZAP PE conjugate better resolves intra-clonal bimodality. It is important to document bi-modal ZAP-70 expression and correlate it with cytogenetic findings as it may be due to a mixed karyotype. In the first patient (Fig.3) determined to have inter-clonal ZAP-70 bimodality, two cytogenetic sub-populations were detected: 13q14 deletion and trisomy 12. Fluorescent in situ hybridization on sorted cells revealed that the CD20 bright population carried the 13q14 deletion (unpublished observation). The other patient showed intra-clonal ZAP-70 bimodality; trisomy 12 was the only observed cytogenetic abnormality. However, the two populations were easily seen and detected. The larger cells could represent transformation. Further studies are indicated to explore this possibility and its possible impact on clinical course.
In conclusion, the combined patient/normal donor tube provides reliable results as associated with the IGHV mutational status with all the five methods of analysis studied. The highest statistical significance was achieved using either 1E7.2 AF488 or SBZAP PE anti-ZAP-70 antibody clones with the percentile method, ND T-cell/clone ratio, and the clone/ ND B-cell ratio. The combined patient/normal donor tube method gives flexibility for each laboratory to choose the antibody clone and their own method of ZAP-70 expression analysis. It also gives an option for those who want to use more than one method of analysis to confirm their ZAP-70 results. Our findings suggest that the combined patient/normal donor tube method is superior to the use of two independent anti-ZAP-70 non-mixed single tubes. The combined mixed tube provides a robust internal control (normal donor T- and B-cells) for all methods, even when there are few remaining normal lymphocytes in the patient specimen. Patients and ND control cells are subjected to the same permeabilization and anti-ZAP-70 staining conditions as the target CLL cells. We would conclude that a combined one-tube and a single ZAP-70 antibody reagent is sufficient. This allows for multiple methods of ZAP-70 analysis. And it would now seem that internal ND positive and negative controls would be routinely available. This assay could be further developed by the addition of one or more of the other prognostic markers in CLL such as CD38, CD49d, CD69 or CD26.
Acknowledgments
We acknowledge the internal reviewers of this paper by Drs. Steven Bauer and Bharat Joshi .We also acknowledge the nursing and data management skills of Susan Soto and Therese White in this study. In addition, we acknowledge the friendly support provided by the Phlebotomy Service (Department of Transfusion Medicine, NIH). This research was supported in part by the Intramural Research Program of the NHLBI, NIH.
References
- 1.Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. 1999;94:1840–1847. [PubMed] [Google Scholar]
- 2.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;94:1848–1854. [PubMed] [Google Scholar]
- 3.Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L, Dohner K, Bentz M, Lichter P. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343:1910–6. doi: 10.1056/NEJM200012283432602. [DOI] [PubMed] [Google Scholar]
- 4.Oscier DG, Gardiner AC, Mould SJ, Glide S, Davis ZA, Ibbotson RE, Corcoran MM, Chapman RM, Thomas PW, Copplestone JA, et al. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutations of the p53 gene are independent prognostic factors. Blood. 2002;100:1177–84. [PubMed] [Google Scholar]
- 5.Krober A, Seiler T, Benner A, Bullinger L, Bruckle E, Lichter P, Döhner H, Stilgenbauer S. V (H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood. 2002;100 (14):1410–6. [PubMed] [Google Scholar]
- 6.Shanafelt TD, Geyer SM, Kay NE. Prognosis at diagnosis: integrating molecular biologic insights into clinical practice for patients with CLL. Blood. 2004;103:1202–10. doi: 10.1182/blood-2003-07-2281. [DOI] [PubMed] [Google Scholar]
- 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;90:391–399. [PubMed] [Google Scholar]
- 8.Rosenwald A, Alizadeh AA, Widhopf G, Simon R, Davis RE, Yu X, Yang L, Pickeral OK, Rassenti LZ, Powell J, et al. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. J Exp Med. 2001;194:1639–48. doi: 10.1084/jem.194.11.1639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.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;348:1764–75. doi: 10.1056/NEJMoa023143. [DOI] [PubMed] [Google Scholar]
- 10.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 leukemia. Lancet. 2004;363(9403):105–11. doi: 10.1016/S0140-6736(03)15260-9. [DOI] [PubMed] [Google Scholar]
- 11.Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG, Neuberg DS, Flinn LW, Rai KR, Byrd JC, et al. 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;351:893–901. doi: 10.1056/NEJMoa040857. [DOI] [PubMed] [Google Scholar]
- 12.Gachard N, Salviat A, Boutet C, Arnoulet C, Durrieu F, Lenormand B, Leprêtre S, Olschwang S, Jardin F, Lafage-Pochitaloff M, et al. For the GEIL. Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method. Hematologica. 2008;93:215–223. doi: 10.3324/haematol.11622. [DOI] [PubMed] [Google Scholar]
- 13.Degheidy HA, Venzon DJ, Farooqui MZH, Abbasi F, Arthur DC, Wilson WH, Wiestner A, Stetler-Stevenson MA, Marti GE. Methodological Comparison of Two Anti-ZAP-70 Antibodies. Cytometry Part B Clinical Cytometry. 2011;80B:300–308. doi: 10.1002/cyto.b.20591. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Degheidy HA, Venzon DJ, Farooqui MZH, Abbasi F, Arthur DC, Wilson WH, Wiestner A, Stetler-Stevenson MA, Marti GE. Improved ZAP-70 Assay Using Two clones, multiple methods of analysis and clinical correlation. Cytometry Part B Clinical Cytometry. 2011;80B:309–317. doi: 10.1002/cyto.b.20593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, Hillmen P, Keating MJ, Montserrat E, Rai K, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008;111:5446–5456. doi: 10.1182/blood-2007-06-093906. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Rassenti L, Kipps TJ. Clinical utility of assessing ZAP-70 and CD38 in chronic lymphocytic leukemia. Clinical cytometry PartB. 2006;70B:209–213. doi: 10.1002/cyto.b.20129. [DOI] [PubMed] [Google Scholar]
- 17.Bakke AC, Purtzer Z, Leis J, Huang J. A robust ratiometric method for analysis of ZAP-70 expression in chronic lymphocytic leukemia (CLL) Clinical cytometry Part B. 2005;70B (4):227–234. doi: 10.1002/cyto.b.20079. [DOI] [PubMed] [Google Scholar]
- 18.Rossi FM, Del Principe MI, Rossi D, Irno Consalvo M, Luciano F, Zucchetto A, Bulian P, Bomben R, Dal Bo M, Fangazio M, et al. 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. doi: 10.1186/1479-5876-8-23. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Wiestner A, Rosenwald A, Barry TS, Wright G, Davis RE, Henrickson SE, Zhao H, Ibbotson RE, Orchard JA, Davis Z, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with un-mutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. 2003 Jun 15;101(12):4944–51. doi: 10.1182/blood-2002-10-3306. [DOI] [PubMed] [Google Scholar]
- 20.Rosenwald A, Chuang EY, Davis RE, Wiestner A, Alizadeh AA, Arthur DC, Mitchell JB, Marti GE, Fowler DH, Wilson WH, et al. Fludarabine treatment of patients with chronic lymphocytic leukemia induces a p53-dependent gene expression response. Blood. 2004;104:1428–34. doi: 10.1182/blood-2003-09-3236. [DOI] [PubMed] [Google Scholar]
- 21.Shankey TV, Forman M, Scibelli P, Cobb J, Smith CM, Mills R, Holdaway K, Bernal-Hoyos E, Van Der Heiden M, Popma J, et al. An optimized Whole Blood Method for Flow cytometric Measurement of ZAP-70 Protein Expression in Chronic Lymphocytic Leukemia. Cytometry part B (clinical cytometry) 2006;70B:259–269. doi: 10.1002/cyto.b.20135. [DOI] [PubMed] [Google Scholar]