- Research note
Evaluation of CD38 expression in Sudanese patients with chronic lymphocytic leukemia
BMC Research Notesvolume 11, Article number: 815 (2018)
The objective of this study was to evaluate the cluster of differentiation-38 (CD38) expression in Sudanese patients with chronic lymphocytic leukemia (CLL) and to determine its association with clinical and laboratory characteristics of the disease.
We conducted a cross-sectional study on 99 patients diagnosed with CLL in Khartoum Oncology Hospital in Khartoum, Sudan. Immunophenotyping and CD38 expression levels were measured with four-color flowcytometry. The results of physical examination and blood analyses were used for assigning a modified Rai clinical staging system. The collected data were analyzed using the Statistical Package for the Social Science, version 22 (SPSS Inc., Chicago, IL, USA). According to our findings, the frequencies of 7%, 20%, and 30% cutoff levels of CD38 expressions were 68.7%, 41.4%, and 36.4% respectively. CD38 cutoff level of 7% showed a significant association with hemoglobin concentration (P = 0.04), whereas other cutoff levels showed insignificant results. All the three cutoff levels showed insignificant associations with the other clinical and laboratory variables. In conclusion, the CD38 expression at a cutoff level of 7% seems to be more valuable clinically than higher cutoff levels in Sudanese CLL patients.
Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative malignancy characterized by an accumulation of cluster of differentiation-5 (CD5) monoclonal B cells in both primary and secondary lymphoid tissues . The distinct immunophenotype of CLL lymphocytes is essential to confirm the diagnosis . The 1994 scoring system was based on the evaluation of five parameters: CD5, CD23, FMC7, an intensity of kappa/lambda chains, and CD22/CD79b. The B-CLL score would range between five (typical B-CLL cases) and three (less typical B-CLL cases). Lower scores (0–2) would exclude the diagnosis of B-CLL .
CD38 is a type II transmembrane glycoprotein that acts as a complex ectoenzyme, a receptor molecule, and a signaling factor in lymphocytic cells . It participates in many cellular activities that include cell adhesion, signal transduction, and calcium regulation . CD38 expression, as well as zeta-chain-associated protein kinase-70 (ZAP70) and the mutation status of immunoglobulin variable region heavy chain (IgVH), are important prognostic indicators in CLL. The advantage of CD38 is its stability over time and its easy measurement [6, 7].
The expression of CD38 by CLL cells is associated with an aggressive clinical presentation that is confirmed by many studies [8,9,10,11,12,13]. The progression-free intervals in CD38 +ve patients are shorter, and they die sooner when compared with the CD38 −ve patients . This observation encouraged many clinical centers to adopt the determination of CD38 percentage expression as part of the regular investigations of CLL patients .
Flowcytometry is a useful tool for population analysis. It enables measurements of multiple characteristics in single cells. Light scattering can detect differences in size and internal complexity, and the fluorescence emitted from labeled antibodies can locate cell surface and intracellular antigens .
The analysis of both CD38 and ZAP-70 would provide valuable information in the diagnostic work-up of B-CLL patients . CD38 expression also correlates with the IgVH mutation status [17, 18]; however, the two parameters were found to be independent prognostic factors in B-CLL [19, 20]. Besides, CD38 is an independent predictor of progression-free survival (PFS) in Binet stage A patients .
Many articles described the dynamic expression of CD38 that indicates the proliferative activity of the leukemic cells at the time of analysis [22, 23]. The expression could be a parallel indicator of clonal evolution, which ultimately determines the patient’s prognosis . On the other hand, the best threshold for CD38 positivity is unclear. Early reports suggested 30% as a cutoff level of interest, but lower figures (7%, 20%) might be more clinically relevant .
There is a lack of data regarding the prevalence of CD38 positivity or its clinical application in the management of CLL patients in Sudan. We carried out this study to estimate the level of CD38 expression in CLL patients, to find its association with baseline clinical and laboratory parameters at presentation, and to determine a cutoff level for a CD38 appearance that is more likely to be associated with the clinical and laboratory findings of CLL patients.
We conducted a cross-sectional study at Khartoum Oncology Hospital/Sudan, during the period from September 2016 to February 2017. The study included 99 newly diagnosed, untreated B-CLL patients (69.7% males, 30.3% females), diagnosed according to the International CLL Workshop Criteria  and staged according to the modified Rai system . A structured questionnaire was used to collect clinical and demographic data. Physical examination was performed to determine the dimensional diameters of lymph nodes in the neck, axilla, supraclavicular, inguinal, and femoral regions. The size of the liver and spleen were assessed by chest radiography and abdominal ultrasound. Three ml of peripheral blood (PB) were withdrawn from each participant, collected in EDTA tubes, and preserved at room temperature (22–24 °C). All the samples were processed within 6–24 h of collection. Complete blood count (CBC) was obtained by automated hematology analyzer (Sysmex XE-2100™, Kobe, Japan). Four color flowcytometer (COULTER EPICS XL-MCLTM Flowcytometer—Miami, Florida—USA) with SYSTEM II software was used to determine the immunophenotyping and CD38 expression for the study population. The instrument set up was checked daily using QC check beads Flowcytometry (Beckman Coulter, Miami, USA).
The immunophenotyping (IPT) of lymphocytes (from lysed whole peripheral blood samples) was carried out to confirm the diagnosis of CLL using the following monoclonal antibodies (Beckman Coulter); CD45, CD5, CD19, CD20, CD22, CD23, kappa and lambda light chains, FMC7, and CD79b. A marker was considered positive at a cutoff level of 20%. The Matutes scoring system allocates one point each for the expression of weak SmIg, CD5, CD23, and absent or low expression of CD79b and FMC7 [3, 28].
The CD38 was analyzed in peripheral blood using anti CD38 monoclonal antibodies (Mo Ab), (from Beckman Coulter, Miami, USA). The cell surface staining method was applied according to the following protocol: Twenty microliters labeled Mo Ab were dispensed into appropriately labeled tubes. A sample of 100 µl was added containing no more than 1 × 104 leukocytes/ml. Each tube was vortexed for 5 s. The tubes were incubated at room temperature in a dark place for 10 min. One milliliter of the RBCs lysis solution was added to each tube and then incubated at room temperature in a dark place for 10 min. Centrifugation at 1500g (3200 rpm) for 3 min was done, 3.5 ml PBS added, and centrifugation at 1500g (3200 rpm) for 3 min repeated. Samples were suspended in PBS, tubes vortexed and analyzed using flowcytometer. CD38 was measured by flowcytometry and plotted against B cell marker CD19 expression. The CD38 expression was considered positive at three cutoff levels; 7%, 20%, and 30%.
The collected data were analyzed using the software program of the Statistical Package for Social science for Windows (SPSS), version 22 (SPSS Inc., Chicago, IL, USA). Both quantitative variables (mean and standard deviation) and qualitative variables (number and percentage) were described. Chi square test was used for Comparison of categorical variables. Statistical significance was defined for a P-value less than 0.05.
The study was approved by the Ethical Research Committee of the Sudan Medical Specialization Board (SMSB, 8/2016). Official letters were sent to the Director of Khartoum Oncology Hospital. Informed written consent was obtained from each participant before sampling. The study followed the general principles of medical research and the stated guidelines of the Ethical Committee.
Ninety-nine CLL patients, including 69 (69.7%) males and 30 (30.3%) females, participated in this study. Their mean age (SD) was 62.0 (11.14) year, with a median of 63 (range 36–95) years. The clinical, hematological and flowcytometry findings are presented in Tables 1 and 2. The majority of the enrolled patients presented with lymphadenopathy (71.7%) and splenomegaly (52.5%). Complete blood count results showed anemia (72.7%), and thrombocytopenia (40.4%). The study results showed that nearly two-thirds of the participants (64.7%) were a high-risk group, 30.3% were intermediate risk group, and only 5.0% were low-risk group (Table 1). Using the scoring system, the majority of our patients had a score of 5.0 (53.5%), followed by a score of 4.0 (31.3%), (Table 1).
The diagnosis of B-CLL was confirmed by flowcytometry. It revealed a typical immunophenotype; (kappa (κ)/lambda (λ) staining) weak, CD5+, CD19+, CD20 weak, CD23+, FMC7 (a CD20 epitope) and CD79b were absent or weakly expressed) (Table 2).
There was marked variability in the percentage expression of CD38 (range 0.09–96.1), with a median 13.00 and mean (SD) expression of 36.6 (37.92). The CD38 expression at cutoff levels of 7%, 20%, and 30%, were 68.7%, 41.4%, and 36.4% respectively (Table 3). CD38 cutoff level of 7% showed a significant association with the different classes of hemoglobin concentration (P = 0.04). The association with the other variables (age, sex, lymphadenopathy, splenomegaly, hepatomegaly, TWBC, platelets, and modified Rai staging systems was statistically insignificant (P > 0.05, Table 3).
The CD38 is a glycoprotein expressed on the surface of many white blood cells, including lymphocytes of CLL, and it has been linked to aggressiveness and poor prognosis of the disease. The determination of CD38 expression and IgVH mutation status are ordinary laboratory investigations in CLL patients . In the clinical setting, CD38 is an independent predictor of progression-free survival (PFS) rate in Binet stage “A” patients . However, the cutoff value for CD38-positivity that is most likely to predict a poor prognosis has been a source of extensive discussion.
In our study, CD38 expression at cutoff levels of 7%, 20%, and 30%, were 68.7%, 41.4%, and 36.4% respectively. A major finding is a significant association between CD38 positivity at a cutoff level of 7% and hemoglobin concentration. Since the modified Rai staging system classifies CLL patients with hemoglobin concentration < 11.0 g/dl as a high-risk group, CD38 expression at a cutoff value of 7% could be more useful clinically in detecting the high-risk group than the other higher cutoff levels. Similar findings were described by Kröber et al.  and Falay et al. . They reported that the cutoff value of 7% is an important prognostic marker that can be used in the classification of CLL patients into different prognostic groups.
The present study showed an insignificant association between CD38 positivity and many of the clinical and laboratory parameters that included age, gender, lymphadenopathy, splenomegaly, hepatomegaly, WBC count, platelet count, and modified Rai staging system. This might be related to the late presentation of most of our patients; as about two-thirds of them are a high-risk group at the time of diagnosis, 30% were an intermediate-risk group, and only 5% are a low-risk group. In contrast to our findings, a significant association was found between advanced disease and surface expression of CD38 in > 30% of B-CLL cells . Contradictory results were also reported by Damle et al. who showed that high levels of CD38 (> 30%) are associated with unmutated IgVH genes , whereas the mutated IgVH genes that indicate good prognosis, were found in patients with < 30% CD38-positive cells. Our finding that CD38 positivity in CLL at a cutoff level of 30% was 36% goes in line with the published literature that reported comparable results [32,33,34,35,36].
In conclusion, the CD38 expression at a cutoff value of 7% is significantly associated with low hemoglobin concentration, and therefore, more likely to predict poor outcomes than the higher CD38 cutoff levels in Sudanese CLL patients. Further studies are highly recommended for determining the “progression-free survival” and the most likely predictors of prognosis in Sudanese patients.
The present study has many limitations. The sampling method that depended on the voluntary participation of participants, the late presentation of our patients compared to other studies, and the lack of follow-up information, should all be considered for interpretation of the results. The follow-up information is particularly relevant because it gives valuable information about the prognosis regarding survival rate and response to treatment.
chronic lymphocytic leukemia
cluster of differentiation
immunoglobulin variable region heavy chain
zeta-chain-associated protein kinase-70
total white blood cells count
Bockstaele F, Verhasselt B, Philippé J. Prognostic markers in chronic lymphocytic leukemia: a comprehensive review. Blood Rev. 2009;23:25–47.
Matutes E, Attygalle A, Wotherspoon A. Diagnostic issues in chronic lymphocytic leukemia (CLL). Best Pract Res Clin Hematol. 2010;23:3–20.
Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A, Que TH, Catovsky D. The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia. 1994;8(10):1640–5.
Mehta K, Shahid U, Malavasi F. Human CD38, a cell-surface protein with multiple functions. FASEB J. 1996;10(12):1408–17.
Deaglio S, Vaisitti T, Zucchetto A, et al. CD38 as a molecular compass guiding topographical decisions of chronic lymphocytic leukemia cells. Semin Cancer Biol. 2010;20(6):416–23.
Chevallier P, Penther D, Avet-Loiseau H, et al. CD38 expression and secondary 17p deletion are important prognostic factors in chronic lymphocytic leukemia. Br J Haematol. 2002;116(1):142–50.
Deaglio S, Aydin S, Grand MM, et al. CD38/CD31 interactions activate genetic pathways leading to proliferation and migration in chronic lymphocytic leukemia cells. Mol Med. 2010;16(3–4):87–91.
Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. 1999;94(6):1840–7.
Del Poeta G, Maurillo L, Venditti A, et al. Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood. 2001;98(9):2633–9.
Ibrahim S, Keating M, Do KA, et al. CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood. 2001;98(1):181–6.
Jelinek DF, Tschumper RC, Geyer SM, et al. Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukemia. Br J Haematol. 2001;115(4):854–61.
Morabito F, Mangiola M, Oliva B, et al. Peripheral blood CD38 expression predicts survival in B-cell chronic lymphocytic leukemia. Leuk Res. 2001;25(11):927–32.
Durig J, Naschar M, Schmucker U, et al. CD38 expression is an important prognostic marker in chronic lymphocytic leukemia. Leukemia. 2002;16(1):30–5.
Matrai Z. CD38 as a prognostic marker in CLL. Hematology. 2005;10(1):39–46.
Brown M, Wittwer C. Flow cytometry: principles and clinical applications in hematology. Clin Chem. 2000;46(8 Pt 2):1221–9.
Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profi le. Blood. 2003;101(12):4944–51.
Shanafelt TD, Geyer SM, Kay NE. Prognosis at diagnosis: integrating molecular biologic insights into clinical practice for patients with CLL. Blood. 2004;103:1202–10.
Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP-70 expression as a surrogate for immunoglobulin variable-region mutations in chronic lymphocytic leukemia. N Engl J Med. 2003;348:1764–75.
Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW, Stevenson FK, et al. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood. 2002;99:1023–9.
Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG, et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med. 2004;351:893–901.
Hock BD, McKenzie JL, McArthur L, et al. CD38 as a prognostic marker in chronic lymphocytic leukemia at a single New Zealand center: patient survival in comparison to age and sex matched population data. Intern Med J. 2010;40:842–9.
Calissano C, Damle RN, Hayes G, et al. In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia. Blood. 2009;114(23):4832–42.
Damle RN, Temburni S, Calissano C, et al. CD38 expression labels an activated subset within chronic lymphocytic leukemia clones enriched in proliferating B cells. Blood. 2007;110(9):3352–9.
Ding W, Nowakowski GS, Knox TR, et al. Bidirectional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression. Br J Haematol. 2009;147(4):471–83.
Hoffbrand AV, Higgs DR, Keeling DM, Mehta AB. Postgraduate hematology. 7th ed. New York: Wiley; 2016.
National Comprehensive Cancer Network (NCCN) clinical practice guidelines in oncology: Non-Hodgkin’sLymphomas. Version 4.2014. https://www.nccn.org/about/nhl.pdf.Accessed: 30 Dec 2017.
Hallek M, Cheson BD, Catovsky D, Aligaris-Cappio F, Dighiero G, Döhner H, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International workshop on chronic lymphocytic leukemia updating the national cancer institute-working group 1996 guidelines. Blood. 2008;111(12):5446–56.
Moreau EJ, Matutes E, Hern RP, Morilla AM, Morilla RM, Owusu-Ankomah KA, Seon BK, Catovsky D. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J ClinPathol. 1997;108(4):378–82.
Sagatys EM, Ling Zhang L. Clinical and Laboratory Prognostic Indicators in Chronic Lymphocytic Leukemia. Cancer Control. 2012;19(1):18–25.
Letestu R, Levy V, Eclache V, et al. Prognosis of Binet stage A chronic lymphocytic leukemia patients: the strength of routine parameters. Blood. 2010;116(22):4588–90.
Kröber A, Seiler T, Benner A, Bullinger L, Brückle E, Lichter P, et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood. 2002;100(4):1410–6.
Falay M, Ceran F, Gunes AK, Dagdas S, Ayli M, Ozet G. CD38 expression and variation as a prognostic factor chronic lymphocytic leukemia. Clin Lab. 2016;62(7):1287–93. https://doi.org/10.7754/Clin.Lab.2015.151125.
Schroers R, Griesinger F, Trumper L, Haase D, Kulle B, Klein-Hitpass L, et al. Combined analysis of ZAP 70 and CD 38 expression as a predictor of disease progression in B cell chronic lymphocytic leukemia. Leukemia. 2005;19:750–8.
Assem M, Abdel Hamid T, Kohla S, Arsanyos S. The prognostic significance of combined expression of ZAP-70 and CD38 in chronic lymphocytic leukemia. J Egypt Natl CancInst. 2009;21(4):287–97.
El-Kinawy NS, Sharaf HM, Abd El-Hamid M. Prognostic significance of del 17p, ZAP-70 and CD38 as independent indicators for B-CLL: correlation to response to treatment and disease outcome. Egypt J Med Hum Genetics. 2012;13:173–81.
Gogia A, Sharma A, Raina V, Kumar L, Gupta R, Kumar. R prevalence of ZAP-70 and CD 38 in Indian chronic lymphocytic leukemia patients. Indian J Cancer. 2013;50(4):333–6.
EAA conceived the study, participated in data collection, performed the statistical analysis, interpreted the results, and revised the manuscript. NHE participated in data collection, participated in the statistical analysis, and drafted the manuscript. TAE, OAA, and EAF participated in the data collection, carried out the laboratory work, and prepared the results. EMA participated in study conception, supervised the laboratory work, and revised the manuscript. THM participated in the supervision, data interpretation, and drafting the manuscript. All authors read and approved the final manuscript.
We are very grateful to the staff of the Khartoum Oncology Hospital-Sudan, for their valuable assistance. We extend our gratitude and thanks to all members of the Flow Cytometry Laboratory for Leukemia & Lymphoma Diagnosis in Khartoum for their great support.
The authors declare that they have no competing interests.
Availability of data and materials
The individual data are available in the archives of the Khartoum Oncology Hospital-Sudan and can be obtained from the corresponding author on request.
Consent for publication
Ethics approval and consent to participate
The study was approved by the Ethical Committee of the Sudan Medical Specialty Board (SMSB, 8/2016). Official letters were sent to the Director of Khartoum Oncology Hospital. Informed consent was obtained from each participant before data collection.
This research did not receive any fund or financial support.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.