Combined analysis of ZAP-70 and CD38 expression in sudanese patients with B-cell chronic lymphocytic leukemia

Objective To investigate the ZAP-70 and CD38 expressions and their combined expressions in Sudanese B-CLL patients and their relationships with clinical and hematological characteristics as well as the disease staging at presentation. Results In the present cross-sectional descriptive study, analysis of ZAP-70 expression showed that 36/110 (32.7%) patients positively expressed ZAP-70 and insignificant higher presentation in intermediate and at advanced stages as well as no correlation was seen with hematological parameters and clinical features compared with negatively ZAP-70, on the other hand, 41/110 (37.3%) were CD38+ and no significant correlation was shown with the stage at presentation, clinical characteristics (except Splenomegaly, P = 0.02) and hematological parameters. However, in combined expressions of both ZAP-70 and CD38 together, 20/110 (18.2%) were concordantly ZAP-70+/CD38+, 53/110 (48.2%) concordantly ZAP-70−/CD38− and 37/110 (33.6%) either ZAP-70+ or CD38+, and these three groups showed insignificant correlation with clinical (except Splenomegaly, P = 0.03) and hematological parameters, and the stage at presentation. Our data showed the combined analysis of these two markers, lead to classify our patients into three subgroups (either concordant positive, negative or discordant expressions) with statistically insignificant correlation with clinical presentation (except Splenomegaly), hematological parameters and stage at presentation of B-CLL patients. Electronic supplementary material The online version of this article (10.1186/s13104-019-4319-8) contains supplementary material, which is available to authorized users.


Introduction
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by progressive accumulation of monoclonal, small, mature-appearing CD5 + B-cells in the peripheral blood, bone marrow and secondary lymphoid tissue [1]. CLL is the most common leukemia in adults in western countries and it is more common in males [2,3]. B-CLL is a heterogeneous disorder characterized by a variable clinical course [4]. In a continual effort to identify patients with poor prognosis and to facilitate the clinical management of B-CLL [5].
Staging systems have delineated the clinical presentation and natural history of B-CLL and have allowed predicting survival and treatment requirements [6,7]. However, the staging systems lack the ability to distinguish prospectively patients with early stage B-CLL that will rapidly progress to aggressive disease from patients destined to remain in early stage for a long period of time [4].
The presence or absence of somatic mutations in the (IgVH) of B-CLL cells has been described as one of the most powerful prognostic factors distinguishing two disease subsets [8,9]. The cases with mutated (IgVH) genes exhibit a favorable clinical course and they may never require treatment [10]; while patients with unmutated (IgVH) genes are characterized by a reduced survival and responsiveness to chemotherapy [8,11,12]. However, determination of (IgVH) mutation is based on DNA sequencing which is not always available for routine clinical use. So, several surrogate markers [13], that correlate with (IgVH) mutational status have been identified. Moreover, CD38 and ZAP-70 expressions have been proposed as less expensive and time-consuming surrogates for (IgVH) mutational status [14,15]. More recently the combined of ZAP-70 and CD38 expressions analysis provided complementary prognostic information to identify three patient subgroups with good, intermediate and poor prognosis [16]. Many studies showed that (CD38 − /ZAP-70 − ) group of patients with good prognosis, (CD38 + /ZAP-70 + ) group with poor prognosis, and group (CD38 + /ZAP-70 − ) or CD38 − /ZAP-70 + ) with intermediate prognosis [16,17].
To the best of our knowledge, this is the first study conducted in Sudan to investigate the combined expressions of both ZAP-70 and CD38 in B-CLL patients and their Correlation with clinical features, hematological parameters and at a stage of presentation. The aim of our study to evaluate the effect of ZAP-70 and CD38 independently as well as combined expression of both together in B-CLL patients and their influence in clinical features, hematological parameters and at a stage of presentation, accordingly our patients are divided into three groups.

Methods
This study was a prospective cross-sectional descriptive study, conducted in Khartoum state, Sudan, in the period from April 2017 to April 2018. A total of 110 blood samples were collected from patients with B-cell CLL. Patients were obtained at Flow Cytometry Laboratory for Leukemia & Lymphoma Center, Khartoum, Sudan, where they were referred for immunophenotypic diagnosis.
All patients were diagnosed based on clinical history, physical examination, complete blood count, immunophenotypic criteria and B lymphocytes ≥ 5×10 9 /l, according to IWCLL [17]. The stage of the CLL was assessed by Rai and Binet [6,7] classification. All patients were newly diagnosed without any previous B-CLL treatment; as explained in our previous work [18].

Determination of blood count and immunophenotyping
Samples were collected in EDTA tubes, complete blood counts were performed by using automated hematological analyzer (SYSMEX-KX-21N, Japan).
The diagnosis of CLL was confirmed for each patient by flow cytometry (EPICS XL Beckman Coulter Flow Cytometer, Miami, FL, USA), standard protocol of Beckman Coulter [19] was used in fluorescent dye labeled monoclonal antibody for CD45, CD5, CD3, CD19, CD20, CD22, CD23, FMC7, CD79b, kappa, and lambda light chain. A marker was considered positive at a cutoff level of ≥ 30% according to the BCSH guideline [20]. A diagnostic scoring system was assessed by Matutes et al. [21,22]. Absolute B lymphocyte count was obtained by flow cytometry. Expression of ZAP-70 and CD38were performed by flow cytometry, as previously described [18,23,24].

Statistical analysis
Patient's data was collected by a structural interview questionnaire and from patient's medical records and analyzed by using the (SPSS), version-23. The analysis was done for quantitative variables of B-cell CLL to compare means and variance of means by using T-test, ANOVA and Kruskal-Wallis and Mann-Whitney test as well as correlations with Pearson and Spearman.

Discussion
ZAP-70 and CD38 were used as surrogate markers for the mutated and unmutated (IgVH) to facilitate the clinical management of B-CLL.
According to meta-analysis done by Liu et al. [30] the role of ZAP-70 expression in the prognosis of B-CLL is unaffected by region. Discrepancies may be due to different [protocols, the antibody used and the cutoff value was used (10% or 20%)] [27,31].
Hus et al. [26], Schroers et al. [16], D'arena et al. [27], Del Giudice et al. [25] and Waheed et al. [28] found that there were significant correlations of CD38 with the stage at presentation but our study found no such associations (Additional file 1: Table S3), same as the study that reported by Abdelgader et al. [24], and Gogia et al. [29], but noteworthy, Abdelgader et al. [24] only found that there was significant association between CD38 and hemoglobin concentration by using cutoff level 7% [24] and Schroers et al. [16]. Hus et al. [26] however, found there were significant differences in hematological parameter with expressions of ZAP-70 and CD38. In our study, the only exception was Splenomegaly (P = 0.021) with CD38 expression and no significant correlations of ZAP-70 and CD38 expressions with age, sex, hematological parameters and clinical findings at presentation were found (Additional file 1: Table S1-S3), which was inconsistent with Abdelgader et al. [24], Gogia et al. [29], Waheed et al. [28].
The correlation of ZAP-70 with CD38 showed a strong association (P = 0.003) (Additional file 2: Figure S1), which agree with the results of the literature. Accordingly, our patients were categorized into three subgroups, two concordant (ZAP-70 + CD38 + ), (ZAP-70 − CD38 − ), and discordant group where only ZAP-70 or CD38 is positive. Our separate analysis of both discordant groups (ZAP-70 − CD38 + ) and (ZAP-70 + CD38 − ) revealed that there were no clinical or biological differences so they were treated as one discordant group.
With the exception of Splenomegaly (P = 0.037), regarding age, sex, clinical findings, and hematological parameters means, there were no significant correlations between concordant ZAP-70 and CD38 expressions or with discordant expression of both (Tables 1, 2 and 3), same as Gogia et al. [29], Waheed et al. [28] reported, and in contrast to Hus et al. [26], Del Giudice et al. [25] who found that there were significant differences in the most hematological parameter between concordant groups.
Noticeably, Splenomegaly was correlated with positive expression of CD38 but not ZAP-70, and the combination of both (ZAP-70 + CD38 + ) was still significant which may imply the significance of concordant analysis of ZAP-70 and CD38 as a better prognostic marker better than each one independently. Frequencies of concordant and discordant expressions of ZAP-70 and CD38 in some important studies are shown in Additional file 1: Table S4.
Conclusion, our study showed CD38 + was significantly associated with Splenomegaly. While expressions of ZAP-70 and CD38 were not statistically associated with significantly more advanced stages at Rai and Binet of B-CLL patients at presentation. In addition, the combination of these two markers together leads to classifying our patients into three subgroups (concordant positive, negative and discordant groups) which is statistically insignificant with clinical presentation (except Splenomegaly), hematological parameters and stage at presentation of B-CLL patients.

Limitations
Limitations are worth to mention such as sampling method which depends on voluntary participation, patients were not followed up for progression, survival rates and response to treatment after diagnosis confirmation. Finally, our study does not include studying the prognostic value of ZAP-70 or CD38 independently or even combined the expression of both together, indeed