The Study of Different Chromosomal Aberrations, CD38 and ZAP-70 in Chronic Lymphocytic Leukemia Patients

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1 THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol. 18 (2), 2011 Page: The Study of Different Chromosomal Aberrations, CD38 and ZAP-70 in Chronic Lymphocytic Leukemia Patients 1 Hassab AH, 1 Elbordiny MM, 2 Elghandour AH, 1 Sorour AF, 1 Swelem RS Departments of 1 Clinical Pathology, and 2 Internal Medicine, Faculty of Medicine, Alexandria University, Egypt. Chronic lymphocytic leukemia (CLL) follows an extremely variable clinical course with overall survival times ranging from months to decades. The clinical staging systems do not allow one to predict if and at what rate there will be disease progression in an individual patient diagnosed with early stage disease. There has been intensive work on clinical and biological factors of potential prognostic relevance that may add to the classic assessment provided by the staging systems. Among these are: Laboratory parameters reflecting the tumor burden or disease activity such as lymphocyte count, lactate dehydrogenase (LDH) elevation, bone marrow infiltration pattern or lymphocyte doubling time (LDT), serum markers such as soluble CD23, β2- microglobulin (β2-mg) or thymidine kinase (TK), and genetic markers of tumor cells such as genomic aberrations, the mutation status of the variable segments of immunoglobulin heavy chain genes (VH), or surrogate markers for these factors (CD38, ZAP-70, LPL). Thirty patients were included in our study, the investigation included CD38 expression, ZAP-70 expression, and interphase Fluorescence In Situ Hybridization (FISH) for the detection of trisomy 12, del 13q14.3, del 17p13, and del 11q22.3. Our results showed positive statistical significant correlation between ZAP-70 expression and CD38 expression with some of the chromosomal aberrations encompassing bad prognosis as ATM and p53. Also CD38 expression was positively correlated with trisomy 12 and p53 deletions. Chromosomal aberrations were found to be present in 76.6% of our patients with 13q deletion as the most frequent abnormality in our patients (46.7%), followed by trisomy 12 (36.7%), then ATM and p53 deletion (26.7%) each. Another interesting finding in our study is the fact that 100% of the ZAP-70 positive patients were of bad prognosis, 58.3% of the CD38 positive cases, 81.8% of the positive trisomy 12 cases, 100% of the ATM deletion, 62.5% of the p53 deletion, and 64.3% of the 13q- cases were also of bad prognosis, which indicates that ZAP-70, trisomy 12, and ATM deletion are powerful indicators of prognosis. We conclude that FISH for the detection of the most important chromosomal aberrations in CLL is an important laboratory parameter that is recommended for assessment and correlation with simultaneous evaluation of ZAP-70 and CD38 expression which could help in the prediction of outcome of CLL patients. Chronic lymphocytic leukemia (CLL) is considered the most common primary B-cell leukemia. It was traditionally considered as a disease of elderly patients with a generally long survival rate lasting up to 30 years from the time of first diagnosis. The duration and outcome of the disease is highly variable. The first period of follow up is usually free of any symptoms. Disease progression may appear years later in the form of lymphadenopathy or organ involvement, but the rapid transformation to prolymphocytic leukemia or Richter s syndrome is also a well-known complication (Byrd et al., 2005; Tsimberiodu & Keating, 2005). In CLL, few patients experience clinically a slow progression in the course of disease, while disease progression with fatal outcomes is observed in majority of cases (Mehes, 2005; Capello et al., 2004; Messer et al., 2004). The standard clinical procedures to estimate prognosis are the clinical staging systems developed by Rai et al. (1975) and Binet et al. (1981). These systems define early (Rai 0, Binet A), intermediate (Rai I/II, Binet B) and advanced (Rai III/IV, Binet C) stage disease. However, heterogeneity in the course of the disease among individual patients within a single stage group is observed. Most importantly, the clinical staging systems do not allow the prediction of disease

2 78 Different Chromosomal Aberrations, CD38 and ZAP-70 In CLL Patients progression in individual patients diagnosed at an early stage of the disease. There is intensive work on clinical and biological factors of potential prognostic relevance that may be added to the classic assessment provided by the staging systems. Laboratory parameters such as serum marker, cytogenetic abnormalities, and mutation of immunoglobulin hypervariable region may reflect the tumor burden or disease activity. These include lymphocyte count, lactate dehydrogenase (LDH) elevation, bone marrow infiltration pattern or lymphocyte doubling time (LDT) (Rozman et al., 1984; Montserrat et al., 1986; Kröber et al., 2002); serum markers such as soluble CD23, β2- microglobulin (β2-mg) or thymidine kinase (TK), (Di Giovanni et al., 1989; Hallek et al., 1999) and genetic markers of tumor cells such as genomic aberrations, gene abnormalities (p53 and ATM), the mutation status of the variable segments of immunoglobulin heavy chain genes (IgVH), or surrogate markers for these factors (CD38, ZAP-70, LPL, etc.) (Damle et al., 1999; Döhner et al., 2000; Oscier et al., 2002; Lin et al., 2002; Orchard et al., 2003; Dewald et al., 2003). CLL cells may have several characteristics that are associated with relatively aggressive disease. One such characteristic is the absence of somatic mutations in rearranged IgVH genes (Hamblin et al., 2002). Currently unmutated IgVH genes are the strongest predictor of aggressive disease (Hamblin et al., 1999; Maloum et al., 2000; Rassenti et al., 2004). It is important to note that patients with unmutated IgVH genes usually have an advanced stage of CLL and unfavorable cytogenetic features, require therapy, and have a short survival. In contrast, patients with leukemic cells that have mutant IgVH genes usually present in an early clinical stage, frequently have 13q14 chromosomal deletions do not have alterations of p53, do not require therapy, and have a long survival (Crespo et al., 2003). The role of ZAP-70 (zeta associated protein) as a surrogate marker for IgVH mutation was reported to help identify patients with a more aggressive clinical course (Crespo et al., 2003; Wiestner et al., 2003). ZAP-70 is a member of the Syk-ZAP-70 protein tyrosine kinase family. It is expressed by normal T cells and natural killer cells and it plays a critical role in the development and differentiation of these cell types (Crespo et al., 2003). There is no ZAP-70 expression on normal B lymphocytes, but it was detected on B-CLL cells. It was suggested that the expression of ZAP-70 could not only predict IgVH mutational status, but it could also serve as a prognostic factor in B-CLL and its expression would be stable during the course of the disease (Crespo et al., 2003). It was found that CLL B cells that had unmutated IgVH genes generally expressed levels of ZAP-70 protein that were comparable to those expressed by normal blood T cells (Crespo et al., 2003; Chen et al., 2002). In contrast, CLL B cells that expressed mutated IgVH genes generally do not express detectable levels of ZAP-70 protein. As such, leukemia cell expression of ZAP-70 can be used as a surrogate marker for Ig mutational status, (Crespo et al., 2003; Wiestner et al., 2003) which can segregate patients that have significantly different tendencies for disease progression (Capello et al., 2004). CD38 (also referred to as T10 antigen) was initially characterized in 1980 as a T-cell differentiation antigen (Chiorazzi & Ferrarini, 2001). In the following years, several studies showed that CD38 expression is not limited to T cells but is widely expressed on different hematopoietic and non hematopoietic tissues (Stilgenbauer et al., 2001). CD38 was initially proposed as a surrogate marker for the absence of IgV gene rearrangement (Dale et al., 1999; Deaglio et al., 2006). CD38 expression can undisputedly be considered as an independent and reliable negative prognostic marker in CLL. CD38 + patients are

3 THE EGYPTIAN JOURNAL OF IMMUNOLOGY 79 characterized by an unfavorable clinical course with a more advanced stage of disease, poor responsiveness to chemotherapy, a shorter lapse in time before initial treatment is required, and a shorter survival rate (Deaglio et al., 2006). Both ZAP-70 and CD38 expression were shown to predict the clinical course of the disease, while ZAP-70 expression appeared to be more predictive than CD38 expression and more relevant in defining the cases of B-CLL responsive or refractory to first line chemotherapy. A simultaneous evaluation of ZAP-70 and CD38 expression allowed distinguishing the patients groups with the most favorable prognosis as well as those with the worst (Tsiodras et al., 2000). Aberrations were more recently reported in 50-80% of the CLL cases. Of these abnormalities we have; 17p-, 11q-, 13q-, and trisomy 12, which are found either separate, or more than one chromosome abnormality in some patients. 13q- is associated with a favorable prognosis, while 17p-, 11q-, and trisomy 12 are of bad prognosis (Damle et al., 2002). The aim of the present study was to study the expression of ZAP-70, CD38 in a group of patients with B-CLL using flowcytometry and correlate the results with the clinical data, laboratory parameters and chromosome abnormalities including trisomy 12, del 13q14.3, del 17p13, and del 11q22.3 detected by fluorescence in situ hybridization(fish). Patients and Methods The study included 30 cases of CLL under treatment and referred to Alexandria Main University Hospital. A written consent was taken from all patients included in the study. All patients were subjected to full history taking and complete clinical examination. The morphological diagnosis of B-CLL was confirmed by flowcytometry with the immunophenotype of typical B cell CLL that includes coexpression of weak monotypic surface immunoglobulin, CD5, CD19, CD23, and weak or absent CD79B, CD22 and FMC7 and Ig light chain (kappa or Lambda). All patients were staged according to the Rai staging system. Peripheral blood samples were obtained during routine follow up visits to our department. Measurement of CD38 and ZAP-70 In the present study, the direct immunofluorescence technique was employed using labeled Abs according to the manufacturer s instructions and also cytoplasmic staining was applied for CD38 and ZAP- 70 respectively. Immunofluorescence on viable leukemic cells in suspension was analyzed using (FACS calibur, Becton Dickinson, San Jose, USA) equipped with Cell Quest software (BD). 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. Analysis of cell surface antigen CD38 The expression of CD38 was studied on viable cells using fluorescein labeled MoAb CD38-FITC clone: T16, (Ref A07778), product of DAKO (Denmark), during analysis by flowcytometry a gate was set around the required cell population which is the population showing CD5 + /CD19 +. Analysis of cell cytoplasmic antigen ZAP-70 ZAP-70 is an intracytoplasmic antigen, so its detection requires an extra step of permeablization. Cells were fixed and permeablized using 4% paraformaldehyde and 2% tween 20. This allows gentle fixation and permeablization of cells with preservation of 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 , product of DAKO, Denmark. FISH was performed using the Vysis CLL FISH Probe Kit (Downers Grove, IL0, Abbot Diagnostics) South Pasadena, CA) including DNA probes to determine deletion status of probe targets for locusspecific identifier (LSI) TP53 (containing tumor protein p53 gene, located on chromosome (17p), LSI ATM (containing ataxia telangiectasia mutated gene, located on chromosome 11q), and LSI D13S319 (containing marker D13S319, located on chromosome 13q), as well as determining trisomy 12 with CEP12 (D12Z3 alpha satellite, located on chromosome 12). The Vysis CLL FISH Probe Kit includes LSI 13q34 (containing lysosomal associated membrane protein 1 gene, located on chromosome 13q) as a quality control probe, the probe is supplied in the form of two vials. The first vial contains probe set 1: LSI D13S319 Spectrum Orange/13q34 Spectrum Aqua/ CEP 12 Spectrum Green Probes and the second vial contains probe set 2; Vysis LSI TP53 Spectrum Orange/ATM

4 80 Different Chromosomal Aberrations, CD38 and ZAP-70 In CLL Patients Spectrum Green Probes. Slides were prepared from material fixed in methanol-acetic acid. In short, lymphocytes were separated by gently layering the sample on equal volume of Ficoll-hypaque. The cells were then washed, fixed with Carnoy s fixative; the fixed material was dropped on positively charged slides labeled with the patient s name, date, type of sample, and the diagnosis. Hybridization and detection of hybridization signals were performed according to the manufacturer's protocols. At least two technologists scored the same case. For each probe, at least 100 nuclei were evaluated. Images of FISH were captured through the Cytovision software (Applied Imaging, Newcastle upon Tyne, England). Peripheral blood samples from healthy individuals were used as a control. Means and standard deviations (SD) of the percentage of nuclei with hybridization signals were calculated. Results were considered abnormal if the percent of nuclei with the abnormal hybridization signal was > 2 SD from the mean. For trisomy 12 it was considered positive if cells showing three signals in > 4% of the counted cells, ATM, p53 and 13q14 deletions if cells show >10%, >9% and >10% cells with deletions respectively. Serum β-2 microglobulin (β-2 M) level measured by (IMMULITE Beta 2 Microglobulin kit, DPC, USA) according to the manufacturer's instructions using solid-phase 2-site chemiluminescent enzyme immunoassay on the IMMULITE Automated Analyzer (DPC, Los Angeles, USA) and Serum LDH level measured by Dimension Rx L Max, USA. Siemens. Statistical Analysis Data analysis was carried out using Statistical Package of Social Sciences (SPSS) version 15. Qualitative data expressed in frequency and percentages was analyzed using Fisher Exact test (FEp) and Monte Carlo test (MCp). Also normally distributed quantitative data of the studied groups expressed in mean and standard deviation (SD) was analyzed using student t-test (t) and F-test (ANOVA), while the abnormally distributed data expressed in range and median was analyzed using non parametric tests such as Mann-Whitney (U) and Kruskal Wallis tests (χ 2 ). Correlation coefficients (r(p)) were used to find the correlation between two parameters in the same group. Results Analysis of clinical data. The diagnosis of CLL (n=30) was based on clinical, morphologic and immunophenotypic criteria. Fourteen (46.7%) patients were males and 16 (53.3%), were females with mean age of 56.1 years, (ranging from years). Clinical data analysis revealed that, 10 patients (33.3%) had hepatosplenomegaly and lymphadenopathy, 6 (20%) had splenomegaly and lymphadenopathy, 3 (10%) had only splenomegaly, 1 (3.3%) had hepatosplenomegaly, 7 (23.3%) had only lymphadenopathy and 3 (10%) had neither hepatosplenomegaly nor lymphadenopathy. Time from diagnosis to start of treatment ranged from immediate start of treatment in 23 patients (76.7%) 0 months and up to 2 years in the remaining 7 patients (23.3%). In our study, 46.7% of the patients had Rai stages 0 to II,16.7% Rai stage III and 36.7 Rai stage IV, 40% had entered into complete remission when followed up for one year. Complete blood count (CBC) CBC showed that from the 30 patients enrolled in our study 24 (80%) patients had leucocytosis with absolute lymphocytosis, 3 (10%) patients had absolute lymphocytosis only and 3 (10%) of the patients with normal counts. Hemoglobin was normal in 8 (26.7%) patients and 22 (73.3%) patients had anemia, mostly normochromic normocytic. Thrombocytopenia was found in 18 (60%) patients and 12 (40%) patients had normal platelet counts. ZAP-70 and CD-38 expression According to ZAP-70 and CD-38 expression the patients were divided into 2 groups according to the cut-off value for ZAP-70 and CD38 (20%). Four (13.3%) patients were positive for ZAP-70, while 12 (40%) patients had positive CD38 expression. LDH & β-2 -microglobulin LDH values were considered higher than normal (cut-off: 190 U/L) in 21 (70%) patients, while β-2 -microglobulin showed abnormal high results (cut-off: 1556 ng/ml) in 25 (83.3%) patients. (Table 1, Figure 1).

5 THE EGYPTIAN JOURNAL OF IMMUNOLOGY 81 Table 1. Distribution according to ZAP 70, CD38, LDH and B 2-microglobulin N (%) Min. Max. Mean ± SD Median ZAP-70 % 30 (100.0) ± Normal 26 (86.7) ± Abnormal 4 (13.3) ± CD38 % 30 (100.0) ± Normal 18 (60.0) ± Abnormal 12 (40.0) ± LDH (U/L) 30 (100.0) ± Normal 9 (30.0) ± Abnormal 21 (70.0) ± β 2-microglobulin (ng/ml) 30 (100.0) ± Normal 5 (16.7) ± Abnormal 25 (83.3) ± Figure 1. 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 82 Different Chromosomal Aberrations, CD38 and ZAP-70 In CLL Patients Interphase FISH to evaluate deletions in chromosome bands 17p13, 11q22.3, 13q14, and trisomy of band 12q13 Interphase FISH showed that 7 (23.3%) patients had no aberrations regarding the studied chromosomal loci. 14 (46.7%) had 13q14 deletion, 11 (36.7%) patients had trisomy 12, 8 (26.7%) patients had 17p13 deletion, and 8 (26.7%) patients had 11q22.3 deletion (Table 2, Figures 2, 3 & 4). A significant difference was found between ZAP-70 positive and negative groups in terms of 11q23 deletion (P=0.006). Similarly, a positive statistically significant difference was found between both groups of ATM deletion as regards the prognosis (P=0.010) and LDH serum levels (P=0.020), also there was a positive significant difference between CD38 positive and negative cases in terms of p53 deletion (P =0.041). No statistically significant difference was otherwise found between the different groups (Tables 3). Correlation between CD38, ZAP70, Chromosomal aberrations and other factors in the studied groups There was a statistically significant positive correlation between ZAP-70 and the poor-risk cytogenetic subset, encompassing ATM deletion (P =0.005), and P53 deletion (P=0.019). In contrast, we found no association between ZAP-70 expression and trisomy 12 (P =0.143) or 13q14 deletion (P =0.952). CD38 was positively correlated with ZAP- 70 (P =0.008), trisomy 12 (P =0.003), and p53 deletion (P =0.002), but was negatively correlated with age (P =0.039). A positive correlation was found between trisomy 12 and P53 deletion (P <0.001), and between ATM deletion with WBC count (P =0.019) and (Absolute lymphocytic count) ALC (P = 0.021). A statistically significant negative correlation was found between 13q14 deletion and both Hb level (P =0.025) and platelet counts (P =0.021) (Table 4). Correlation between the positive cases CD38, ZAP70, Chromosomal aberrations and other factors in the studied groups A statistically significant positive correlation was found between trisomy 12 positive cases and p53 deletion (P =0.046), while a negative correlation was found with both WBC (P =0.045) and ALC (P =0.039). ATM deletion positive cases revealed a positive correlation with WBC (P=0.007) and ALC (P =0.007). Positive CD38 cases were significantly correlated with ZAP-70 (P =0.018) and trisomy 12 (P =0.001). With regards to the clinical course, a significant correlation was found between higher P53 deletion and advanced Rai stages (P <0.001) (Table 5).

7 THE EGYPTIAN JOURNAL OF IMMUNOLOGY 83 Table 2. Distribution of the patients according to chromosomal aberrations N (%) Min. Max. Mean ± SD Median Trisomy 12 % 30 (100.0) ± Normal 19 (63.3) ± Abnormal 11 (36.7) ± q22.3 deletion % 30 (100.0) ± Normal 22 (73.3) ± Abnormal 8 (26.7) ± p13 deletion % 30 (100.0) ± Normal 22 (73.3) ± Abnormal 8 (26.7) ± q14 deletion % 30 (100.0) ± Normal 16 (53.3) ± Abnormal 14 (46.7) ±

8 84 Different Chromosomal Aberrations, CD38 and ZAP-70 In CLL Patients

9 THE EGYPTIAN JOURNAL OF IMMUNOLOGY 85 Table3. Comparison between the normal and abnormal cases in the studied patients in terms of ZAP-70, CD38 and chromosomal aberrations with the chromosomal aberrations and LDH. Trisomy 12 % ATM deletion % P53 deletion % 13q14 deletion % ZAP-70 CD38 Trisomy 12 ATM deletion P53 deletion 13q14 deletion Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Min.-Max Median U= P U= P Test of sig. U=0.000 * U= P U= P U= P P <0.001 =0.090(NS) =0.305(NS) =0.136(NS) =0.407(NS) =0.345(NS) Min.-Max Median Test of sig. U=7.500 * U= P U= P P =0.006 U=0.000 * U= P U= P P <0.001 =0.137(NS) =0.353(NS) =0.203(NS) =0.44(NS)0 Min.-Max Median U= Test of sig. U= * U= U= P=0.041 U=0.000 * U= P <0.001 P=0.560(NS) P=0.069(NS) P=0.620(NS) P=0.233(NS) Min.-Max Median Test of sig. U= P U= P U= P U= P U= P =0.233(NS) =0.261(NS) =0.846(NS) =0.438(NS) =0.100(NS) U=0.000 * P <0.001 Min.-Max Mean ± SD ± ±77.94 ± ± ±85.50 ± ± ±89.23 ± ± Test of sig. t = t = t = t = 2.474* t = t = P = 0.497(NS) P = 0.547(NS) P = 0.137(NS) P = P = 0.222(NS) P = 0.675(NS) t : Student t-test; U : Mann-Whitney;* : Statistically significant at P 0.05; NS=not significant. LDH (U/L)

10 86 Different Chromosomal Aberrations, CD38 and ZAP-70 In CLL Patients Table 4. Correlation between CD38, ZAP-70, chromosomal aberrations and other factors in the studied patients CD38 ZAP- 70 Trisomy- 12 Chromosomal aberration Complete blood count Age ATM P53 13q14 WBCs Hb Platelet Absolute lymphocyte LDH B 2- microglobulin Stage Follow up CD38 % * (0.008) * (0.003) (0.132) * (0.002) (0.177) * (0.039) (0.883) (0.738) (0.850) (0.901) (0.818) (0.281) (0.466) (0.735) ZAP-70 % (0.143) ** (0.005) * (0.019) (0.658) (0.612) (0.819) (0.895) (0.691) (0.675) (0.843) Trisomy- 12% (0.771) ** (<0.001) (0.202) (0.903) (0.297) (0.639) (0.884) (0.167) (0.676) ATM del.% (0.796) (0.875) * (0.019) (0.167) (0.893) * (0.021) (0.082) (0.584) P53 del. % (0.724) (0.506) (0.371) (0.129) (0.493) (0.336) (0.129) q14 del. % (0.381) (0.759) * (0.025) * (0.021) (0.745) (0.063) (0.083) Data in the table are expressed as r (p) where r is the correlation coefficient while p is the value of significance Statistically significant at P 0.05; NS=not significant.

11 THE EGYPTIAN JOURNAL OF IMMUNOLOGY 87 Table 5. Correlation between the positive cases in CD38, ZAP-70, chromosomal aberrations and other factors in the studied patients CD38 ZAP-70 Trisomy- 12 Chromosomal aberration Complete blood count Age ATM P53 13q14 WBCs Hb Platelet Absolute lymphocyte LDH B 2-microglobulin Stage Follow Up CD38 % (n=12) * (0.018) * (0.001) (0.595) (0.057) (0.821) (0.058) (0.933) (0.611) (0.553) (0.901) (0.573) (0.433) (0.271) (0.343) ZAP-70 % (n=4) (0.875) (0.163) (0.665) (0.300) (0.296) (0.676) (0.830) (0.301) (0.202) (0.675) - Trisomy- 12% (n=11) (0.337) * (0.046) (0.727) (0.093) * (0.045) (0.832) (0.594) * (0.039) (0.809) (0.443) (0.792) (0.523) ATM % (n=8) (0.917) (0.727) (0.723) ** (0.007) (0.816) (0.863) * (0.007) (0.878) (0.523) (0.517) (0.369) P53 % (n=8) (0.786) (0.648) (0.569) (0.669) (0.232) (0.493) (0.882) (0.523) ** (<0.001) q14 del.% (n=14) (0.640) (0.828) (0.806) (0.066) (0.789) (0.900) (0.531) (0.300) (0.378) Data in the table are expressed as r (p) where r is the correlation coefficient while p is the value of significance Statistically significant at P 0.05; NS=not significant.

12 88 Different Chromosomal Abberations, CD38 and ZAP-70 In CLL Patients Combined analysis of ZAP70 and CD38: Combined analysis of ZAP-70 and CD38 yielded discordant results in 10 (33.3%) patients; whereas 17(56.6%) patients were concordantly negative and 3(10%) patient were concordantly positive for ZAP-70 and CD38 expression. No statistically significant difference was found between any of the 4 groups in terms of all the above parameters except for a positive significant difference found between the first (+/+) and both third (-/+) and fourth (-/-) groups as regards ATM deletion (P =0.021, P =0.022 respectively). A significant difference was found between ZAP-70 positive and negative groups in terms of 11q23 deletion (P =0.006). Similarly, a positive statistically significant difference was found between both groups of ATM deletion as regards the prognosis (P=0.010) and LDH serum levels (P=0.020), also there was a positive significant difference between CD38 positive and negative cases in terms of p53 deletion (p=0.041). No statistically significant difference was otherwise found between the different groups. No statistically significant correlation was found between follow up with age and sex. On classifying the studied group into early (stages 0/I) and advanced (II, III, IV) Rai stages, no statistical significant difference was found between the two groups as regards the four chromosomal aberrations under study. A positive statistical significant difference was found between both groups in terms of B2-microglobulin (P =0.008), but not in LDH, ZAP-70, CD38, and time from diagnosis to start of treatment (Treatment-free interval (TFI)). From the 23 patients exhibiting chromosomal abnormalities, 10 (43.5%) had one aberration, 8 (34.8%) had 2 aberrations, and 5 patients (21.7%) had 3 aberrations. The most frequent chromosomal aberration was 13q14 deletion found in 14 patients; in 5 patients as a sole abnormality. Among the other 9 patients, 3 patients were accompanied by trisomy 12, 3 by ATM deletion, 2 by both ATM deletion and trisomy 12, and one by trisomy 12 and 17p13 deletion. On dividing the studied group according to chromosomal aberrations into favorable (13qas a sole abnormality or no alteration), and unfavorable (p53 deletion, trisomy 12, and ATM deletion), no statistical significant difference was found as regards ZAP-70 expression (P =0.832) and CD38 expression (P =0.059). No statistical significant difference was found in relation to LDH serum levels, β2- micoglobulin, TFI, and age between the favorable and unfavorable groups. Follow up outcome was significantly different between both groups (P =0.024), where 77.8% of the patients in the unfavorable group didn t achieve CR versus 33.3% of those in the favorable group. Discussion The objective of the present study was to assess the expression of ZAP-70 and CD38 and some chromosomal aberrations including. 17p-, 11q-, 13q-, and trisomy 12 using FISH on leukemic cells of B-CLL patients and to correlate the finding with their clinical data. As regards ZAP-70 expression, our study showed that only 4 (13.3%) patients were positive, whereas 12 (40%) patients were positive for CD38 expression. Data obtained in this study showed that CD38 was positively associated with ZAP-70 expression, trisomy 12 and p53 deletion with a significant difference between positive and negative cases as with respect to p53 deletion. Our results agreed with those revealed by Ibrahim, et al.(2001) who found CD38% positivity in 44% of the patients enrolled in

13 THE EGYPTIAN JOURNAL OF IMMUNOLOGY 89 the study but no significant difference was found between CD38 positive and negative groups in terms of staging, splenomegaly, age, sex, WBC counts, or absolute lymphocyte count. In contrast, there was an association between the number of nodal sites involved and CD38 expression. CD38 positive patients also had significantly lower hemoglobin levels and higher levels of β-2 microglobulin unlike data obtained in this study. This may be due to the effect of chemotherapy received by the patients, and the small number of patients enrolled in our study. Results revealed by Del Guidice et al., (2005) regarding ZAP-70 positivity (28% of the cases, n=57) and its relation with other prognostic parameters agreed with ours in the positive correlation with CD38 %, but were opposite to our results in the absence of significant association between ZAP-70 and deletion 11q, deletion 17p), but were slightly more common in ZAP-70 positive group, also there was a positive correlation with atypical morphology and advanced disease stage. In addition a significant difference was found between both groups in terms of treatmentfree interval (TFI). The study also proved that CD38 expression, ZAP-70 and clinical stages are independent factors for TFI. Moreover, a negative correlation was found between ZAP- 70 and 13q- (Del Guidice et al., 2005).These results may be different from ours because of the difference in the number of patients enrolled in the study, the number of positive cases for ZAP-70, and all of our patients were under treatment. Ertault-Daneshpouy et al. (2008) studied ninety four B-CLL unselected patients in order to assess ZAP-70 expression in B-CLL cells used flowcytometry and examined its relationship with CD38 expression and the median time from diagnosis to initial therapy. ZAP-70 expression levels in B-CLL cells varied widely (0.3-99%). The median time to therapy was significantly shorter for the 54 patients with 20% or more ZAP-70 + cells (30 months) than for the 40 patients with less than 20% ZAP-70+ cells (median time to treatment not reached) which was opposite to our results where no significant difference was found between positive and negative ZAP-70 cases regarding TFI). On the other hand, similar to our results; CD38 and ZAP-70 expressions were significantly associated. The median interval from diagnosis to initial therapy was 16.2 months for ZAP-70 + CD38 + patients, 60 months for ZAP-70 + CD38 - or ZAP-70 - CD38 + patients, and was not reached for ZAP-70 - CD38 - patients. Ertault-Daneshpouy et al. (2008) concluded that the association of ZAP CD19 + CD5 + B-CLL cells and percentage of CD38 + CD19 + CD5 + B-CLL cells evaluated by flowcytometry provide reliable methods that could be introduced into a routine diagnostic B-CLL panel to predict outcome. Del Guidice et al., (2005) also proposed a risk model that combines ZAP-70 and CD38 to identify patients who are likely to progress. When both markers were positive; treatmentfree interval (TFI) was 12 months, when both were negative, TFI was 54 months, a median TFI of 26 months was observed in patients who had discordant results (Del Guidice et al., 2005). In our study; combined analysis of ZAP-70 and CD38 allowed us to separate our patients into 4 subgroups: ZAP-70 - /CD38 - (n=17), ZAP-70 + /CD38 + (n=3), and 2 discordant ZAP-70/CD38 (n=10), where a significant difference was found between ZAP-70 + /CD38 + group and the discordant ZAP-70 - /CD38 + group in terms of CD38 expression, ATM deletion, and between ZAP /CD38 + group and ZAP-70 - /CD38 - in terms of ATM deletion. The double positive group patients (n=3) started treatment immediately after being diagnosed with no TFI, those who belonged to the double negative group had TFI ranged from (0-24 months), and those with discordant results had a range of (0-15 months), also the double positive group showed significantly

14 90 Different Chromosomal Abberations, CD38 and ZAP-70 In CLL Patients higher ATM deletion and CD38 expression than the other groups, and all its cases (n=3) were of bad prognosis (100%) opposite to ZAP-70 - /CD38 + patients (44.4%) and ZAP /CD38 - patients (58.8%), which shows how the combined analysis of both may be more informative than either of them alone. On studying chromosomal abnormalities, our study showed alterations in 23 (76.7%) of the 30 patients under study. ATM deletion showed positive correlation with ZAP-70, WBC count and absolute lymphocyte count which is expected as all are bad prognostic signs. Also a positive significant difference was revealed between positive and negative cases of ATM deletion as regards LDH serum levels and response to treatment, were in the abnormal cases 100% of the cases had bad prognosis, while in the normal group 45.5% had bad prognosis. On the other hand, there was no significant correlation between ATM deletion and CD38, or any of the other chromosomal aberrations, B2-microglobulin and staging. 11q22 deletions have been associated with aggressive clinical courses of the disease, (Nascimento et al., 2006) a fact also observed in our study since there was a significant difference between the 2 ATM groups regarding the prognosis. In our study, P53 was found to be deleted in 26.7% of the cases. P53 deletion showed positive correlation with ZAP-70, trisomy 12, CD38 and only the positive cases of p53 deletion showed positive correlation with the Rai staging system. No significant difference was found between positive and negative cases regarding all the studied parameters. Trisomy 12 positive cases showed a negative correlation with the WBC count and with the absolute lymphocyte count. This finding is opposite to what should be expected as trisomy 12 should be associated with increased counts as it is a bad prognostic parameter. However, our results may be explained by the fact that these counts are after treatment and are due to the effect of chemotherapy received by the patient. On the other hand a positive significant correlation was found between trisomy 12 and CD38. 13q- showed no statistical significant difference or correlation with any of the studied parameters except for a negative significant correlation with level of hemoglobin and platelet counts. The region of 13q14.3 telomeric to the retinoblastoma (RB1) gene is often deleted in CLL and is thought to have a tumor suppressor function. Structural abnormalities of chromosome 13 have generally been associated with early clinical stage, typical morphology and as good prognosis as cases with normal karyotyping. (Nascimento et al., 2006) Our results agreed with those revealed by Nascimento et al. (2006) who found that 51.7% of his patients presented chromosomal abnormalities: 10 (34.5%) with a single aberration and 5 (17.2%) with 2 anomalies. The most frequently observed aberration was 13q14 deletion observed in 10 patients. No statistical significant difference was found between favorable and unfavorable cytogenetic alteration groups in relation to age, Binet clinical stage, CD38 expression, and ZAP-70 expression, but a statistical significant difference was found between gender and prognosis, which was opposite to our study where no significant difference was found. Similar results were obtained by others (Dö hner et al., 2000; Oscier et al., 2002; Sindelarove et al., 2005; Aoun et al., 2004) except for a positive significant correlation between trisomy 12 and LDH serum levels, and Del Principle, et al., (2006) who found similar chromosomal abnormalities expression, and also proved a statistical significant correlation between ZAP-70 expression >20% and the poor risk cytogenetic subsets, while 13q- was significantly correlated with CD38 < 30%.

15 THE EGYPTIAN JOURNAL OF IMMUNOLOGY 91 On the other hand, Ripolle s et al, (2006) found a significant correlation between chromosomal anomalies and age. They found no statistical significant difference when morphology was analyzed, but the most frequent abnormality among patients with typical morphology was 13q-, and it was the most frequent abnormality seen in initial Rai stage (0-I), while patients with advanced stages (II-IV) had more frequent ATM deletion. So, their results show that 13q- as a sole abnormality is a primary change that occurs in early stages and is associated with stable disease and long survival. ATM deletion is a secondary change, in more than half of the patients; it presents together with 13q- or +12 and occurs in advanced stages. It is associated with progression and short survival. As a better way to study the relationship between chromosomal aberrations and the other prognostic factors in CLL, we separated the patients into two cytogenetic groups: favorable (isolated 13q- or no alteration) and unfavorable outcomes (12q+, 17p-, 11q-). We found no significant difference between both groups in relation to age, LDH serum levels, beta2-microglobulin, TFI, lymphadenopathy, hepatomegaly and splenomegaly. On the other hand, there was a significant difference between both groups regarding the prognosis where 33.7% of the patients in the favorable group were of bad prognosis versus 77.8% of the unfavorable group patients. Although no significant difference was found between both groups in terms of ZAP-70 expression and CD38, but all our four ZAP-70 positive cases belonged to the unfavorable group, and from the 12 positive CD38 cases; 8 patients (66.6%) were in the unfavorable group. Another interesting finding in our study is the fact that 100% of the ZAP-70 positive patients were of bad prognosis, 58.3% of the CD38 positive cases, 81.8% of the positive trisomy 12 cases, 100% of the ATM deletion, 62.5% of the p53 deletion, and 64.3% of the 13q- cases were also of bad prognosis, which indicates that ZAP-70, trisomy 12, and ATM deletion are powerful indicators of prognosis. 13q14 deletion which should be accompanied by good prognostic outcome showed 64.3% bad prognosis, and this could be contributed to the fact that from the 14 positive 13q- cases; 64.28% (n=9) were accompanied by other chromosomal anomalies and so, lost its good prognostic effect. Clinical stages have been the basis for assessing prognosis in patients with CLL. However, a number of biological parameters, particularly serum markers, cytogenetics, IgVH mutations, CD38 and ZAP-70 expression in leukemic cells, are important independent prognostic markers. ZAP-70 and IgVH mutations basically provide similar prognostic information and therefore they can substitute each other. It is not difficult to make a prognostication system for patients with CLL in which clinical stages are complemented, by biological markers (Ibrahim et al., 2001). References 1. Aoun P, Blair HE, Smith LM, Dave BJ, Lynch J, Weisenburger DD, Pavletic SZ, Sanger WG. (2004) Fluorescence in situ hybridization detection of cytogenetic abnormalities in B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma. Leuk Lymphoma 45(8): Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, Vaugier G, Potron G, Colona P, Oberling F, Thomas M, Tchernia G, Jacquillat C, Boivin P, Lesty C, Duault MT, Monconduit M, Belabbes S, Gremy F. (1981). A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer, 48: Byrd JC, Stilgenbauer S, and Flinn IW (2004). Chronic lymphocytic leukemia. Hematology Capello D, Guarini A, Berra E, Mauro FR, Rossi D, Ghia E, Cerri M, Logan J, Foà R, Gaidano G. (2004). Evidence of biased immunoglobulin

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