Clinical Staging and Flowcytomteric CD38 and Zap 70 Prognostic Indicators in Sudanese Patients with Chronic Lymphocytic Leukemia

Background: The clinical course of chronic lymphocytic leukemia is highly variable. The determination of ZAP70 and CD38 is increasingly utilized as prognostic factor for chronic lymphocytic leukemia. The aim of conducting this study was to investigate the frequency of CD38 and ZAP70 expression among Sudanese Chronic lymphocytic leukemia (CLL) patients and to relate them to the Binet and Rai clinical staging systems. 
Method: A total of 93 patients (mean age; 62.29 ± 11.68, sd) were enrolled in this cross-sectional study. CD38 and ZAP70 expression levels were measured with four color flowcytometry using the cut-off values of 20% for ZAP70 and 30% for CD38 expression. Staging was assessed by using clinical examination and CBC for all patients. Data were analyzed using the Statistical Package for Social science for Windows (SPSS), version 22. 
Results: There were 93 CLL patients and the median age of the group was 63 years (36–95 years). About 71% of the patients presented with lymphadenopathy, 53.8% with splenomegaly, 73.1% with anemia, and 45.2% with thrombocytopenia. There was higher frequency of Binet stage C and Rai stage IV (62 [66.6%] patients and 34 [36.5%] patients, respectively). In addition, CD38 and ZAP70 showed higher frequency among Binet and Rai advance stages. ZAP70 and CD38 positivity were detected in 21 patients (22.6%) and 31 patients (33.3%), respectively. There was no statistically significant association between ZAP70 and CD38 and clinical staging systems (P-value > 0.05). 
Conclusion: No significant association was observed between Flowcytometric (CD38 and Zap70) Prognostic Indicators and clinical staging systems. 
Keywords: chronic lymphocytic Leukemia, Flowcytometry, ZAP70, CD38, clinical staging systems


Introduction
Sudan is the third largest country in Africa with a total population of around 40 million people [1]. It borders seven countries and its capital is Khartoum. Sudan is a miniature representation of the diversity found in most African countries [2,3].
The country is composed of 18 states; approximately 66% of the population lives in rural areas [4], and the percentage of poverty is around 46.5% [5]. The country suffers from a marked shortage in health workforce worsened by poor distribution over the DOI 10.18502/sjms.v15i1. 6704 Page 44 drug-resistant disease [1]. The Rai and Binet [2,3] clinical staging systems are valuable in classifying CLL patients into broad prognostic subgroups. Clinical stages, however, have some limitations and this has led to a search for novel parameters with improved predictive power. Prognostic predictions in B-CLL at early clinical stage are based on biological disease parameters, such as ZAP-70 and CD38 protein levels, genomic aberrations as well as immunoglobulin variable heavy chain gene (IgVH) mutation status [4]. DNA microarray studies have shown that B-CLL cells with unmutated IgVH genes can be distinguished from those with mutated IgVH genes by the differential expression of a small number of genes, one of which encodes the 70-kDa zeta associated protein (ZAP-70) [5,6]. ZAP-70, a member of the Syk-ZAP-70 protein tyrosine kinase family, is a key signaling molecule for T lymphocytes and natural killer cells.
While ZAP-70 is not expressed in normal B lymphocytes, it is associated with increased intracellular signaling via the immunoglobulin receptor in B-CLL cells [7,8]. Considering three recently published studies [9][10][11], ZAP-70 is the most promising surrogate marker for the IgVH mutation status. In contrast to the technically demanding IgVH analysis, ZAP-70 protein expression is conveniently measured by flow cytometry [9,12,13].
CD38 is a type II transmembrane glycoprotein that acts as a complex ecto-enzyme and receptor molecule with signaling functions in B-CLL cells [14]. Detection of CD38 cell surface expression can be conveniently performed by flowcytometry, and, together with ZAP-70 [11], CD38 analysis may prove a valuable adjunct in the diagnostic workup of B-CLL patients. CD38 expression also has been suggested as a surrogate marker for the two important IgVH mutated and unmutated subgroups of B-CLL [15]. At present, both ZAP-70 and CD38 are regarded as independent prognostic variables in B-CLL [9,16]. The main objective of this study is to determine the frequency of CD38 and ZAP-70 expression and the relationship between clinical staging system and flowcytomtery prognostic markers (CD38 and Zap-70) in Sudanese patients with CLL.

Demographic Data
The 93 patients enrolled in this study include 63 (67.7%) male and 30 (32.3%) female with a median age of 63 (36-95 years). At the time of diagnosis, mean of patient ages was 62.29 ± 11.68, sd. Most of the patients 55 (59.1%) were in the age group 56 -75. (Table 1).

Staging systems
The study results showed that, there was higher frequency of Binet

Discussion
In out some of the limitation in these traditional staging systems [9]. Not all early-stage patients fall into the same prognostic category. A study from Vroblová et al [22] showed that the classic staging systems by Rai and Binet are not able to determine an individual patient's ongoing clinical course at the time of diagnosis, particularly in early stages.
Most of our patients presented in advance stages (Bient stage C and Rai stage IV (71.2%, 40.4% respectively)). This may be explained by; poor health services distribution and lack of screening program and awareness. Rai and Binet staging system showed highly significant association with lymphadenopathy (p = 0.01, 0.00 respectively), Haemoglobin concentration (p = 0.00 for both) and platelet count (p = 0.00 for both).
splenomegaly showed highly significant association with Rai staging system (p = 0.00) while no significant association was observed with Binet staging system (p = 0.20). No significant association was observed between both Rai and Binet staging systems and age, sex, hepatomegaly and TWBCs count (p > 0.05), (Table 2).
Various studies have reported ZAP-70 positivity ranging from 25% to 57% and CD38 positivity in CLL ranging from 29% to 60% as given in (Table 5).  systems. Further studies are recommended to develop a standardized flowcytometry protocol that will allow comparison of ZAP-70 and CD38 measurements between different laboratories.