Background The mutational status of immunoglobulin heavy-chainvariable-region (IgVH) genes in the leukemic cells of chroniclymphocytic leukemia (CLL) is an important prognostic factorin the disease. We investigated whether the expression of ZAP-70by CLL cells correlated with the IgVH mutational status, diseaseprogression, and survival.
Methods The expression of ZAP-70 was analyzed in T-cell andB-cell lines and in peripheral-blood samples from 56 patientswith CLL with the use of flow cytometry, Western blotting, andimmunohistochemistry. The results were correlated with the IgVHmutational status and clinical outcome.
Results ZAP-70 was detected by flow-cytometric analysis in cellsof T-cell lineage and in leukemic cells from 32 of 56 patientswith CLL. In all patients in whom at least 20 percent of theleukemic cells were positive for ZAP-70, IgVH was unmutated,whereas IgVH mutations were found in 21 of 24 patients in whomless than 20 percent of the leukemic cells were positive forZAP-70 (P<0.001). Concordant results were obtained when ZAP-70expression was assessed by immunohistochemistry or Western blotting.The level of ZAP-70 expression did not change over time (median,37 months) in sequential samples from 30 patients with CLL.Patients with Binet stage A CLL who had at least 20 percentZAP-70–positive leukemic cells had more rapid progressionand poorer survival than those with less than 20 percent ZAP-70–positivecells.
Conclusions Among patients with CLL, expression of ZAP-70, asdetected by flow-cytometric analysis, correlated with IgVH mutationalstatus, disease progression, and survival.
The staging systems developed by Rai et al.1 and Binet et al.2are standard methods of assessing prognosis in chronic lymphocyticleukemia (CLL). However, since these systems cannot identifystable or progressive forms of the disease, there has been acontinual effort to identify other prognostic factors in CLL.3,4,5,6
About 50 to 70 percent of patients with CLL have evidence ofsomatic hypermutation in the immunoglobulin heavy-chain variable-region(IgVH) genes of the leukemic cells.7,8,9,10,11 These patientsprobably constitute a subgroup in whom the leukemic cells havepassed through the germinal center, the site of IgVH hypermutation.12It is important to note that patients with unmutated IgVH genesusually have an advanced stage of CLL and unfavorable cytogeneticfeatures, require therapy, and have a short survival. In contrast,patients with leukemic cells that have mutant IgVH genes usuallypresent in an early clinical stage, frequently have 13q14 chromosomaldeletions, do not have alterations of p53, do not require therapy,and have a long survival.11,13,14 For these reasons, knowledgeof the mutational status of IgVH is of considerable value inassessing the prognosis in CLL. Most general laboratories, however,are unable to determine IgVH sequences. Moreover, even whenthe technique is available, it is too costly and time consumingto include in the standard workup of CLL. These considerationshave made finding a surrogate for IgVH mutational status inCLL an important priority.
Investigations using DNA microarrays15,16 have shown that CLLcells exhibit a characteristic gene-expression profile in whichthe expression of a small subgroup of genes, including thoseencoding ZAP-70, IM1286077, and C-type lectin, correlates withthe mutational status of IgVH genes.16,17 ZAP-70, a member ofthe Syk–ZAP-70 protein tyrosine kinase family, is normallyexpressed in T cells and natural killer cells and has a criticalrole in the initiation of T-cell signaling.18,19,20,21,22 Thisfinding led us to hypothesize that the expression of ZAP-70could not only predict IgVH mutational status but also serveas a prognostic factor in CLL. We therefore analyzed ZAP-70protein in CLL cells from a series of patients using Westernblotting, immunohistochemistry, and flow cytometry and correlatedthe results with the mutational status of the IgVH genes andthe clinical outcome.
Methods
Patients and Sample Collection
Fifty-six patients who had received a diagnosis of CLL at ourinstitution were selected on the basis of the availability offrozen samples for biologic studies. Lymph-node–biopsyspecimens were available from eight patients. Progression wasdefined as a change to a more advanced clinical stage or theneed for treatment. The time to progression and survival werecalculated from the time of diagnosis. The median age was 60years (range, 37 to 81), and the median duration of follow-up(from diagnosis) was 63 months.
Mononuclear cells from peripheral-blood samples were isolatedon a Ficoll–Hypaque gradient (Seromed).23 In three samples,CD19+ cells were isolated with use of anti-CD19 fluoresceinisothiocyanate, followed by separation with anti–fluoresceinisothiocyanate Microbeads (Miltenyi Biotec). Lymph-node–biopsyspecimens from six additional patients who had received a diagnosisof mantle-cell lymphoma according to the criteria of the WorldHealth Organization24 were included in the analysis.
Preparation of RNA, Complementary DNA, and Genomic DNA
Total RNA was isolated with use of Ultraspec RNA (Biotecx Laboratories)according to the manufacturer's instructions. ComplementaryDNA (cDNA) was synthesized from 1 µg of RNA with the useof Moloney–murine leukemia virus reverse transcriptase(Invitrogen, Life Technologies). High-molecular-weight DNA wasextracted from mononuclear cells isolated on a Ficoll gradientwith use of a salting-out procedure.25
Amplification of IgVH Genes
We amplified cDNA using a set of six heavy-chain variable-region(VH) family-specific primers (VH1 through VH6) that anneal tosequences in the leader region26 along with primers complementaryto the constant region (IgM and IgG).7 When amplification withthese primers failed, an alternative set of primers specificto framework I region and the heavy-chain joining (JH) regionwas used.27 For the amplification of genomic DNA, approximately100 ng of DNA was used in a total volume of 50 µl.11
IgVH Mutational Status
Polymerase-chain-reaction (PCR) products were purified eitherdirectly with use of the Concert rapid PCR purification system(GIBCO BRL), or by gel excision with use of the QIAEX II agarose-gelextraction kit (Qiagen). Products were directly sequenced fromboth strands with use of the Big Dye Terminator Cycle SequencingReady Reaction (versions 2.0 and 3.0, Applied Biosystems) accordingto the manufacturer's instructions. Sequencing analysis andalignments were performed with use of DNAPLOT software and theVBASE data library.28 Samples in which fewer than 2 percentof base pairs differed from those of the consensus sequencewere considered unmutated.8
Western Blotting
Western blotting of cell lysates was carried out as previouslydescribed.23 Cell lysates from the following cell lines werealso included: Jurkat (a T-cell line derived from lymphoblasticlymphoma), JVM-2 (B-cell prolymphocytic leukemia), Granta 519and REC (mantle-cell lymphoma), DHL-16 and DOHH-2 (follicularlymphoma), Ly1.2 and Ly3 (diffuse large-B-cell lymphoma), andNamalwa, Raji, Daudi, and Ramos (Burkitt's lymphoma). Blotswere incubated with anti–ZAP-70 antibody (Upstate Biotechnology)and anti–-tubulin (Calbiochem). Lysates from Jurkat cells,a T-cell line with a high level of expression of ZAP-70 protein,18were used as a positive control. To analyze the influence ofnormal T cells present in the CLL samples, we mixed differentconcentrations of mononuclear cells from healthy blood donorswith REC cells, a B-cell line that does not express ZAP-70.
Immunohistochemistry
We also analyzed the expression of ZAP-70 in paraffin-embeddedsections of lymph-node–biopsy specimens from eight patientswith CLL and six patients with mantle-cell lymphoma using ananti–ZAP-70 antibody (ZAP-70-LR, Santa Cruz Biotechnology).We evaluated the level of ZAP-70 expression in tumor cells,T cells, and residual germinal-center areas.
Flow Cytometry
Mononuclear cells, as well as whole blood, from 56 patientswith CLL and 10 healthy blood donors used as normal controlswere fixed and permeabilized with use of the Fix & Permkit (Caltag Laboratories) according to the manufacturer's instructions.Then 1.5 µg of anti–ZAP-70 antibody per 500,000cells was incubated for 20 minutes at room temperature, washedtwice in phosphate-buffered saline (Biomerieux), incubated for20 minutes with goat antimouse immunoglobulin fluorescein isothiocyanate(Dako), washed, and then incubated with normal mouse serum forfive minutes. Finally, CD3–phycoerythrin, CD56–phycoerythrin,CD19– peridinin chlorophyll protein cychrome 5.5, andCD5–allophycocyanine (BD Biosciences) were added, andthe samples were incubated for 15 minutes. Samples were analyzedwith a flow cytometer (FACS Calibur, BD Biosciences) with agate on the fluorescence 2 detector to ensure that at least1000 T cells and natural killer cells were analyzed in eachsample. In samples from healthy control subjects, at least 5000B cells were also analyzed. Analysis of stained samples wascarried out with use of CellQuest software (BD Biosciences).
Lymphocytes were gated to avoid the inclusion of debris, monocytes,and doublets. The resultant cells were then gated to selectCD3+CD56+ cells (T and natural killer cells), used as an internalcontrol for ZAP-70 expression, and CD19+CD5+ (CLL cells). Biparametricdot graphs of cells that were stained for ZAP-70 and CD3 plusCD56 were independently plotted for T cells and natural killercells and CLL cells. A marker that included T cells and naturalkiller cells (in the upper right quadrant of each graph) wasused to calculate the percentage of CLL cells that were positivefor ZAP-70. The percentage of CD19+ cells that also expressedCD38 was quantified as previously described.8
Statistical Analysis
Correlations between ZAP-70, CD38, and mutational status wereanalyzed with use of Wilcoxon's and Fisher's exact tests anda multiple regression analysis. To identify the level of ZAP-70expression that could best be used to discriminate mutated fromunmutated cells, we used a receiver-operating-characteristicplot.29 Correlations between the clinical characteristics andthe expression of ZAP-70 were also analyzed with use of Wilcoxon'sor Fisher's exact test. Survival and time to progression wereestimated according to the method of Kaplan and Meier and comparedbetween groups by means of the log-rank test. All P values weretwo-sided, and the type I error was set at 5 percent. Statisticalanalyses were performed with use of SPSS software.30
Results
Somatic Mutations in IgVH Genes
All sequences of IgVH genes in the leukemic cells from the 56patients were in-frame rearrangements, and only two prematurestop codons were observed. We found somatic mutations (up to98 percent homology with germ-line sequences) in 21 of 56 patients(38 percent), including 7 that had 95.6 to 97.3 percent homologywith germ-line sequences (Table 1).
Table 1. Main Biologic and Clinical Characteristics of the Patients.
Western Blot Analysis of ZAP-70 Expression
ZAP-70 expression was analyzed in 12 human cell lines correspondingto different stages of B-cell differentiation. Peripheral-bloodlymphocytes from healthy blood donors (data not shown) and theJurkat T-cell line were used as positive controls. The proteinwas not detected in any of the B-cell lines, except the Ramoscell line, in which it was expressed weakly (Figure 1A).
Figure 1. Western Blot Analysis of the Expression of ZAP-70 in a T-Cell Line, a Peripheral-Blood Sample from Patient 17 with Chronic Lymphocytic Leukemia (CLL), and 10 B-Cell Lines (Panel A); Seven Patients with CLL According to the Presence or Absence of Mutations in the Immunoglobulin Heavy-Chain Variable-Region (IgVH) Genes (Panel B); and Normal Peripheral-Blood T Cells (Panel C).
In Panel A, only cells from the T-cell line Jurkat and Patient 17 showed a high level of expression of ZAP-70; the B-cell line Ramos exhibited weak expression. In Panel B, patients without IgVH mutations — Patients 17, 19, and 20 — had a high level of ZAP-70 expression, whereas patients with IgVH mutations had a low level of expression. Patient 45 had a high level of expression of ZAP-70 because of the high percentage of T cells (20 percent) in the sample. In Panel C, various concentrations (0 percent, 2 percent, 5 percent, 10 percent, 15 percent, 20 percent, and 30 percent) of mononuclear cells from healthy blood donors were mixed with REC cells, a B-cell line that does not express ZAP-70. The level of expression of ZAP-70 was barely visible in the sample containing 2 percent T cells and was high in the samples containing more than 10 percent T cells. In each panel, the expression of -tubulin was used as a control.
Of 32 samples from the patients with CLL, 16 had a high levelof expression of ZAP-70 on Western blotting and the remaining16 had a low level of expression (Figure 1B). The presence ofmore than 2 percent T cells in the blood samples influencedthe result, especially when the proportion was 10 percent orhigher (Figure 1C). However, samples from all but two patientswith CLL contained less than 15 percent T cells on Western blotting(mean [±SD], 4.5±5.5 percent); Patient 45 hadmore than 20 percent T cells in the sample.
Immunohistochemical Analysis of ZAP-70 Expression
We analyzed lymph-node–biopsy specimens from eight patientswith CLL, none of whom had IgVH mutations, and six patientswith mantle-cell lymphoma, which is characterized by proliferationof CD5+ B cells but not usually by IgVH mutations. All lymph-node–biopsyspecimens from the patients with CLL were positive for ZAP-70,with a diffuse pattern reflecting the infiltration by leukemiccells. T cells in the biopsy specimens showed stronger immunoreactivitythan the CLL cells. Residual germinal centers were negativefor ZAP-70. All mantle-cell lymphomas were negative for ZAP-70,whereas T cells in the tissue had a high level of expressionof ZAP-70.
Flow-Cytometric analysis of ZAP-70 Expression
In samples of normal blood, flow-cytometric analysis discloseda population with high and homogeneous expression of ZAP-70(Figure 2A). This population corresponded to T cells and naturalkiller cells. In these samples, the proportion of normal B cells(from healthy blood donors) expressing as much ZAP-70 as T cellsand natural killer cells was 0 to 6.5 percent (mean, 4±2.5).No significant differences in ZAP-70 expression were observedbetween CD5– and CD5+ B-cell subpopulations (Figure 2B).
Figure 2. Immunofluorescence and Flow-Cytometric Analysis of the Expression of ZAP-70.
Panel A shows flow-cytometric plots of lysed whole blood. In the left-hand plot, forward and side scatter of lysed blood is represented with a region (R1) drawn around the lymphocytes. The right-hand plot shows side scatter and ZAP-70 staining of the same sample; A1 denotes a population with a low SSC and a high level of expression of ZAP-70. Panel B shows the level of expression of ZAP-70 by normal B cells. The left-hand plot shows the expression of CD5 on gated CD19+ cells, with two regions showing the CD5+ and CD5– B-cell subpopulations. The histogram on the right-hand side represents the expression of ZAP-70 by the CD19+CD5– subpopulation and the CD19+CD5+ subpopulation. Panel C shows the method used to quantify the expression of ZAP-70 by chronic lymphocytic leukemia (CLL) cells. Lymphocytes were R1 gated, and then T cells and natural killer (CD3+CD56+) cells (the R2 region in the leftmost plot) and CLL (CD19+CD5+) cells (the R3 region in the second plot) were selected according to their phenotype. The third plot shows the expression of ZAP-70 after lymphocyte (R1) gating. For the purposes of quantification, markers were placed so that the T cells and natural killer cells (R1 and R2 gated) with a high level of expression of ZAP-70 would appear in the upper right quadrant (fourth plot). Subsequently, CLL cells were plotted, and the cells in the lower right quadrant were quantified as CLL cells with a high level of expression of ZAP-70 (rightmost plot). Panel D shows the level of expression of ZAP-70 by lymphocytes from five representative patients with CLL according to the mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes. The percentage of CLL cells with a high level of expression of ZAP-70 is shown in the lower right quadrant of each plot, after the exclusion of T cells and natural killer cells. FITC denotes fluorescein isothiocyanate.
The method used to quantify the percentage of CLL cells expressingas much ZAP-70 as T cells is shown in Figure 2C. Leukemic cellsfrom 56 patients with CLL were analyzed for ZAP-70 expressionby flow cytometry. In these samples, T cells and natural killercells also displayed high and homogeneous levels of expressionof ZAP-70. In contrast to normal CD5+ B cells, CD5+ CLL cellsfrom 32 of 56 patients expressed high levels of ZAP-70 (Figure 2D).
In all but one patient (Patient 45), there was complete concordanceof ZAP-70 expression as assessed by flow cytometry, Westernblotting, and immunohistochemistry (Table 1). These discrepantresults in Patient 45 (Figure 1B and Table 1) could be explainedby the high number of T cells (20 percent) in the patient'ssample. ZAP-70 expression was also analyzed in 30 of 56 patientsfrom whom two sequential samples were available. The mediantime between the collections of the samples was 37 months (range,11 to 64). None of these 30 patients had significant changesin ZAP-70 expression over time.
Correlation between ZAP-70 Protein and IgVH Mutational Status
There was a strong correlation between the presence of IgVHmutations and the percentage of leukemic cells that expressedZAP-70, as assessed by flow cytometry. In the receiver-operating-characteristicanalysis, a value of 17.5 percent ZAP-70–positive CLLcells was the best cutoff for assigning IgVH mutational status.We used a cutoff value of 20 percent for simplicity. Patientswith unmutated IgVH genes had higher percentages of ZAP-70–positiveCLL cells than did patients with IgVH mutations (48±21percent vs. 6±4 percent, P<0.001). Samples from all21 patients with IgVH mutations contained less than 20 percentZAP-70–positive CLL cells, and all but 3 patients withoutIgVH mutations (91 percent) had 20 percent or more ZAP-70–positiveCLL cells (Figure 3A and Figure 3B). Conversely, all the patientswith increased proportions of ZAP-70–positive cells didnot have somatic mutations, and 88 percent of patients withlow numbers of ZAP-70–positive cells had IgVH mutations.Overall, the probability of the absence of somatic mutationsin the presence of more than 20 percent ZAP-70–positiveCLL cells (positive predictive value) was 100 percent (95 percentconfidence interval, 89 to 100), whereas the probability ofIgVH mutations in the presence of a low percentage of ZAP-70–positivecells (negative predictive value) was 87.5 percent (95 percentconfidence interval, 68 to 97). The sensitivity and specificityof the flow-cytometric analysis were 91 percent (95 percentconfidence interval, 77 to 98) and 100 percent (95 percent confidenceinterval, 84 to 100), respectively.
Figure 3. Correlation of the Level of Expression of ZAP-70 and Immunoglobulin Heavy-Chain Variable-Region (IgVH) Mutational Status (Panel A) and IgVH Sequence Homology (Panel B).
Panel A shows a scatter plot representing the levels of ZAP-70 expression measured by flow-cytometric analysis and the IgVH mutational status of the 56 patients analyzed. When the threshold for categorizing ZAP-70 was established at 20 percent, two subgroups were clearly delineated: patients with unmutated IgVH and a high level of ZAP-70 expression and patients with mutant IgVH and low levels of ZAP-70 expression. Only three patients without IgVH mutations had low levels of ZAP-70 expression. Panel B shows the mean (±SD) (horizontal line and box) level of expression of ZAP-70 according to the degree of homology of each IgVH sequence with the consensus sequence (less than 95 percent, 95 to 98 percent, or more than 98 percent). There were no significant differences in the level of ZAP-70 expression between the subgroup with less than 95 percent sequence homology and the subgroup with 95 to 98 percent sequence homology.
To verify ZAP-70 expression in the three patients in whom IgVHstatus and flow-cytometric results were discordant, Westernblot analysis was performed on isolated fractions of CD19+ cellswith a purity greater than 98 percent (CD19 is a surface markerof B cells). A weak signal was observed in all three samples(data not shown), validating the low levels of expression ofZAP-70 observed on flow cytometry.
ZAP-70 and CD38 Expression
Data for the expression of CD38 were available for 45 patients(CD38 is a marker that has been proposed as a surrogate forIgVH mutational status). Of these 45 patients, 18 had no morethan 30 percent CD38+ cells (Table 1). There were significantdifferences in the mean percentages of CD38+ CLL cells betweenpatients with IgVH mutations and those without IgVH mutations(20 percent vs. 60 percent, P<0.001). In addition, 22 of28 patients without IgVH mutations (79 percent) had at least30 percent CD38+ cells, whereas 12 of 17 (71 percent) with somaticmutations had less than 30 percent CD38+ cells. Moreover, thepercentage of CD38+ cells was at least 30 percent in 21 of 25patients (84 percent) with at least 20 percent ZAP-70–positivecells and less than 30 percent in 14 of 20 patients (70 percent)with less than 20 percent ZAP-70–positive cells. The levelof CD38 expression was also low in the two patients with discordantresults of ZAP-70 expression and IgVH mutations (Table 1). Ina multiple regression analysis, only ZAP-70 expression (P<0.001),and not CD38 expression, maintained its correlation with IgVHmutational status.
Prognostic Importance of the Percentage of ZAP-70–Positive Cells
None of the standard variables were associated with ZAP-70 expression,including Binet's and Rai's clinical stages, the lymphocytecount, and the lymphocyte doubling time. However, an increasedpercentage of ZAP-70–positive cells was associated witha short time to progression. Among 44 patients with Binet stageA, the median time to progression was 29 months for the 26 patientswith at least 20 percent ZAP-70–positive cells, whereasthe median was not reached among the 18 patients with less than20 percent ZAP-70–positive cells (P=0.009) (Figure 4A).
Figure 4. Kaplan–Meier Estimates of the Actuarial Risk of Disease Progression (Panel A) and the Likelihood of Survival (Panel B) among Patients with Binet Stage A Chronic Lymphocytic Leukemia, According to the Level of Expression of ZAP-70.
In Panel A, the median time to progression among 26 patients with a high level of ZAP-70 expression (20 percent or more) was 29 months, whereas it was not reached in 18 patients with a low level of ZAP-70 expression (less than 20 percent) (P=0.009). In Panel B, the probability of survival at 10 years was 90 percent among patients with a low level of ZAP-70 expression. The median survival was 90 months among the patients with a high level of ZAP-70 expression, whereas it was not reached among patients with a low level of ZAP-70 expression (P=0.01). Tick marks indicate censored data.
When survival was calculated from the time of diagnosis, the26 patients with Binet stage A CLL and at least 20 percent ZAP-70–positivecells had a median survival of 90 months, whereas the mediansurvival was not reached in the 18 patients with Binet stageA and less than 20 percent ZAP-70–positive cells (P=0.01)(Figure 4B). However, when all patients were analyzed (Binetstage A, B, and C), the percentage of ZAP-70–positivecells was not significantly correlated with survival (P=0.06).The percentage of CD38+ cells (with a cutoff of 30 percent)did not predict progression or survival (data not shown). Finally,as expected, IgVH mutational status also correlated with progressionand survival (data not shown).
Discussion
The management of CLL is based on each patient's individualrisk, because the disease has a widely variable clinical course.An important determinant of the prognosis is the mutationalstatus of IgVH genes in the leukemic cells, which correlateswith the clinical outcome better than and independently of classicprognostic factors.8,11,31 Methods to identify IgVH mutationsare, however, not widely available in clinical practice. Ourstudy confirms that the leukemic cells without IgVH mutationsin patients with CLL express high levels of ZAP-70, whereasthe leukemic cells with IgVH mutations in patients with CLLhave barely detectable levels of ZAP-70.16,17 Moreover, usingflow cytometry, we found that none of the patients with CLLand at least 20 percent ZAP-70–positive cells had IgVHsomatic mutations, whereas all but three patients with lessthan 20 percent ZAP-70–positive cells had mutated IgVHgenes. Flow-cytometric analysis of ZAP-70 expression was a sensitiveand specific surrogate for mutational status of IgVH genes;this method should be readily available in general laboratories.
The mechanisms accounting for the relation between ZAP-70 expressionand the mutational status of IgVH are unknown. We also haveno explanation for the inconsistency between mutational statusand ZAP-70 levels in three of our patients. A discrepancy betweenZAP-70 expression, as assessed by Western blotting, and theIgVH mutational status was also found in 1 of 22 patients inanother series.17
Apart from CLL cells, ZAP-70 was expressed only in T cells andtumors of T-cell lineage, whereas it was barely detectable innormal CD19+CD5+ cells. It was not detected by Western blottingor immunohistochemistry in any of the B-cell lines or biopsyspecimens of mantle-cell lymphoma. Nevertheless, ZAP-70 messengerRNA has been found in other B-cell lines with the use of reverse-transcriptase–PCR(RT-PCR) analysis.16 Our results indicate that, among B-celland T-cell lymphoproliferative disorders, a high level of ZAP-70expression is restricted to T-cell proliferative diseases anda subgroup of CLL.
The use of flow cytometry to assess the level of ZAP-70 expressionoffers advantages over other systems of analysis. With flowcytometry, it is possible to analyze the percentage of ZAP-70–positivecells selectively in subpopulations of CLL cells, T cells, andnatural killer cells. By contrast, Western blotting and RT-PCRcan overestimate the level of expression of ZAP-70 owing tothe presence of T cells in the sample.16,17
The value of CD38 as a surrogate for IgVH mutations is controversial.32,33Moreover, the level of CD38 expression may vary over the courseof the disease.34 In our series, the level of CD38 expressiondid not correlate with disease progression or survival. In contrast,the level of expression of ZAP-70 did not change over time,and the presence of ZAP-70 was associated with rapid progressionand poor survival.
In conclusion, the expression of ZAP-70 by CLL cells, as ascertainedby flow-cytometric analysis, is a simple and reliable surrogatefor the identification of IgVH mutations. Moreover, ZAP-70 expressionby itself can be used as a prognostic marker. For these reasons,ZAP-70 analysis should be included in the workup of patientswith CLL.
Supported in part by grants from the José Carreras InternationalFoundation against Leukemia (EM/02 and CR/02), Fondo de InvestigacionesSanitarias (02/0250 and 01/1581), and Ministerio de Cienciay Tecnologia (SAF-02-3261). Ms. Crespo is a recipient of a grantfrom Fondo de Investigaciones Sanitarias, Spain.
We are indebted to Professor Freda K. Stevenson for technicaladvice in the analysis of the somatic mutations of IgVH , toMontse Sánchez for immunohistochemical analysis, andto Penelope Elvy and Eoin McGrath for their assistance in thepreparation of the manuscript.
Source Information
From the Department of Hematology (M.C., F.B., A.L.-G., E.M.) and the Hematopathology Unit (N.V., B.B., D.C., M.R., S.M., E.C.), Institut d'Investigacions Biomèdiques August Pi i Sunyer, Hospital Clínic, Barcelona, Spain. Ms. Crespo and Drs. Bosch and Villamor contributed equally to the article.
Address reprint requests to Dr. Bosch at the Department of Hematology, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain, or at fbosch{at}clinic.ub.es.
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(2006). Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia. Blood
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(2006). Clinical Outcomes and Prognostic Factors in Patients With Richter's Syndrome Treated With Chemotherapy or Chemoimmunotherapy With or Without Stem-Cell Transplantation. JCO
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A correction has been published: N Engl J Med 2003;349(5):506.
-tubulin (Calbiochem). Lysates from Jurkat cells, a T-cell line with a high level of expression of ZAP-70 protein,18 were used as a positive control. To analyze the influence of normal T cells present in the CLL samples, we mixed different concentrations of mononuclear cells from healthy blood donors with REC cells, a B-cell line that does not express ZAP-70. 
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