Significance of zeta-associated protein (ZAP-70) and CD38 expression in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a disease with a highly variable clinical course; some patients never need treatment, while others require intensive treatment early after diagnosis. Some new prognostic factors, such as immunoglobulin variable heavy chain (IgVH) mutational status, zeta-associated protein (ZAP-70) and CD38 expression in leukemic cells were introduced to identify attenuated versus progressive types of CLL bearing the potential to facilitate risk-adapted treatment strategies. So, the aim of this work is to evaluate the clinical value of ZAP-70 and CD38 as predictors of disease progression. We assessed the expression of these markers by flowcytometry in 38 patients with CLL and correlated their levels with genetic abnormalities detected by fluorescence in situ hybridization (FISH) and the clinical outcome. We found that 18 patients (47.4 %) were positive for ZAP-70 (> or = 20%) and 16 patients (42.1%) were positive for CD38 (> or = 20%). Positive ZAP-70 and CD38 status were associated with an unfavorable clinical course including high leukocytic count, lymphocytosis, high lactate dehydrogenase (LDH) serum level, advanced disease stage, trisomy 12 and del (11q); negative ZAP-70 and CD38 status were correlated with del (13q). The treatment-free survival time was 30 months for ZAP-70-positive patients and 18 months for ZAP-70-nagative patients (p < 0.01). Combined analysis of ZAP-70 and CD38 yielded discordant results in 10 patients (26.3 %), whereas 16 patients (42.1%) were concordantly negative and 12 patients (31.6%) were concordantly positive for ZAP-70 and CD38 expression. Median treatment-free survival times in patients whose leukemic cells were ZAP-70+CD38+ was 27 months as compared to 100 months in patients with a ZAP-70(-)CD38(-) status. In patients with discordant ZAP-70/CD38 results, the median treatment-free survival time was 40 months. Thus, ZAP-70 and CD38 expression analyses provide complementary prognostic information and allow distinguishing the patients groups with the most favorable prognosis as well as those with the worst. The current findings suggest that both ZAP-70 and CD38 protein expression should be assessed in patients with CLL for the definition of prognostic subgroups.     

The effect of IgVH mutational status on the induction of apoptosis by rituximab in patients with heavily pretreated B-cell chronic lymphocytic leukemia: evidence from a clinical phase I/II trial

We investigated tumor cell apoptosis in vivo in 14 heavily pretreated patients with B-cell chronic lymphocytic leukemia undergoing rituximab monotherapy. Apoptosis induction was more pronounced in patients with mutated IgVH genes than in those with unmutated IgVH genes, independently of the levels of expression of CD20, CD38, and ZAP-70 and of the presence of 17p deletion. Our results suggest an association between IgVH gene mutational status and rituximab-induced apoptosis.     

Investigating the role of CD38 and functionally related molecular risk factors in the CLL NOD/SCID xenograft model

We explored the role of CD38 and functionally associated molecular risk factors in a recently described chronic lymphocytic leukemia (CLL) nonobese diabetic/ severe combined immunodeficient xenograft model. Intravenous injection of peripheral blood mononuclear cells from 73 patients with CLL into 244 mice resulted in robust engraftment of leukemic cells into the murine spleens detected 4 wks after transplantation. Leukemic cell engraftment correlated significantly (P < 0.05) with markers reflecting disease activity, e.g., Binet stage and lymphocyte doubling time, and the expression of molecular risk factors including CD38, CD49d, ZAP-70, and IgVH mutational status. Increased engraftment levels of CD38+ as compared to CD38- CLL cells could be attributed, in part, to leukemic cell proliferation as evidenced by combined immunostaining of murine spleen sections for Ki-67 and CD20. In short-term (24 h) homing assays, CD38+ CLL cells migrated more efficiently to the bone marrow of the recipient animals than their CD38- counterparts. Finally, CD38 expression by the leukemic cells was found to be dynamic in that it was regulated not only by elements of the murine microenvironment but also by co-engrafting non-malignant human T cells. This model could be useful for evaluating the biological basis of CLL growth in the context of the hematopoietic microenvironment as well as preclinical testing of novel compounds.     

Quantification of ZAP-70 expression in chronic lymphocytic leukemia: T/B-cell ratio of mean fluorescence intensity provides stronger prognostic value than percentage of positive cells

Expression of ZAP-70 measured by flow cytometry belongs to the most powerful prognostic parameters in chronic lymphocytic leukemia (CLL). However, many technical factors such as setting of the positivity threshold may significantly influence results.. Quantification using mean fluorescent intensity (MFI) may eliminate the subjective error which is inevitable in the isotype control method. The aim of the present project was therefore to assess the prognostic significance of ZAP-70 using three different methods. Between 2005 and 2010 we measured ZAP-70 expression in 157 patients with CLL (108 males, 49 females, median age 60 years [range, 31-82]; low/intermediate/high Rai risk in 41/48/11%). Expression of ZAP-70 was determined by flow cytometry using phycoerythrin (PE)-conjugated monoclonal antibody, clone 1E7.2. Evaluation was performed by 1) percentage of positive cells compared to isotype control (cut-off 20%), 2) MFI ratio of T-cells/CLL cells (cut-off 3.0); 3) MFI ratio of ZAP-70/isotype control on CLL cells (cut-off 2.5). MFI method with T-cells/CLL cells ratio was the best in the identification of patients with unfavourable outcome: ZAP-70 positive patients had significantly shorter time to treatment (TTT, median 24 vs. 55 months, p=0.0001) and overall survival (OS, median 97 vs 174 months, p=0.0074). The differences in TTT a OS were not significant with the use of isotype percentage and MFI isotype methods. Combined analysis of ZAP-70 with CD38 expression or IgVH mutation status lead to identification of a subgroup with the longest TTT and OS (ZAP-70 and CD38 negative, p<0.0001 and p=0.012; ZAP-70 negative and mutated IgVH genes, p<0.0001 and p=0.0019). In conclusion, our results suggest that measurement of ZAP-70 expression in CLL by MFI using T-cells/CLL cells ratio might be the optimal method for accurate prediction of clinical course. Combined analysis of ZAP-70 with CD38 or IgVH mutation status further refined individual patient′s prognosis.     

慢性淋巴细胞白血病血清可溶性CD23及血小板生成素研究

目的 探讨慢性淋巴细胞白血病(CLL)患者血清可溶性CD23(sCD23)及血小板生成素(TPO)水平及与其他预后指标的相关性.方法 采用酶联免疫吸附试验检测25例CLL患者外周血标本中sCD23及TPO的水平;流式细胞术检测ZAP-70蛋白及CD28的表达.结果 CLL患者TPO水平为67.22～1881.77 ng/L,明显高于正常对照组70.29～147.98 ng/L(P=0.003);CLL患者血清sCD23水平为129.80～405.31 U/ml,也明显高于正常对照组0.65～32.99 U/ml(P=0.000).血清TPO水平与Binet分期、CD28具有显著相关性.Binet A期患者TPO水平为121.92～163.83 ng/L,低于BinetB和C期患者140.57～457.48 ng/L(P=0.014);CD38高表达组TPO水平113.23～199.10 ng/L,高于CD38低表达组141.34～454.92 ng/L(P=0,033).而TPO与ZAP-70表达及sCD23与CD28、ZAP-70表达无明显相关性.另外,血清sCD23及TPO与患者性别、年龄、外周血淋巴细胞计数和乳酸脱氢酶均无相关性.结论 血清TPO水平对CLL预后判断可能具有一定的价值.     

Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia

CD49d/alpha4-integrin is variably expressed in chronic lymphocytic leukemia (CLL). We evaluated its relevance as independent prognosticator for overall survival and time to treatment (TTT) in a series of 303 (232 for TTT) CLLs, in comparison with other biologic or clinical prognosticators (CD38, ZAP-70, immunoglobulin variable heavy chain (IGHV) gene status, cytogenetic abnormalities, soluble CD23, beta2-microglobulin, Rai staging). Flow cytometric detection of CD49d was stable and reproducible, and the chosen cut-off (30% CLL cells) easily discriminated CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (P<.001) and ZAP-70 (P<.001), or with IGHV mutations (P<.001), was independent prognosticator for overall survival along with IGHV mutational status (CD49d hazard ratio, HRCD49d=3.52, P=.02; HRIGHV=6.53, P<.001) or, if this parameter was omitted, with ZAP-70 (HRCD49d=3.72, P=.002; HRZAP-70=3.32, P=.009). CD49d was also a prognosticator for TTT (HR=1.74, P=.007) and refined the impact of all the other factors. Notably, a CD49dhigh phenotype, although not changing the outcome of good prognosis (ZAP-70low, mutated IGHV) CLL, was necessary to correctly prognosticate the shorter TTT of ZAP-70high (HR=3.12; P=.023) or unmutated IGHV (HR=2.95; P=.002) cases. These findings support the introduction of CD49d detection in routine prognostic assessment of CLL patients, and suggest both pathogenetic and therapeutic implications for CD49d expression in CLL.     

Baff serum level predicts time to first treatment in early chronic lymphocytic leukemia

We analyzed the correlation between well‐established biological parameters of prognostic relevance in B‐cell chronic lymphocytic leukemia (CLL) [i.e. mutational status of the immunoglobulin heavy chain variable region (IgVH), ZAP‐70 and CD38 expression] and serum levels of B cell–activating factor (BAFF of the TNF family) by evaluating the impact of these variables on the time to first treatment (TFT) in a series of 169 previously untreated CLL patients in Binet stage A. Higher levels of BAFF were more frequently associated with female gender (P = 0.02), younger age (P = 0.01), Rai stage 0 (P = 0.002), higher platelet count (P = 0.005), mutated IgVH disease (P = 0.002), higher occurrence of normal cytogenetic profile or presence of 13q deletion (P = 0.02), low ZAP‐70‐ (P = 0.003), and CD38‐expression (P = 0.02). Maximally selected log‐rank statistic plot identified a serum BAFF concentration of 0.313 ng/mL as the best cut‐off (P < 0.0001). This threshold recognized two subsets of patients with different TFT (P < 0.0001). Because in multivariate analysis soluble BAFF [Hazard ratio (HR), 8.23; confidence Interval (CI) 95%,3.0–22.6, P < 0.0001] and mutational status of IgVH (HR = 2.60; CI 95% 1.10–6.14, P = 0.03) maintained the discriminating power their combined effect on clinical outcome was assessed. When three groups were considered: ( 1 ) low‐risk (n = 93), patients with concordant IgVH_mut and higher soluble BAFF; ( 2 ) intermediate‐risk (n = 50), patients with IgVH_mut and low BAFF levels or IgVH_unmut and soluble higher BAFF;( 3 ) high‐risk (n = 26), patients with concordant IgVH _unmut and low soluble BAFF, the 2‐yr TFTs were, respectively, 95%, 85%, and 41% (P < 0.0001). In conclusion, our results indicate that in early B‐cell CLL, the biological profile including among other parameters soluble BAFF may provide a useful insight into the complex interrelationship of prognostic variables.     

ZAP-70 expression is associated with enhanced ability to respond to migratory and survival signals in B-cell chronic lymphocytic leukemia (B-CLL)

Molecular markers like IgV(H) mutational status, chromosomal abnormalities, and CD38 and ZAP-70 expression have prognostic value in B-cell chronic lymphocytic leukemia (B-CLL). These may be pathogenetic because of the coincidental expression of ZAP-70 and increased B-cell receptor (BCR) signaling and the signaling function of CD38 in CLL. This study shows that ZAP-70(+) CLL B cells respond in vitro more readily than ZAP-70(-) CLL and normal B cells to chemokine migratory signals through enhanced surface CCR7 expression (P = .009; P < .001) and increased responsiveness to its ligands CCL19 and CCL21, demonstrated by F-actin polymerization (P < .05) and cellular migration (P < .01). In addition, ZAP-70(+) CLL cells exhibit sustained ERK phosphorylation/activation following stimulation with CXCL12 (SDF1-alpha, a survival factor produced by stromal cells) compared with ZAP-70(-) cells (P = .004). Following coculture with nurse-like cells, the survival of ZAP-70(+) but not ZAP-70(-) CLL cells is significantly enhanced by the addition of CXCL12 (P < .05), an effect that is partially blocked by the MEK inhibitor PD98059. These advantageous migratory and survival responses may promote easier access to and greater proliferation in pseudo-germinal centers and explain in part the more progressive nature of ZAP-70(+) disease.     

Quantification of ZAP-70 mRNA by real-time PCR is a prognostic factor in chronic lymphocytic leukemia

Background

Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. Mutational status of immunoglobulin heavy chain variable region (IGHV) appears to be a particularly strong prognostic marker, but it is difficult to perform in a routine clinical laboratory. ζ-chain-associated protein kinase 70?kDa (ZAP-70) protein detected by flow cytometry is a strong surrogate marker of IGHV mutational status; however, it suffers from the lack of standardization.

Methods

We investigated whether ZAP-70 mRNA expression level can be a prognostic factor in CLL. Real-time quantitative polymerase chain reaction was used to analyze ZAP-70 mRNA expression from 102 CLL patients.

Results

The expression of ZAP-70 mRNA was significantly associated with Binet stage (P?P?=?0.003), ZAP-70 protein (P?=?0.018), IGHV mutational status (P?=?0.038), and cytogenetic abnormality of del(17p13) or del(11q22.3) (P?=?0.037) in CLL patients. According to receiver operating characteristic curve analysis for ZAP-70 mRNA and ZAP-70 protein, positive ZAP-70 mRNA (P?=?0.006) was an adverse factor in determining the treatment free survival (TFS). In a multivariate Cox analysis of TFS, ZAP-70 mRNA is not ideal as an independent prognostic factor. However, ZAP-70 mRNA was statistically significant in predicting treatment response.

Conclusion

This study demonstrated the value of determination of ZAP-70 mRNA in providing useful prognostic information in CLL patients. However, ZAP-70 mRNA is not an independent prognostic factor.     

实时定量RT-PCR检测慢性淋巴细胞白血病正调节基因1表达及临床意义

目的 研究慢性淋巴细胞白血病(CLL)正调节基因1(CLLU1)在CLL患者的表达特征,探讨CLLU1在CLL预后中的意义.方法 采用实时定量RT-PCR(qRT-PCR)检测41例CLL患者外周血标本CLLU1的表达.以3-磷酸甘油醛脱氰酶(GAPDH)为内参、2~(-△Ct)方法计算CLLU1的相对定量值.组间对比采用秩和检验,Spearman相关分析CLLU1表达水平与性别、年龄、临床分期、IgVH基因突变、CD_(38)和ZAP-70蛋白的关系.结果 qRT-PCR标准曲线相关系数R值均>0.99,敏感度可以检测到102个拷贝/μgRNA,批内差、批间差分析结果其变异系数(CV)均<5%.41例患者CLLU1表达水平中位数为0.139(0～5256.912).CLLU1表达水平与临床分期、IgVH基因突变、CD_(38)密切相关,Binet B+C期患者CLLU1表达水平明显高于Binet A期患者(P=0.040),无IgVH突变患者的CLLU1表达水平高于IgVH突变患者(P=0.021),CD_(38)阳性患者中CLLU1表达水平较阴性者高(P=0.045).结论 采用qRT-PCR方法检测CLLU1表达敏感可靠.CLLU1表达水平与CLL临床分期和重要预后因素IgVH基因突变及CD_(38)密切相关,并可较准确地预测IgVH突变情况,在CLL的预后中具有重要价值.     

ZAP-70 in B-cell chronic lymphocytic leukemia: clinical significance and methods of detection

B-cell chronic lymphocytic leukemia (B-CLL) represents a heterogeneous disease with highly variable prognosis. Clinical staging systems (Rai, Binet) fail to accurately predict the prognosis of individual patients, especially in early stages. Modern prognostic markers, mainly the mutational status of the variable regions of immunoglobulin heavy chains (IgVH) and genetic aberrations, allow more accurate risk stratification. Assessment of the expression of intracellular protein tyrosine kinase ZAP-70 represents not only a potential surrogate marker for the technically difficult and routinely unavailable assessment of the IgVH mutational status, but might also be an independent prognostic factor. Study of ZAP-70 function in B-cells has broadened our knowledge on the pathogenesis of B-CLL. Routine ZAP-70 assessment has been hindered so far mainly by the lack of harmonization and standardization of the available methods of detection.     

New prognostic markers in chronic lymphocytic leukemia

The prognosis of patients with chronic lymphocytic leukemia (CLL) is extremely variable. Prognostication of patients with CLL has been classically based on clinical parameters. In the last few years, several biologic markers such as cytogenetics, IgVH mutations, CD38 and ZAP-70 expression in leukemic cells have shown to offer important prognostic information. However, before being incorporated into daily practice these markers require standardization and validation in prospective trials. Meanwhile, prognosis of patients with CLL should remain to be based on clinical stages and other easily obtainable clinical parameters. An important area of research is the identification of markers useful for predicting response to therapy. Among them, 17p- reflecting p53 abnormalities is particularly important. Also relevant is 11q- pointing out to ATM defects. The correlation of IgVH mutations, ZAP-70 and CD38 expression with response is unclear and needs further investigation. Finally, there is increasing evidence that response to therapy, particularly when all measurable disease is eradicated, is associated with longer survival.     

Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia

The clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is variable, and novel biologic parameters need to be added to the clinical staging systems to predict an indolent or aggressive outcome. We investigated the 70-kDa zeta-associated protein (ZAP-70), CD38, soluble CD23 (sCD23), and cytogenetics in 289 patients with B-CLL. Both a shorter progression-free survival (PFS) and overall survival (OS) were observed in ZAP-70(+) (P < .001), in CD38(+) (P < .001) and in sCD23(+) patients (P < .001 and P = .013, respectively). ZAP-70(+)CD38(+) or ZAP-70(+) patients with an unmutated IgV(H) status showed both a shorter PFS (P < .001) and OS (P < .001 and P < .001, respectively) as compared with ZAP-70(-)/CD38(-) or ZAP-70(-) patients with mutated IgV(H) genes. Discordant patients showed an intermediate outcome. Note, ZAP-70(+) patients even if CD38(-) or mutated showed a shorter PFS, whereas ZAP-70(-) patients even if CD38(+) or unmutated had a longer PFS. Furthermore, ZAP-70 positivity was associated with a shorter PFS both within normal karyotype (P < .001) and within the poor-risk cytogenetic subset (P = .02). The predictive value of ZAP-70 expression was confirmed in multivariate analysis. Thus, ZAP-70 protein determined by flow cytometry improves the prognostic significance of cytogenetics and appears to be a better predictor of outcomes than IgV(H) gene mutational status. On this line, we recommend and are also interested in conducting a prospective randomized trial of early intervention versus observation for ZAP-70(+) patients.     

Impact of CD39 expression on CD4+ T lymphocytes and 6q deletion on outcome of patients with chronic lymphocytic leukemia

Objective/Background

Chronic lymphocytic leukemia is one of the commonest leukemias affecting adults. CD39 inhibits T-cell and Natural killer (NK) cell responses by hydrolyzing adenosine triphosphate and adenosine diphosphate, suppressing the immune system. We investigated expression of CD39 on CD4+ T Lymphocytes in chronic lymphocytic leukemia (CLL) patients and its relationship with deletion 6q, its association with disease stage and survival.

Discordance of ZAP-70 in patients with chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia has a highly variable clinical course with behavior ranging from indolent to aggressive. Identified prognostic markers include IgV(H) mutational status, and CD38 and ZAP-70 expression. In several studies, ZAP-70 expression correlated with IgV(H) mutational status, and predicted disease progression and overall survival. In addition to its prognostic utility, ZAP-70 expression was found to be constant over time, and did not vary between peripheral blood, bone marrow or lymph node specimens in individual patients. In contrast to these reports, we present three CLL patients with discordant ZAP-70 levels. One demonstrated a change in expression with time; the second and third cases had discordant results in blood, lymph node and bone marrow and between blood and bone marrow specimens, respectively, obtained at the same time.