|Year : 2016 | Volume
| Issue : 4 | Page : 194-199
Serum-soluble APRIL influences survival in patients with chronic lymphocytic leukemia
Ola A El Shora MD 1, Abeer Shahbah2
1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||16-Aug-2016|
|Date of Acceptance||30-Aug-2016|
|Date of Web Publication||20-Jan-2017|
Ola A El Shora
Mostafa Kamel Str., Kafr Elzayat, El Gharbiya
Source of Support: None, Conflict of Interest: None
Background A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor family. APRIL plays an important role in B-cell development; it activates chronic lymphocytic leukemia (CLL) cells by reacting with its receptors, enhancing immune recognition, proliferation, and survival of the leukemia cells.
Aim The aim of this study was to determine the impact of serum-soluble APRIL levels detected at diagnosis in B-CLL patients, and their relationship with disease parameters and patient outcomes.
Patients and methods Serum APRIL levels were estimated in 40 untreated B-CLL patients and 40 apparently healthy controls. Serum was withdrawn at diagnosis of untreated CLL disease and was tested by enzyme-linked immunosorbent assay.
Results APRIL levels in peripheral blood samples were significantly higher in B-CLL patients than in normal participants. There were positive correlations between serum-soluble APRIL level, age, white blood cell count, peripheral blood lymphocyte count, CD38%, ZAP-70, and modified RAI staging. There were negative correlations between serum-soluble APRIL, hemoglobin level, and platelet count. During a 2-year follow-up, APRIL levels correlated with overall survival and disease-free survival.
Conclusion APRIL is a powerful prognostic marker related to parameters of disease activity and staging and, more importantly, to overall survival and disease-free survival.
Keywords: APRIL, chronic lymphocytic leukemia, prognostic marker
|How to cite this article:|
El Shora OA, Shahbah A. Serum-soluble APRIL influences survival in patients with chronic lymphocytic leukemia. Egypt J Haematol 2016;41:194-9
|How to cite this URL:|
El Shora OA, Shahbah A. Serum-soluble APRIL influences survival in patients with chronic lymphocytic leukemia. Egypt J Haematol [serial online] 2016 [cited 2019 Dec 11];41:194-9. Available from: http://www.ehj.eg.net/text.asp?2016/41/4/194/198649
| Introduction|| |
Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm that results from expansion of the mature lymphocyte compartment . It is the most common form of leukemia, accounting for about 22–30% of all leukemias. CLL is considered to be mainly a disease of the elderly, with a median age at diagnosis varying between 64 and 70 years .
Seventy percent of CLL patients require therapy during the course of the disease. The RAI staging system predicts overall survival, but it does not predict patients’ survival in the early stages (0–I), which will progress and require therapy. The drawbacks of this staging system are that a great number of patients with early-stage disease will rapidly deteriorate to an advanced stage that requires therapy and that is unable to futurely differentiate the rapidly progressing patient from the more stable ones who will not progress for years .
The known prognostic parameters based on routine tests involving the blood or bone marrow, such as clinical stage, lymphocyte doubling time, β-2 microglobulin levels, and lactate dehydrogenase level, are useful but they may not accurately predict progression for a given patient ,. Therefore, focus of research in prognostic factors in CLL has changed from clinical to biological factors.
A proliferation-inducing ligand (APRIL) is a member of tumor necrosis factor family ligands that bind to members of the tumor necrosis factor receptor family such as transmembrane activator and calcium modulator and cyclophilin ligand interactor and B-cell maturation antigen. APRIL is present on dendritic cells, macrophages, and T lymphocytes .
APRIL plays an important role in B-cell development; it activates CLL cells by reacting with its receptors, enhancing immune recognition, proliferation, and survival of the leukemia cells. APRIL induces activation of the canonical NF-κB pathway and protects CLL cells from apoptosis ,.
| Patients and methods|| |
A written consent from all patients was taken
The present study was conducted on 40 newly diagnosed CLL patients selected from Hematology Units of Internal Medicine Departments of Tanta University Hospital and Tanta Cancer Institute, from January 2014 to January 2016. In CLL patient group, 25 patients were male (62.5%) and 15 patients were female (37.5%), and their ages ranged from 52 to 77 years with a mean value of 64.60±8.21; 40 apparently healthy participants matched for age and sex with the patient group served as the control group. Patients with newly diagnosed CLL were included in the study; any patient with malignant diseases other than CLL was excluded from the study. B-CLL diagnosis was based on a clinical examination, as well as on morphological and immunophenotyping of peripheral blood (PB) lymphocytes.
Patients were subjected to the following: full history taking and thorough clinical examination, abdominal ultrasonography, and laboratory investigations, including the following:
- Routine investigations:
- Complete blood picture was taken on an ERMA PCE-210N cell counter (for ERMA CELL COUNTER: Japan).
- Immunophenotypingfor the CLL panel was performed using the markers CD5-FITC, CD19-PE, CD20-APC, FMC-7-FITC, CD10-PE, CD23-PE, CD2-FITC, κ-FITC, λ-PE, and CD38-FITC on BD FACS calibur flow cytometry. Samples were analyzed by four-color flow cytometry using the Becton Dickinson FACS Calibur instrument; FACS CALIBUR (Becton Dickinson, San Jose, California, USA). For each analysis, 10 000 events were acquired and analyzed using the Cell Quest software; CELL QUEST SOFTWARE (Becton Dickinson, version 3, verify software House Topsham, ME, USA). An acquisition gate was done based on FSC and SSC that excluded dead cells and debris. Isotype antibody was used to prevent nonspecific binding.
- Specific laboratory test:
Estimation of serum-soluble APRIL by enzyme-linked immunoassay technique (Cat. No. RAF004R; Biovendor Research and Diagnostic, Czech Republic) from PB specimens of untreated B-CLL patients and healthy controls. PB was collected in a red-top tube, allowed to clot, and centrifuged at ∼1000g for 15min, and the serum was separated and stored at −20°C until estimated. The instruction protocol of the manufacturer was followed.
The statistical analysis of the results was carried out using SPSS version 22 for Windows. Qualitative data were described using number and percent. Quantitative data were described using mean and SD for normally distributed data. For normally distributed data, comparison between two independent populations was done using independent t-test. Correlations between two quantitative variables were assessed using Pearson’s coefficient. Receiver operating characteristic curve was used to detect sensitivity, specificity, negative predictive value, and positive predictive value. Kaplan–Meier analysis is a method of calculating survival of a patient population in which the increments are the actual survival times of the patients.
| Results|| |
Correlation between serum-soluble APRIL and studied parameters
To assess the prognostic value of soluble APRIL, an analysis was performed to correlate the serum levels of APRIL with parameters usually recognized as having prognostic significance, such as age, hemoglobin level, platelet count, white blood cell count, PB lymphocytes, CD38%, and ZAP-70 ([Table 1] and [Table 2]).
|Table 1 Clinical characteristics in chronic lymphocytic leukemia patient group|
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|Table 2 Comparison between chronic lymphocytic leukemia patient group and control group as regards laboratory data and serum-soluble APRIL levels|
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There were positive correlations between serum-soluble APRIL level, age, white blood cell count, PB lymphocyte count, CD38%, and ZAP-70. There were negative correlations between serum-soluble APRIL level, hemoglobin level, and platelet count, as shown in [Table 3].
Sensitivity and specificity percentage of the serum-soluble APRIL in the diagnosis of CLL patients
Serum-soluble APRIL had 97.5% sensitivity, 93% specificity, 92.8% positive predictive value, and 97% negative predictive value when the cutoff value was 42.5 ng/ml ([Table 4] and [Table 5] and [Figure 1]).
|Table 4 Association between serum APRIL levels and staging of studied chronic lymphocytic leukemia patients|
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|Table 5 The sensitivity and specificity regarding the level of serum-soluble APRIL|
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|Figure 1 Receiver operating characteristic (ROC) curve for serum APRIL levels.|
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Survival of B-CLL patients according to APRIL levels
Patients with APRIL levels above cutoff (42.5 ng/ml) had significantly shorter overall survival (15.5 vs. 18 months) and significantly lower disease-free survival (5 vs. 11.5 months) at 24 months versus those with APRIL levels below cutoff (P<0.001) ([Figure 2] and [Figure 3]).
|Figure 2 Kaplan–Meier procedure showing the impact of serum APRIL level on overall survival in chronic lymphocytic leukemia (CLL) patients. High serum APRIL level above cutoff correlated with reduced OS.|
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|Figure 3 Kaplan–Meier procedure showing the impact of serum APRIL level on disease-free survival in chronic lymphocytic leukemia (CLL) patients. High serum APRIL level above cutoff correlated with reduced disease-free survival (DFS).|
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| Discussion|| |
B-CLL is a B-cell neoplasm, characterized by indolent course, with progressive splenic and lymph node enlargement associated with PB chronic lymphocytosis. This disease is related to accumulation of monoclonal B cells with the morphology of small mature lymphocytes, which is mainly due to inhibition of apoptosis rather than enhanced cell proliferation . ZAP-70 and CD38 expression and unmutated IgVH genes are considered the most useful markers in detecting patients with a more aggressive clinical course ,,.
Identification of novel markers that are predictive of overall survival and disease-free survival may further improve the knowledge of the pathophysiology of B-cell CLL. In the present study, we analyzed the cohort of 40 patients with untreated B-CLL, aiming to assess prognostic significance of APRIL levels.
The present study was conducted on 40 newly diagnosed CLL patients, of whom 25 patients were male (62.5%) and 15 patients were female (37.5%), and their ages ranged from 52 to 77 years with a mean value of 64.60±8.21; 40 apparently healthy participants matched for age and sex with the patient group served as the control group.
As regards clinical picture, hepatosplenomegaly was present in 24/40 (60%) patients, and lymphadenopathy was present in 26/40 (65%) patients. These results were in agreement with those described by OBrien and Keating .
In the present study, according to modified RAI staging system, 40 CLL patients were categorized into three classes: low risk, four patients (10%); intermediate risk, 15 patients (37.5%); and high risk, 21 patients (52.5%). Most patients were included in the high-risk group with lymphocytosis together with anemia or thrombocytopenia. This was in contrast to the study by Bosch and Montserrat  who reported that most CLL cases were low risk, which may be attributed to the delay in seeking medical advice.
In the present study, the level of serum-soluble APRIL in CLL patients was significantly higher in comparison with the normal control group; this was in accordance with Planelles et al.  and Junak et al.  who reported that serum-soluble APRIL level was higher in CLL patients than in healthy individuals.
In this study, no significant association was detected between serum-soluble APRIL and sex, which was in agreement with that reported by Junak et al. , who found no association between sex and serum-soluble APRIL level.
In the current study, there was a positive correlation between age and serum-soluble APRIL level; this was in contrast to the study by Junak et al.  who reported that there was no correlation between age and serum-soluble APRIL level.
In the current study, there was a negative correlation between hemoglobin level, platelet count, and serum-soluble APRIL level; this was in agreement with the study by Junak et al.  who reported that there was a negative correlation between serum-soluble APRIL level, hemoglobin, and thrombocytopenia.
In the present study, there was a positive correlation between white blood cells, PB lymphocytosis, and serum-soluble APRIL level, which was in agreement with the study by Junak et al.  who reported that there was a positive correlation between leukocytosis, PB lymphocytosis, and serum-soluble APRIL level.
There was also a positive correlation between serum-soluble APRIL, ZAP-70, and CD38%; this was in contrast to the studies by Haiat et al. and Junak et al.  who found no association between CD38% and serum-soluble APRIL.
Serum-soluble APRIL was related to disease activity parameters and staging of CLL (higher levels of serum APRIL in high modified RAI stage). Higher levels of serum-soluble APRIL were related to shorter overall survival and associated with worse outcome of patients when compared with lower levels; this was in agreement with the studies by Planelles et al.  and Junak et al. . However, it was in contrast to the study by Tecchio et al. , we did not observe a predictive role of sAPRIL in Overall Survival (OS), but we did observe it in Time To First Treatment (TTFT).
APRIL may constitute an autocrine pathway that is thought to play an important role in B-CLL cell survival. It is possible that deregulated expression of APRIL may contribute to reduced levels of apoptosis found in B-CLL. Kern et al.  showed that APRIL is able to protect B-CLL cells against spontaneous and drug-induced apoptosis.
| Conclusion|| |
There was a positive correlation between APRIL levels, age, white blood cell count, PB lymphocyte count, CD38%, and ZAP-70, and a negative correlation between serum-soluble APRIL, hemoglobin level, and platelet count. APRIL levels above cutoff were associated with shorter overall survival and disease-free survival. Therefore, it may be useful in prognosis, and this may suggest its potential role as a prognostic factor.
Our findings suggest that patients with ZAP-70- and CD38-positive expression levels in conjunction with high serum APRIL levels may require aggressive therapeutic approaches.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]