The Clinical Utility of LPL and ZAP-70 Expression in Assessment of Prognosis in Patients with B-Cell Chronic Lymphocytic Leukemia

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1 Med. J. Cairo Univ., Vol. 77, No. 1, March [2]: , The Clinical Utility of LPL and ZAP-70 Expression in Assessment of Prognosis in Patients with B-Cell Chronic Lymphocytic Leukemia MOHAMAD R. ALMASRY, M.D.; HALA MAHMOUD, M.D. and SAHAR ELWAKIL, M.D.* The Departments of Internal Medicine and Clinical Pathology*, Faculty of Medicine, Cairo University. Abstract Background: It was found that B-CLL patients with unmutated immunoglobulin heavy chain variable region (IgVH) have poor prognosis and clinical outcome. The difficulty of performing the mutation status of IgHV in the routine diagnostic workup of B-CLL patients, prompts the search for surrogate markers particularly gene expression profile in B- CLL cells. Objective: To investigate the role of LPL and ZAP-70 expression in assessment of prognosis and survival in a group of B-CLL patients and correlate the results with other prognostic variables. Methods: The study included 47 B-CLL patients who were subjected to clinical staging which was done by Binet and Rai scoring systems. The expression of LPL and ZAP-70 was measured by real-time quantitative PCR (RQ-PCR) and flow-cytometric analysis respectively. The patients were followed over 24 months for proper estimation of treatment free survival (TFS) and disease free survival (DFS). Results: The results of our study showed positive LPL and ZAP-70 expression in 46.8% and 48.9% of B-CLL patients respectively. There was significant correlation between LPL and ZAP-70 positive expression in our patients (p<0.0001). The positive expression of both genes is associated with advance in clinical staging with significant correlation between LPL & ZAP-70 positive expression and shortening of the TFS and DFS and subsequent classification of most of the LPL + & ZAP-70 + cases as patients with poor prognosis. Conclusion: Our study showed that expression of LPL and ZAP-70 has a significant role in determining the prognosis in B-CLL patients, being positive expression of LPL and ZAP-70 is associated with poor prognosis with shortening of TFS and DFS. Also, both genes expression is of great value in selection of B-CLL patients in early clinical stages that are in need to start chemotherapy to avoid progression to aggressive forms of the disease. Key Words: ZAP-70 LPL B- CLL. Introduction CHRONIC lymphocytic leukemia (CLL) is a disease caused by the accumulation of monoclonal CD5 + B cell lymphocytes with a characteristic immunophenotype in the bone marrow (BM), peripheral blood and lymphoid tissues. B-CLL predominates in the elderly and has an extremely variable clinical course. The clinical diagnosis of B-CLL requires an absolute lymphocytosis with a lower threshold of greater than 5000 matureappearing lymphocytes/ gl. Three main phenotypic features define B-CLL: The predominant population shares B-cell markers (CD19, CD20 and CD23) with the CD5 antigen, in the absence of other pan- T-cell markers; the B cells are monoclonal with regard to expression of either x or A, light chains and the B cells characteristically express surface immunoglobulin, CD79b, CD20 and CD22 with low density. These characteristics are generally adequate for a precise diagnosis of B-CLL and they also distinguish B-CLL from other disorders such as prolymphocytic leukemia, hairy-cell leukemia, mantle-cell lymphoma and other lymphomas that can mimic B-CLL [1]. The classical Rai [2] or Binet [3] staging systems provided a basis for therapeutic stratification by allocating B-CLL cases into 3 major risk groups (low, intermediate and high), according to tumor burden and the presence of anemia and thrombocytopenia. Asymptomatic patients with a low tumor burden (Binet stage A) do not benefit from treatment with chlorambucil. However, the disease in half of these patients will progress and both staging systems fail to initially identify such patients. Accurate identification of these patients is therefore increasingly important. Normal CD5-positive peripheral blood B cells are characterized by rearranged immunoglobulin (IG) genes with unmutated variable region (V) gene segments [4]. In about 50% of B-CLL patients, it appears that the IGV genes of malignant cells 207

2 208 The Clinical Utility of LPL & ZAP-70 Expression in CLL have undergone somatic hypermutation [5]. It has shown that the presence or absence of immunoglobulin V gene mutations predicts the natural history of the disease. It was found that the presence of unmutated V gene is associated with poor prognosis even within the group of patients in Binet stage A. this suggests that there are two morphologically indistinguishable types of B-CLL; one arise from relatively less undifferentiated (immunologically native) B cells with unmutated heavy chain genes and has a poor prognosis; the other evolves from the more differentiated B cells (memory B cells) with somatically mutated heavy chain genes and has a good prognosis [6]. Despite these clinical and molecular differences, studies on gene expression profiling of B-CLL cells showed that B-CLL is characterized by a common gene expression signature which is irrespective of Ig mutational status and differs from other lymphoid cancers and normal lymphoid subpopulations, suggesting that B-CLL cases share a common mechanism of transformation and/or cell of origin [7], but despite sharing a common signature, CLL cells express few hundreds of genes. In fact these differentially expressed genes can be relevant prognostic factors. Among these genes, over expression of lipoprotein lipase (LPL) and Zeta-chain-associated protein (ZAP-70) are observed in the aggressive unmutated cases [8]. Aim of the study: The objective of the current study is to evaluate the significance of LPL and ZAP-70 expression in the prognosis and survival in a group of patients with B-CLL and correlation of the results with other prognostic factors and the clinical stages of the disease. Patients and Methods The current study included 47 patients (30 males and 17 females) with newly diagnosed B- CLL; they have attended the hematology unit of Internal Medicine department of Kasr Al-Aini hospital, Cairo University. The patients were diagnosed according to the standard morphologic and immunophenotypic criteria. These include peripheral lymphocytes >5x10 9 /L, BM lymphocytes >30%, negative CD3, CD7 and positive CD5, CD19, CD23 and negative FMC7. All patients were subjected to proper history taking and detailed physical examination emphasized on the number of groups of lymph nodes (LN) enlarged, the diameter of the largest one, the presence or absence of hepatomegaly and/or splenomegaly and the presence or absence of any extra lymphatic involve- ment. 15 healthy blood donors used as controls regarding ZAP-70 expression in their normal Bcells and for normal expression levels of LPL. All patients were subjected to CT chest and abdomen for proper staging which was done by Binet and Rai staging systems. The chemotherapy protocol for all patients was mainly based on Fludarabine monophosphate in a dose of 25mg/ m 2 /day for 5 days and all patients completed 6 cycles. The response to therapy was evaluated according to response criteria given by Cheson et al. [9]. The included patients were followed over 24 months duration for proper estimation of treatment free survival (TFS) which is the time from the diagnosis till the start of chemotherapy and disease free survival (DFS) which is the time from remission till new relapse. Three patients died during the course of our study due to illnesses related to the disease. The included patients were investigated by the following laboratory tests: Complete hemogram, BM aspirate and biopsy, serum level of β 2 microglobulin (β 2 m) measured by using Dade Behring turbiditimer (Level is expressed as mg/l), LPL expression level by realtime quantitative PCR (RQ-PCR) and Flowcytometric analysis of ZAP-70 expression level. All samples were collected in a single day for each patient after informed consent. EDTA blood sample was collected for complete hemogram and flowcytometric analysis of ZAP-70. Another EDTA sample on a special tube was used for separation of mononuclear cells for measurement of LPL expression by RQ-PCR. Finally serum sample was collected for β 2 m determination. LPL gene expression by RQ-PCR: Total RNA was extracted using RNA Bee (Campro Scientific). cdna was synthesized from 1ug of RNA using random hexamer priming. cdna prepared from 50ng of RNA was used for all PCR amplifications. The expression level of LPL gene was determined by RQ-PCR. The amplification of RQ-PCR was performed with the ABI PRISM 7700 Sequence Detector using 50ul mix containing 20uM deoxyribonucleo side triphosphate (dntp; Amersham Pharmacia Biotech); 15pmol of both forward and reverse primers for the reference gene Porphobilinogen Deaminase (PBGD); 15pmol of forward and 45pmol reverse primer for LPL gene measured with 1xSYBR Green I dye (Applied Biosystems). The cycling condition for the SYBR Green I reactions were 10 minutes at 95 C followed by 45 cycles of denaturation for 15 seconds at 95 C, annealing at 60 C for 30 seconds and extension at

3 Mohamad R. Almasry, et al C for 30 seconds, the annealing and extension temperature for LPL was 62 C. To quantify the relative expression level of LPL gene in B-CLL, the values were normalized for the endogenous reference PBGD and compared with a calibrator that was chosen for its high expression level of LPL gene. LPL reference sequences NM calibrator cell line HL 60 as follow; FW5 CCGC- CGACCAAAGAAGAGAT-3, RV 5 TTCCTGT- TACCGTCCAGCCAT-3. Reference gene PBGD reference sequences NM calibrator cell line HPB as follow; FW 5 GGCAATGCGGCTG- CAA-3, RV 5' GGGTACCCACGCGAATCAC- 3 and probe 5 CATCTTTGGGCTGTTTTCTTC- CGCC-3'. Flow-cytometric analysis of ZAP-70 expression: 100ul of peripheral blood mononuclear cells of B-CLL patients and controls were stained for 20 minutes at room temperature with CD 19 specific monoclonal antibodies conjugated with phycoerythrin (PE-anti-CD19, MCA 1019 PE Serotec U.K) in the dark. The cells were washed twice and fixed with 4% paraformaldehyde in phosphate-buffered saline and then permeabilized with saponin in Hank s balanced salt solution for 5 minutes at 4 C. The cells were washed and then stained for 45 minutes at 4 C with a monoclonal antibody specific for ZAP-70 (Becton Dickinson). The samples were washed and analyzed by flow-cytometry (BEAK- MAN Coulter Epics XL). The flow-cytometer equipped with argon laser 488nm, software system II version 30. In each case, the dot-plot was gated on the lymphoid gate on the side scatter-forward scatter (SSC-FSC) plot. The expression of ZAP-70 was measured on CD 19 positive cells, patients expressed ZAP-70 on or more than 20% of their CD19 positive cells were considered as ZAP-70 positive. The use of 20% as cutoff value in our analysis was arbitrary and allowed comparison with Rassenti et al. [10]. In each experiment, we used control cells from the healthy donors, CLL cells from one patient with high level of ZAP-70 expression and CLL cells from another patient with low level of ZAP-70 expression. Flourochrome conjugated isotype control monoclonal antibodies of irrelevant specificity were used in all experiments to monitor for nonspecific staining. Flow-cytometric analysis of ZAP- 70 expression is shown in Fig. (1). (A) (B) (C) (D) (E) (F) (G) Fig. (1): Flow-cytometric analysis of ZAP-70 expression in B-CLL patients. Flow-cytometry dot plots using a two-colour method to detect ZAP-70 expression in normal B lymphocytes from healthy blood donor and B-CLL lymphocytes from B-CLL patients. A: A flow-cytometric plot of the forward-angle light scatter and side-angle scatter of blood mononuclear Cells from a healthy donor. Gate is drawn around the cells having the light scatter characteristics of lymphocytes. B: 0.1% expression of ZAP-70 in CD 19 positive cells of normal lymphocytes of healthy blood donor. C: A histogram of staining for ZAP-70 in gated CD19-B cells from healthy blood donor. D, E, F & G: Representative histograms of staining for ZAP-70 in gated CD 19 CLL B cells from four patients with CLL.

4 210 The Clinical Utility of LPL & ZAP-70 Expression in CLL Statistical analysis: Data were statistically described in terms of range, mean, standard deviation ( ± SD) and frequencies (Number of cases) and relative frequencies (percentage) when appropriate. Comparison of quantitative variables between good and poor prognostic groups was done using Mann Whitney U test for independent samples. For comparing categorical data, Chi-square ( χ 2 ) test was performed. Yates correction was used in stead when the expected frequency is less than 5. A probability value (p value) less than 0.05 was considered statistically significant. Receiver operator characteristic (ROC) analysis was done to determine the optimum cut-off level of ZAP-70 in predicting poor prognosis. All statistical calculations were done using computer programs Microsoft Excel version 7 (Microsoft Corporation, IL, USA) and SPSS (Statistical Package for the SocialScience; SPSS Inc., Chicago, IL, USA) statistical program. Results The study included 47 B-CLL patients (30 males and 17 females). All patients were staged clinically by Binet and Rai staging systems. According to Binet staging; the patients were classified as follow; 23 patients (48.9%) were stage A, 18 (38.3%) were stage B and 6 (12.8%) classified as stage C; however with Rai staging; 5 patients (10.6%) were stage 0, 24 (51.1 %) were stage I, 12 (25.5%) were stage II, 4 (8.5%) were stage III and only 2 patients (4.3%) classified as stage IV. The expression of LPL and ZAP-70 genes was assessed in all patients with the cut-off values were 0.7 and 20% respectively. Based on the cut-off point of the LPL expression, the patients were classified into two main groups; group I included 22 B-CLL patients with positive expression of LPL and group II contained 25 B-CLL patients with negative expression of LPL. In group I, hepatomegaly and splenomegaly were found in 77.3% and 81.8% of cases respectively with significant correlation with positive LPL expression with p values and respectively. On clinical staging, 18.2% of patients were Binet stage A, 59.1 % were stage B and 22.7% were stage C; with Rai staging, the patients were classified from stage I to stage IV as follow; 31.8%, 45.5%, 18.2% and 4.5% respectively. This means that there is significant correlation between positive LPL expression and advance in clinical staging. In LPL+ patients, ZAP-70 was positively expressed in 19 (86.4%) patients with significant correlation between positive expression of both differentially expressed genes (p<0.0001). On histopathological examination, the nodular pattern was predominant in patients with LPL positive expression, being elicited in 54.5% of cases, followed by the diffuse pattern in 22.7% of cases, the interstitial pattern in 13.6% of cases and lastly the interstitial/nodular pattern in only 9.2% of patients. There was significant correlation between positive LPL expression and shortening of both TFS and DFS with all p values < The dead patients (n=3) were classified clinically as Binet stage C and Rai stage III, and their genetic profile showed positive expression for LPL and ZAP-70. This finding suggests the potential relationship between positive LPL & ZAP-70 expression and advance in clinical staging and subsequently aggressive forms of the disease. The descriptive characteristics of patients of group I are presented in Table (1). Table (1): The descriptive characteristics of LPL positive patients (n=22). The variable Mean ± SD Sex 13 males / 9 females Age (years) ±8.65 No. of LN enlarged 2.91±0.811 LN diameter (cm) 3.159±0.931 ZAP-70 (%) 32.36± Hb (gm/dl) ± 1.81 WBCs (K/uL) 58.73± Lymphocytes (%) 74.77± Prolymphocytes (%) 6.86± 1.49 BM lymphocytes (%) 46.55±4.95 β 2 microglobulin (mg/l) 5.868±0.53 Platelet count (K/uL) 223.4± Treatment free time (months) 3.32±2.476 Disease free survival (months) 6.86±2.274 In group II, hepatomegaly and splenomegaly were found in 28% and 48% of patients with negative expression of LPL respectively. On clinical staging, 76% of patients were Binet stage A, 20% were stage B and only 4% were stage C; with Rai staging, most of the cases were stage 0 (20%) and stage I (68%) with only 2 cases (8%) were stage II and one patient (4%) classified as stage IV. These data signify that most of the LPL negative cases are within early phases in the clinical staging systems. In LPL patients, ZAP-70 was negatively expressed in 21 (84%) patients with significant correlation between negative expression of both genes (p<0.0001). On histopathological examination, the interstitial pattern was predominant in patients with LPL negative expression; being elicited in 56% of cases followed by the interstitial/ nodular pattern in 32% of cases, nodular pattern in 8% of patients and lastly the diffuse pattern in only one (4%) patient. There was significant cor-

5 Mohamad R. Almasry, et al. 211 relation between negative LPL expression and prolongation of both TFS and DFS with all p values < The descriptive characteristics of patients of group II are presented in Table (2). Table (2): The descriptive characteristics of LPL negative patients (n=25). The variable Mean ± SD Sex 18 males / 7 females Age (years) 60.76±7.81 No. of LN enlarged 1.65± LN diameter (cm) 1.840± ZAP-70 (%) 16.28±7.861 Hb (gm/dl) 11.46± WBCs (K/uL) 45.76± Lymphocytes (%) 60.80± Prolymphocytes (%) 4.84± BM lymphocytes (%) 40.84±3.826 β 2 microglobulin (mg/l) 3.564± 1.3 Platelet count (K/uL) 247.4±73.29 Treatment free time (months) 9.68±3.891 Disease free survival (months) 8.68± We studied the significance of all variables including clinical staging, LPL and ZAP-70 expression levels in determining the prognosis of all B-CLL patients included in the study. According to the descriptive statistics, the patients are classified into good prognosis group (n=34) and poor prognosis group (n=13). There was no significant correlation between the age of the patients and the prognosis. The presence of hepatomegaly is more predictive for poor prognosis (p=0.148) than splenomegaly (p=0.053). In patients with good prognosis, 64.7% were Binet stage A, 38.3% were stage B and 12.8% were stage C; while in patients of poor prognosis, only 7.7% were stage A, 53.8% were stage B and 38.5% were classified as stage C. Furthermore, by Rai staging the patients with good prognosis were classified from stage 0 to stage IV as follow: 14.7%, 55.9%, 26.5%, 2.9% and 0% respectively; while patients with poor prognosis were classified as follow: 0%, 38.5%, 23.1%, 23.1% and 15.4% respectively. The interstitial histopathological pattern was prevalent in patients with good prognosis (44.1%), while the nodular (38.5%) and diffuse (23.1%) patterns were predominant in patients with poor prognosis. The value of LPL and ZAP-70 expression in determination of the patient s prognosis is thoroughly investigated in both groups. In 34 patients with good prognosis, only 13 (38.2%) patients were LPL positive and 21 (61.8%) patients were LPL negative. In 17 patients with poor prognosis, 9 (69.2%) patients were LPL positive and 4 (30.8%) patients only were LPL negative. There was significant correlation between LPL negativity and good prognosis and between LPL positivity and poor prognosis with the overall p value= On the other hand, in patients with good prognosis, ZAP-70 was positively expressed in only 14 (41.2%) patients and negatively expressed in 20 (58.8%) patients, while in patients with poor prognosis, 9 (69.2%) patients were ZAP-70 positive and 4 (30.8%) patients were ZAP-70 negative. There was significant correlation between ZAP- 70 negativity and good prognosis and between ZAP-70 positivity and poor prognosis with the overall p value= The descriptive statistics of patients of both good and poor prognosis are summarized in Tables (3,4) respectively. Table (3): The descriptive statistics of patients with good prognosis (n=34). The variable Mean ± SD Age (years) 62.41±7.660 LPL ± ZAP ± No. of LN enlarged 1.91 ± 1.24 LN diameter (cm) 2.162± Hb (gm/dl) ±0.91 WBCs (K/uL) 50.53± 13.9 Lymphocytes (%) 65.12± 15.6 Prolymphocytes (%) 5.53±2.08 BM lymphocytes (%) 42.74±4.94 β 2 microglobulin (mg/l) 4.329± 1.51 Platelet count (K/uL) ±78.9 Treatment free time (months) 7.71 ±4.36 Disease free survival (months) 8.91 ± 1.42 Table (4): The descriptive statistics of patients with poor prognosis (n=13). The variable Mean ± SD Age (years) 62.62± LPL ±0.164 ZAP ± No. of LN enlarged 2.92±0.760 LN diameter (cm) 3.231±0.665 Hb (gm/dl) 9.662±2.211 WBCs (K/uL) 55.23±21.46 Lymphocytes (%) 73.15± Prolymphocytes (%) 6.46± 1.45 BM lymphocytes (%) 45.54±5.53 β 2 microglobulin (mg/l) 5.462± Platelet count (K/uL) ± Treatment free time (months) 4.08±4.32 Disease free survival (months) 5.00± 1.00

6 212 The Clinical Utility of LPL & ZAP-70 Expression in CLL Discussion B-CLL is heterogenous with a continuous spectrum of disease; at one extreme are patients who have an almost normal life expectancy with no need for treatment; at the other are patients who die of drug resistant disease as early as 2 years after initial diagnosis. Therefore, reliable prognostic markers are of utmost importance for determining if early treatment is meaningful for all younger patients or at least for a high risk subgroup of such patients. Addition to the current clinical staging systems such as the Rai and Binet score for prognostic assessment of CLL patients, the assessment of biological and genetic markers at diagnosis has provided more accurate prediction of disease outcome [11]. The presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of CLL B cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Igunmutated B-CLL) often have progressive disease whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated B-CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. The difficulty of performing the mutation status of IGHV in the routine diagnostic workup of CLL patients, prompts the search for surrogate markers particularly gene expression profile in CLL cells. So, the aim of our study was to assess the expression levels of two differentially expressed genes namely; LPL and ZAP-70 in a group of B-CLL patients and to evaluate the significance of both genes expression in determining the prognosis and survival of the included B-CLL patients. LPL is a heparin-releasable enzyme bound to glycosaminoglycan components of the capillary endothelium and is particularly abundant in muscle, adipose tissue and macrophages. With apolipoprotein CII, LPL mediates the hydrolysis of triacylglycerol components of circulating chylomicrons and very-low-density-lipoproteins and plays a central role in lipid metabolism and transport [16]. Mutations in the LPL gene are frequently associated with dyslipidemia and atherosclerosis [17]. LPL is not expressed in normal purified B and T lymphocytes, but it has been found to be expressed and secreted by natural killer (NK) cells where it was shown to modulate their cytotoxic activity [18]. The reasons for high LPL expression in unmutated CLL B cells are not known, however; because unmutated CLL B cells have been shown to be more responsive to stimulation through their antigen receptors, it has been speculated that LPL could play a role in lipid raft formation or stabilization, a biologic process known to be important in B- cells activation [19]. ZAP-70 is a receptor associated protein tyrosine kinase, usually found in T lymphocytes and NK cells, but not in normal B cells. High levels of this protein are detected in the majority of unmutated B-CLL cases [12]. Chen et al., demonstrated that CLL B cells that express ZAP-70 are more likely to respond to IgM cross-linking with increased tyrosine phosphorylation and calcium influx than ZAP-70 negative CLL B cells [13]. This suggests that the expression of ZAP-70 in B-CLL allow more effective IgM signaling in CLL B cells, a feature that could contribute to the more aggressive course observed in these forms. The expression of ZAP-70 can be assessed by different methods, such as western blotting, real-time quantitative PCR (RQ-PCR), flow-cytometry and immunohistochemistry. Flow-cytometry is the most useful technique, as it is a precise and fast methodology extensively used in routine diagnosis of hematological malignancies [14]. The assessment of ZAP-70 in neoplastic CLL B cells by flow-cytometry implies multiparametric staining to indepently identify CLL B cells from normal residual T and NK cells. For this reason, antigens able to recognize CLL B cells and both T and NK cells are used in combination with an intracellular staining for the ZAP-70 protein [8]. Also, this technique is simplified by using directly labeled anti-zap-70 monoclonal antibodies and/or flourochrome-conjugated secondary antibodies specifically recognizing the isotype of the anti-zap-70 monoclonal antibody [15]. The results of our study showed positive LPL and ZAP-70 expression in 46.8% and 48.9% of patients respectively. There was significant correlation between both genes expression in our B- CLL patients (p<0.0001). The positive expression of both genes is associated with advance in clinical staging with significant correlation between LPL & ZAP-70 positive expression and shortening of the TFS and DFS and subsequent classification of most of the LPL + & ZAP-70+ cases as patients with poor prognosis. Conversely, the negative expression of both genes categorized the patients in the early phases of clinical staging with significant prolongation of TFS and DFS and subsequent classification of most of the LPL & ZAP case as patients with good prognosis. Our study reported that the predominant histopathological patterns in patients with poor prognosis are similar to the

7 Mohamad R. Almasry, et al. 213 patterns elicited in ZAP-70 + & LPL+ patients, also the predominant patterns in patients with good prognosis are similar to the patterns detected in LPL& ZAP patients One of the interesting find ings in our study that despite four patients categorized clinically as Binet A & Rai I, they showed positive expression profile for both genes and one of them was included in the poor prognosis group. This reflects the prognostic significance of positive LPL and ZAP-70 expression in selection of B-CLL patients especially in early clinical phases that are in need to start early treatment to avoid progression to aggressive disease forms. The role of ZAP-70 in evaluation of IgVH mutational status in B-CLL patients was previously investigated. Wiestner et al., reported that ZAP- 70 expression correctly predicted IgVH mutation status in 93% of patients and the authors concluded that both ZAP-70 and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis [20]. Another study conducted on 42 B-CLL patients showed that 94% of patients with unmutated IgVH were ZAP-70 positive and 92% of patients with mutated IgVH were ZAP-70 negative [21]. The value of ZAP-70 expression together with other prognostic factors in prediction of the clinical outcome in B- CLL patients was previously studied. Stamatopoulos et al studied the expression of ZAP-70, CD38 and LPL in a cohort of 108 B-CLL patients and evaluated the association with IgVH mutational status, overall survival (OS) and TFS. The study showed that ZAP-70 expression was significantly associated with OS (p=0.0021) & TFS (p<0.0001). ZAP-70+ patients had significantly shorter median TFS (24 months) than ZAP-70 patients (157 months) (p<0.0001). Moreover, the measured ZAP- 70 expression has greater prognostic power than IgVH mutational status and the other prognostic markers tested [22]. The reliability of measurement of ZAP-70 by flow-cytometry which was the method selected in our study was previously investigated. Crespo et al., analyzed the ZAP-70 expression in T cell and B cell lines and in peripheral blood samples from 56 B-CLL patients by flow-cytometry, western blotting and immunohistochemistry with correlation of the results with IgVH mutational status and clinical outcome. ZAP-70 was detected by flowcytometric analysis in cells of T cell lineage and in leukemic cells from 32 of 56 patients with B- CLL. Concordant results were obtained when ZAP- 70 expression was assessed by the other two methods and the authors concluded that among patients with B-CLL, expression of ZAP-70 as detected by flow-cytometric analysis, correlated with IgVH mutational status, disease progression and survival [8]. The value of the differentially expressed LPL gene as a novel prognostic marker in B-CLL is recently studied. The prognostic significance of LPL expression detected in our B-CLL patients is concomitant with the findings presented by Xu et al., who investigated LPL expression in 58 Chinese B-CLL patients and its correlation with other prognostic factors, including IgVH mutational status, Binet stages, ZAP-70 protein and CD38 expression levels. The authors reported that LPL expression level was significantly correlated with Binet stages (p=0.036), ZAP-70 expression (p=0.001) and the higher LPL expression level was also associated with higher ZAP-70 expression level and more aggressive Binet stage [23]. Furthermore, Nuckel et al., studied the prognostic role of LPL and ADAM29 genes expression in a cohort of 133 B- CLL patients and correlated the results with clinical outcome and other known prognostic factors. The study showed that LPL provided prognostic information in both early stage (Binet A) and patients with more advanced disease (Binet B and C). Conversely, high ADAM29 expression was predictive of a long treatment free interval in Binet stage A but did not retain its prognostic significance in Binet B and C patients; collectively the data confirm a role for LPL as a novel prognostic indicator in B-CLL [24]. The prognostic significance of LPL and ZAP- 70 genes compared to other commonly differentially expressed genes in B-CLL was studied. Brooijmans et al selected ten genes including LPL and ZAP-70 and tested with RQ-PCR in unpurified samples from 130 B-CLL patients. The selected genes showed prognostic significance, however; in multivariate logistic regression analysis, expression levels of LPL, ZAP-70, ADAM29 and SEPT 10 were the most predictive for IgVH mutational status. In univariate analysis, the LPL was the best predictor and for survival, expression of LPL was the strongest prognostic factor. Finally the study concluded that LPL expression is a predictor for survival in B-CLL and for this purpose is as good as IgVH mutational status and even more reliable than ZAP-70 expression when tested in unpurified B-CLL samples [25]. Also, Van Bockstaele et al., used a quantitative PCR method for determining LPL mrna expression and analyzed samples of lysed whole blood and CD19 selected cells from 50 B-CLL patients. The authors investigated the associations of LPL and ZAP-70 expression with OS and TFS. As shown in our study, the results

8 214 The Clinical Utility of LPL & ZAP-70 Expression in CLL revealed that LPL expression was significantly associated with both OS and TFS in log-rank tests (both p value=0.002) and LPL-positive patients had a significantly shorter median TFS time (23 months) than LPL-negative (88 months). The study concluded that expression of LPL deserves to be a valuable prognostic marker in B-CLL [26]. In conclusion, the clinical course of individual B-CLL is highly variable, with life expectancies ranging from months to decades. Importantly, a significant subset of patients presents with low grade B-CLL, but will nevertheless develop a more aggressive and life-threatening disease. As these patients may potentially benefit from early treatment, it is crucial to assess patient s prognosis at diagnosis, allowing individual risk-adapted therapy. For instance, the expression of LPL and ZAP-70 is much more laborious to investigate than is IgVH mutational status. We concluded that expression of LPL and ZAP-70 has a significant role in determining the prognosis in B-CLL patients, being positive expression of LPL and ZAP-70 is associated with more advanced clinical stages and poor prognosis with shortening of TFS and DFS. Also, both genes expression is of great value in selection of CLL patients in early clinical stages that are in need to start chemotherapy to avoid progression to aggressive forms of the disease. References 1- DIGHIERO G.: Unsolved issues in CLL biology and management. Leukemia, 17: , RAI K.R., SAWITSKY A., CRONKITE E.P., et al.: Clinical Staging of chronic lymphocytic leukemia. Blood, 46: , BINET J.L., LEPOPRIER M., DIGHIERO G., et al.: A clinical staging system for chronic lymphocytic leukemia: Prognostic significance. Cancer, 40: , OSCIER D.G., THOMPSETT A., ZHU D., et al.: Differential rates of somatic hypermutation in V (H) genes among subsets of chronic lymphocytic leukemia defined by chromosomal abnormalities. 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