Expression of CD38 and ZAP-70 in Chronic Lymphocytic Leukemia and Their Relation to Clinical Stage

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1 Journal of Medical Research Institute JMRI, 2007; Vol. 28 No.2: (174-80) Expression of CD38 and ZAP-70 in Chronic Lymphocytic Leukemia and Their Relation to Clinical (1) Amani Sorour, (2) Manal ElSorady Abstract Background: Chronic lymphocytic leukemia (CLL) is a disease with a variable clinical course. In aggressive disease, the CLL cells usually express an unmutated immunoglobulin heavy-chain variable region gene (IgVH) and the 70-kD zeta-associated protein (ZAP-70), whereas in indolent disease, the CLL cells usually express mutated IgVH but lack expression of ZAP-70. Also, CD38 appears to be a useful prognostic marker which was thought to identify IgVH mutation but is now viewed as an independent marker of the clinical course and outcome in CLL. Patients and Methods: We evaluated 63 patients with CLL for ZAP-70 and CD38 expression. 22 of them were newly diagnosed, while 41 had started therapy before enrollment in our study. We investigated the association between the results and clinical stage, blood counts and the time from diagnosis to initial therapy. Results and Conclusions: Both ZAP-70 and CD38 expression were shown to correlate with the total leukocyte counts and absolute lymphocyte counts in our CLL patients. CD38 expression was also shown to correlate with advanced stages of the disease in the untreated group. However, in the treated group, the time from diagnosis to initial therapy did not differ significantly between ZAP- 70+ve and -ve or CD38 +ve and -ve patients. To confirm this finding, we need to expand our study on a larger number of patients. Key Words: Zap-70; CD38; B-CLL; IgVH; prognostic marker. Introduction Chronic lymphocytic leukemia (CLL) is a disease characterized by a heterogeneous clinical course. Some patients have aggressive disease and require therapy within a relatively short time after diagnosis, whereas others have indolent, asymptomatic disease and need no therapy for many years. (1) Because of the difficulty in predicting the course of disease at the time of diagnosis and the uncertainty of the value of early treatment, therapy is currently recommended only for patients with symptomatic or progressive disease. (2) There has been intense search for prognostic markers in early-stage disease that might facilitate risk-adapted treatment (3) strategies. Although cytogenetic abnormalities, especially trisomy 12 and deletions of 11q and 17p, have been shown to correlate with shorter survival, these changes may not be present in early disease and may only be acquired during disease progression. (4) 1 Clinical pathology department, 2 Hematology unit, Internal medicine department, Faculty of Medicine- Alexandria University. The presence or absence of somatic mutations in the variable region of the B-cell receptor heavy chain gene (IgVH) has been shown to distinguish between two disease subsets conferring important prognostic information. Patients whose CLL cells express mutated IgVH genes have a significantly longer median survival than those with Ig- unmutated CLL. This survival difference may reflect a distinct biologic behavior of the leukemic clone. (5) Therefore, it would be beneficial to incorporate this distinction between Ig-mutated and Ig-unmutated CLL into the clinical diagnosis of CLL patients. However, most clinical diagnostic laboratories do not have the routine ability to sequence IgVH genes as this analysis is both time consuming and expensive, making it doubtful to be established as a clinical test available to all patients with CLL. (6) Several studies reported a strong association between the expression of ZAP-70 in CLL cells and the presence of unmutated IgVH genes. ZAP-70 is an intracellular tyrosine kinase which has a critical role in T-cell receptor signaling. Moreover, its expression in CLL cells appears to be constant over time. Therefore, it might be used at the time of diagnosis to identify patients who are at increased risk for early disease (7, 8) progression. Similarly, CD38 which is a membrane protein that marks cellular activation and maturation and that has signaling

2 Sorour and El-Sorady activity, often correlates with the presence of IgVH mutations. (9) However, since this finding has varied among different studies, CD38 is now viewed as an independent marker of outcome, with clinical value of its own in foreshadowing the likely course of CLL. (10) The aim of the present study was to assess the clinical value of ZAP-70 and CD38 expression as markers of disease progression in CLL. Patients and Methods Sixty-three patients with CLL who presented to the hematology unit of the Alexandria Main University Hospital between September 2005 and May 2007, were included in our study. Twenty-two patients were newly diagnosed (group I), while forty-one patients had initiated therapy before enrollment in our study. (group II). The morphological diagnosis of B-CLL was confirmed by flowcytometry revealing CD19+, CD20+, CD5+ and CD23+ immunophenotype. All of our patients were staged according to the Rai staging system. (11) As regards the treated patients they received either chlorambucil with or without steroids or purine nucleoside analogues. Peripheral blood samples were usually obtained during routine follow-up visits to our unit, with informed consent. Measurement of CD38 and Zap-70: In the present study, the direct immunofluorescence technique was employed using labelled Abs according to the manufacturer s instructions and also cytoplasmic staining was used for CD38 and Zap-70 respectively. Immunofluorescence on viable leukemic cells in suspension was analyzed using Becton Dickinson, FACS Calibre flow cytometer equipped with Cell Quest software USA. Analysis of cell surface antigen CD38: Viable cells were incubated with fluorescein labelled MoAb CD38-FITC clone: T16, (Ref A07778), product of DAKO, Denmark. In short, 10 μl ofcd38moabwere incubated for 10 minutes with 100μl of EDTA blood followed by 2 washes using phosphate buffered saline (PBS) and then centrifuged at 2000 rpm for 2 minutes each at room temperature. Lysis solution was then added and incubated for 10 minutes in the dark, and centrifuged for 5 minutes at 2000 rpm, the supernatant was discarded. The washing steps were repeated. After the last wash, the cells were analyzed by flowcytometry, during analysis a gate was set around the required cell population which is the population showing CD5 + /CD19 +. The cut-off point of positivity was considered when 20% or more cells stained with the antibody in excess of the background fluorescence in the negative control. (12) Analysis of cytoplasmic antigen Zap -70: Zap-70 is an intracytoplasmic antigen, so its detection requires an extra step of permealization. Cells were fixed and permealyzed using 4% paraformaldhyde and 2% Tween 20. This allows gentle fixation and permealization of cells with preservation of the light scatter properties making distinction of different cell populations possible and therefore giving accurate results. The expression of Zap-70 was determined using monoclonal antibody Zap- 70(G-4) PE: sc-17760, product of DAKO, Denmark. The cut-off point of positivity was considered when 20% or more cells stained with the antibody in excess of the (8, 13) background fluorescence in the negative control. Statistical Analysis of the Data Statistical analysis of the data obtained in the present study was carried out using statistical package for social sciences (SPSS) version 15. Qualitative data were expressed in frequency and percentages and were analyzed using Chi-square test. Also normally distributed quantitative data of the studied groups were expressed as mean & standard deviation and were analyzed using student t-test, while the not normally distributed data were expressed in range and median and were analyzed using non parametric tests such as Mann-Whitney. Pearson correlation coefficient was used to find the correlation between two parameters in the same group. Results This study included 63 patients with CLL who presented to the hematology unit in the Main Alexandria University Hospital during the period from September 2005 to May Twenty-two of them were newly diagnosed, while forty-one were on chemotherapy in the form of either chlorambucil with or without steroids or were receiving cycles of fludarabine and cyclophosphamide, (FC protocol). Ten of the newly diagnosed patients were males, while twelve were females. Their ages ranged from 45 to 70 years. (Mean = ± 6.36 years) Regarding the patients on chemotherapy, nineteen were males while twenty-two were females. Their ages ranged from 33 to 72 years. (Mean = ± 8.04 years). There was no significant difference between treated and untreated patients as regards sex. On the other hand, treated patients had a significantly younger age than newly diagnosed patients. (p = 0.015) (Table 1). Staging of newly diagnosed patients according to the Rai staging system showed that 68.2% were in early stages (0-II) and 31.8% were in advanced stages (III-IV). As regards patients receiving chemotherapy, 51.2% were in stage (0-II) and 48.8% were in stage (III-IV). On comparing both patient groups as regards the expression of CD38, patients on chemotherapy showed JMRI, 2007; Vol.28 No 2: (174-80)

3 significantly higher levels (mean = ± 22.14%) (p=0.002) than newly diagnosed patients (mean= ± 18.77%), (p=0.002).on the other hand, no significant differences were found between both groups as regards expression of ZAP-70.(mean value of group I 6.95± 8.61%, mean value of group II 8.71±12.71%) (Table 2). When we compared our patients according to their pattern of ZAP-70 and CD38 expression, we found no significant differences between positive and negative cases for both markers as regards age and sex distribution.. Similarly, there were no significant differences as regards clinical staging between positive and negative cases for ZAP-70 and CD38 in both groups of patients (treated and untreated). However, in group I patients, there was a significant positive correlation between CD38 expression and the stage of the disease.(r=0.438*,p=0.041) (Table 3). There were no significant differences between positive and negative cases for either CD38 or ZAP-70 in group I patients as regards hemoglobin levels or platelet counts. CD38+ve patients had significantly higher total leukocytic counts and absolute lymphocyte counts (mean = ± /mm3, / mm3 respectively) than CD38-ve patients (mean = ± 50318/mm3, 47820±38125/mm3 respectively)(p=0.019, 0.04). Similarly, ZAP 70+ve patients (Mean= ± 94947/mm3, ± /mm3) than ZAP-70-ve patients (Mean= ± 25130, 38605/ mm3 ± 24679/ mm3 respectively) (p=0.002, respectively)(table 4). There was a significant positive correlation between CD38 expression and both the total leukocytic counts and absolute lymphocyte counts in group I patients. (r= 0.84*, p= 0,001 and r=0.881*, p= respectively) ZAP- 70 expression also showed a similar correlation with both the total leukocytic counts (r =0.909*, p 0.001) and dgggggggggggggggggggggggggggggggggg the absolute lymphocyte counts(r=0.871*,p 0.001)(Table 3). However, in group II patients, no significant differences were noted between CD38 +ve and CD38 ve or ZAP-70+ve and ZAP-70-ve patients as regards their blood counts. On the other hand, there was a significant positive correlation in this group between ZAP-70 expression and both the total leukocytic counts (r=0.39*,p=0.012) and the absolute lymphocyte counts (r =0.395*, p=0.011) (Table 5). We examined the relationship between ZAP-70 and CD38 expression and the time from diagnosis to initial therapy in group II patients. No significant differences were noted between ZAP- 70+ve patients (mean= 0.86±2.27 months) and ZAP- 70-ve (mean= 2.56±4.44 months) patients as regards the time from diagnosis to initiation of therapy. (p= 0.372) Similarly, CD38 +ve patients (mean= 1.35 ± 2.67 months) showed no significant differences than CD38-ve patients (mean=1.88±3.52 months) as regards time from diagnosis to initial therapy (p=0.805). The correlation between ZAP-70 and CD38 expression (Figures 1 and 2) showed that in 44 patients (69.8%), there was a complete concordance of ZAP-70 and CD38 expression: 6 patients (9.5%) were positive for ZAP-70 and CD38 and 38 patients (60.3%) showed a ZAP-70-ve/CD38-ve phenotype. 5 patients (7.9%) were characterized by ZAP-70+ve/CD38-ve phenotype and 14 patients (22.2%) showed a ZAP-70-ve/CD38+ve phenotype. Figure III is a representative flow cytometeric dot blot of a patient of CLL having CLL cells positive for both CD38 and Zap-70 expression. As regards the correlation between CD38 and ZAP- 70 expression in both groups of patients, there was a positive correlation, although being statistically insignificant. (r= 0.337, p = and r = 0.156, p = respectively). Table (1): Comparison of the two groups according to demographic data. Sex untreated (n=22) Treated (n=41) Test of sig. Male No. (%) 10 (45.5) 19 (46.3) Female No. (%) 12 (54.5) 22 (53.7) χ 2 =0.005 P=0.946 Age Min.-Max Mean ± SD 59.55± ±8.04 Median t=2.502 * p=0.015 χ 2 : Chi-square test t: Student t-test *: Statistically significant at p? 0.05

4 Sorour and El-Sorady Table (2): Comparison between the two studied groups according to CD38, ZAP-70 and stage. Untreated Treated Test of sig. No. % No. % Early (0 II ) Advanced (III IV) CD 38(%) Range Mean ± SD ± ± ZAP-70(%) Range Mean ± SD 6.95 ± ± * Staging was done according to the Rai staging system χ 2 : Chi square test U: Mann-Whitney test *: Statistically significant at p? 0.05 χ 2 =1.682 p = U = * p = U = p = Table (3): Correlation between CD38 and ZAP-70 with complete blood count in the untreated group (number of cases = 22). CD38 ZAP-70 White blood cells (WBCs) count r * * p <0.001 <0.001 Absolute lymphocytic count r * * p <0.001 <0.001 r * p r: Pearson coefficient *: Statistically significant at p? 0.05 Table (4) :Comparison between positive and negative cases of CD38 and ZAP-70 in the untreated group according to complete blood count and stage of the disease. CD38 ZAP-70 -ve (n = 19) +ve (n = 3) -ve (n = 18) +ve (n = 4) Hb(g/dl) Range Mean ± SD ± ± ± ± 3.29 Median t(p) (0.614) (0.453) WBCs /mm3 Range Mean ± SD ± ± ± ± Median U(p) * (0.019) * (0.002) Platelets/mm3 Range Mean ± SD ± ± ± ± Median t(p) (0.779) (0.627) Lymphocytes %. Range Mean ± SD ± ± ± ± Median t(p) (0.362) (0.432) Absolute lymphocytic count /mm3 Range Mean ± SD ± ± ± ± Median U(p) * (0.040) * (0.002) Early(0-II) 14 (93.3) 1 (6.7) 13 (86.7) 2 (13.3) Advanced(III,IV) 5 (71.4) 2 (28.6) 5 (71.4) 2 (28.6) FEp t: Student t-test U: Mann-Whitney test FEp : p value for Fisher Exact test *: Statistically significant at p? 0.05 JMRI, 2007; Vol.28 No 2: (174-80)

5 Table (5): Correlation between CD38 and ZAP-70 with white blood count, absolute lymphocyte count and stage in the treated group (number of cases = 41). CD 38 ZAP 70 (WBCs) White blood cells count Absolute lymphocytic count r * P r * P r P r: Pearson coefficient *: Statistically significant at p? 0.05 Fig. (1): Correlation between CD38 and Zap-70 in newly diagnosed patients (number of cases = 22). Fig.(2): Correlation between CD38 and Zap-70 in treated patients (number of cases = 41). Fig.(3): A representative figure for a patient with CLL cells positive for the expression of ZAP-70 and CD38. Dot-plot showing the majority of cells are double positive for ZAP-70 & CD38 expression in the upper right quadrant

6 Sorour and El-Sorady Discussion The B-cells of patients with chronic lymphocytic leukemia (CLL) can be segregated into one of at least two major subsets based upon the mutational status of the expressed immunoglobulin heavy chain variable region genes (IgVH). Patients with CLL cells that express unmutated IgVH tend to have a relatively aggressive clinical course when compared with patients who have CLL cells that express IgVH with somatic mutations. (14) ZAP-70, (a zeta associated protein of 70 KDa) is a receptor-associated protein tyrosine kinase expressed by T lymphocytes and natural killer cells but not by normal B cells. It is generally expressed by CLL B cells with unmutated IgVH genes and thus its expression may help to identify patients with a more aggressive clinical course. (15,16) Rosenwald et al demonstrated that expression analysis of the ZAP-70 gene could correctly predict the IgVH mutation status in 93% of patients. (15) The expression of ZAP-70 in CLL may allow for more effective IgM signaling in CLL B cells, a feature that could enhance the survival and / or proliferation of these cells, thus accounting in part for the relatively aggressive clinical behavior associated with CLL cells that express unmutated (17) IgVH. Expression of CD38 as determined by flow cytometry has also been shown to have prognostic significance in CLL. (18) Initially, it was proposed that CD38 might serve as a surrogate marker for IgVH mutational status. (19) Subsequent studies have not always shown this relationship. (10) Some of the differences may be due to technical aspects of the CD38 assays and the choice of an optimal cutoff point for the number of CD38 +ve cells. (20) When we compared our patients in both groups according to their ZAP-70 status, we found that there were no significant differences between positive and negative cases as regards the clinical stage. ZAP-70 +ve patients had significantly higher total leukocytic and absolute lymphocyte counts than ZAP-70-ve patients. ZAP-70 expression showed a significant positive correlation with both the total leukocytic and absolute lymphocyte counts. Similarly, CD38 expression by leukemic cells showed a significant correlation with both the total leukocytic counts and the absolute lymphocyte counts in both groups of our patients. It also correlated significantly with the stage of the disease in the newly diagnosed patients as 66.6% of the CD38+ve patients in this group were in advanced stages, while only 26.3% of the CD38-ve patients had stage III or IV disease. These findings are similar to those of Hus and colleagues who demonstrated that positive expression of both ZAP-70 and CD38 by leukemic cells correlated with higher total leukocytic and absolute lymphocyte counts as well as stage of the disease in their patients. (21) On the other hand, the correlation between the stage of the disease and both ZAP-70 and CD38 in the treated group was insignificant in our patients. This may be due to the effect of therapy in modifying the disease stage in patients responding to treatment. Although Rassenti et al (1) found that the median time from diagnosis to initial therapy in their group of patients who were ZAP-70 positive was significantly shorter than their ZAP-70 negative patients, we could not demonstrate this in our group of treated patients. This may be due to the smaller number of patients included in our study. There was a positive correlation between ZAP-70 expression and CD38 expression in our patients, although it was statistically insignificant. The clinical significance of CD38 expression in CLL remains controversial. According to Oscier et al, CD38 expression can not be used as an independent prognostic factor, but rather it gives additional information in (22) patients with an established mutational status. Domingo-Domenech et al found a link between CD38 expression and clinical and biological data, but in (23) multivariate analysis it lost statistical significance. Another confounding issue is that while some investigators have reported that CD38 expression by the leukemic clone is stable and unaffected by chemotherapy, others showed that it may change during the course of the disease, and an increase of CD38 expression may herald disease progression. (24) In conclusion, both ZAP-70 and CD38 expression were shown to correlate with both total leukocyte counts and absolute lymphocyte counts in our patients. Also, CD38 expression correlated with the disease stage in the untreated patients. However, on comparing the time to initiation of therapy in both ZAP-70 positive and negative patients and CD38 positive and negative patients in group II patients, no significant differences were found between both groups. This may be due to the relatively smaller number of patients included in our study as compared to other studies which found that positive ZAP- 70 expression to be associated with a shorter time to disease progression. References 1. Rassenti LZ, Huynh L, Toy TL, 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: Dighiero G, Binet JL. When and how to treat chronic lymphocytic leukemia. 3. N Engl J Med 2000; 343: Byrd JC, Waselenko JK, Keating M et al. Novel therapies for chronic lymphocytic leukemia in the 21 st century. Semin Oncol 2000; 27: Nascimento MC, Yamamoto M, Rodrigues MM et al. 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