|Year : 2014 | Volume
| Issue : 3 | Page : 128-133
The prognostic value of CXCR4 and pCXCR4 in B-lineage acute lymphoblastic leukemia in adults
Amina M Elnaggar1, Rania A Ghonaim1, Tarek A El-Gohary2, Maha Atfy MD 1
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Egypt
2 Department of Medical Oncology & Hematology, Faculty of Medicine, Zagazig University, Egypt
|Date of Submission||15-Oct-2013|
|Date of Acceptance||13-Sep-2013|
|Date of Web Publication||31-Dec-2014|
Department of Clinical Pathology, Hospitals of Zagazig University, PO Box 44519, Sharkeyia
Source of Support: None, Conflict of Interest: None
Background CXC chemokine receptor 4 (CXCR4) is activated by phosphorylation (pCXCR4) and is essential for the migration of hematopoietic precursors to bone marrow. Data regarding the prognostic impact of CXCR4 in patients with B-acute lymphoblastic leukemia (B-ALL) are sparse and limited to the pediatric population.
Patients and methods Fifty adult patients with B-ALL were included in the study. CXCR4 was assessed by flow cytometry and pCXCR4 by immunocytochemistry. All patients received a hyper-CVAD regimen.
Results Thirty-one patients had positive CXCR4 expression, of whom only 18 (58%) had positive pCXCR4 expression. The complete response rate for patients with positive expression of pCXCR4 was significantly inferior to those with negative expression (61.1 vs. 93.7%; P < 0.01). In the multivariate analysis, pCXCR4 expression was associated with a worse disease-free survival ( P < 0.027) and overall survival (P < 0.024). Positive expression of pCXCR4 was not associated with an increased incidence of extramedullary disease or had any relation to the degree of maturity of B-ALL.
Conclusion The current study indicates that adult B-ALL patients with positive expression of pCXCR4 have an inferior response to chemotherapy, disease-free survival, and overall survival compared with patients with negative expression.
Keywords: acute lymphoblastic leukemia, CXC chemokine receptor 4, flowcytometry, immune-cytochemistry, PCXCR4, prognosis
|How to cite this article:|
Elnaggar AM, Ghonaim RA, El-Gohary TA, Atfy M. The prognostic value of CXCR4 and pCXCR4 in B-lineage acute lymphoblastic leukemia in adults. Egypt J Haematol 2014;39:128-33
|How to cite this URL:|
Elnaggar AM, Ghonaim RA, El-Gohary TA, Atfy M. The prognostic value of CXCR4 and pCXCR4 in B-lineage acute lymphoblastic leukemia in adults. Egypt J Haematol [serial online] 2014 [cited 2020 Jan 18];39:128-33. Available from: http://www.ehj.eg.net/text.asp?2014/39/3/128/148238
| Inroduction|| |
Acute lymphoblastic leukemia (ALL) is a malignant clonal disorder of bone marrow lymphopoietic precursor cells with progressive medullary and extramedullary accumulation of lymphoblasts that lack the potential for differentiation and maturation  .
In the marrow and lymphoid tissues, ALL cells are in close contact with accessory cells (stromal cells and/or 'nurse-like cells') that constitute a distinct microenvironment. In vitro, these cells support long-term leukemia cell survival  .
By inference, the leukemia cell microenvironment may also enhance the survival of ALL cells in vivo and possibly account for the noted resistance of ALL cells to many forms of cancer chemotherapy , .
Chemokines are a superfamily of chemoattracting, cytokine-like proteins of target cells. They are the major regulators of cell trafficking and adhesion; they regulate trafficking of leucocytes by directing the migration of circulating leukocytes to sites of inflammation or injury. Chemokines usually bind to multiple receptors, and the same receptor may bind to more than one chemokine  .
CXC chemokine receptor 4 (CXCR4) has received much attention because it is the receptor for stromal-derived factor 1α known as CXCL-12. CXC4R-CXCL12 axis is essential for the migration of normal cells to the BM microenvironment  .
Among hematopoietic malignancies, CXCR4 is best studied in patients with AML, wherein CXCR4 over-expression is an independent predictor of a poor prognosis. In contrast, data on the prognostic value of CXCR4 expression in B-ALL is scarce, especially in adults. CXCR4 is activated by phosphorylation, and it is believed that this process is primarily ligand-dependent. This view is supported by the antitumor efficacy of AMD3100, an antagonist of CXCL12 binding  . The aim of this work was to study the expression levels of CXCR4 and phosphorylate-CXCR4 and their association with clinical and laboratory parameters in adult patients with B-ALL.
| Patients and methods|| |
Fifty patients with newly diagnosed adult B-ALL were included in the study after obtaining approval from the Institutional Review Board of Zagazig University Hospitals. This study was conducted from January 2011 to March 2012. The participants included 28 male and 22 female patients, with a median age of 33 years (range from 18 to 55 years).
In addition, 20 individuals matched for age and sex were selected as controls. All participants were informed adequately about the aim of the study and consented to donate samples for research purposes. Samples were obtained after informed consent and in accordance with procedures approved by the human ethics committee. All patients received a hyper-CVAD regimen  .
Five milliliter blood volumes were withdrawn aseptically into a sterile disposable syringe from every patient and control, and then divided as follows: 1 ml blood was collected in a sterile EDTA vacutainer tube for flow cytometry (FC) analysis; 3 ml blood was collected in a vacutainer tube containing the preservative free heparin and mixed immediately to avoid clotting at a concentration of 20-30 U/ml for immunocytochemistry. A sample of 1 ml of bone marrow was generally adequate. Samples were heparinized to prevent clotting, and were kept under sterile conditions until further conventional karyotype analysis. Samples were collected at the time of presentation before therapy was initiated.
| Methods|| |
Participants enrolled in this study were subjected to the following: full history taking, clinical examination, complete blood count with examination of a Leishman-stained smear, liver and kidney function tests, serum lactate dehydrogenase, B2 microglobulin, and radiological examinations.
Immunophenotyping of blast cells was performed using the whole blood lysing technique.A panel of monoclonal antibodies directly conjugated with fluorescein isothiocyanate, PE, Percp, were used; the monoclonal antibodies included CD3, CD5, CD7, CD10, CD13, CD19, CD20, CD22, CD33, CD34, CD79a, TDT, HLA-DR, and MPO, and appropriate isotypic controls IgG1 and IgGa2 were used in all cases. All monoclonal antibodies were purchased from Becton-Dickinson BD (San Diego, California, USA). The cutoff of the positive marker was defined above 20% for lymphatic and myeloid markers. Immunophenotypic characterization was performed according to the criteria of the European Group for the Immunological Characterization of Leukemia  . The mean fluorescent intensity ratio was calculated as the mean fluorescent intensity (MFI) signal antibody or peptide-incubated condition/the MFI control condition.
Assessment of total CXCR4 expression
It was performed in all samples using FC: fluorescein isothiocyanate-conjugated monoclonal antibodies against CXCR4 were used. Manual gated CD10+/19+ and CD34+ cells were used to determine ALL cell purity and B-cell purity in patients and control samples, respectively. In all cases, appropriate isotype controls were included. The MFI of the chemokine receptor expression was determined on the CD19+B lymphocytes. The MFI ratio was determined by dividing the mean fluorescence of the chemokine receptors by the mean fluorescence of the isotype control of the respective sample. The samples were analyzed by FC (FACSCalibur, BD Biosciences, San Jose, CA, USA), and analysis was performed with CellQuest software (BD Biosciences, San Jose, CA, USA).
Conventional karyotype analysis using the G-banding technique was performed on bone marrow aspirate specimens  . In addition, fluorescence in-situ hybridization studies were performed to assess for the presence of a Philadelphia chromosome using the LSI BCR/ABL probe: (Vysis, Downers Grove, Illinois, USA).
Assessment of activated (phosphorylated) CXCR4 by immunocytochemistry
Mononuclear cells were purified from the peripheral blood by Ficoll-Hypaque gradients (Biochrom, Berlin, Germany). Separated lymphocytes were washed twice with a fluorescence-activated cell sorter washing buffer (1% PBS, 2% fetal calf serum, 0.05% sodium azide, 0.5 mol/l ethylenediamine tetra-acetic acid) according to Perper et al.  The supernatant was then discarded and the cell count adjusted between 3000 and 5000 cell/cm 2 . A test for viability was performed  .
The technique of immunocytochemistry
It is a two-step immunocytochemistry staining technique based on an HRP-labeled polymer that is conjugated with secondary antibodies  . Phosphosite-specific rabbit antibodies for the S339-phosphorylated form of the CXCR4 receptor were used. At least 500 cells were counted for each monoclonal antibody. Negative cells are homogeneously green in color. Positive staining appears as distinct brown black positivity on the cells; the results are expressed as the percentage positivity of cells. The cutoff was at least 10 positive cells/100 cells for pCXCR4.
Analysis of data was performed using SPSS computer program (version 16.0; SPSS Inc., , Chicago, Illinois, USA). The Kaplan-Meier method was used to estimate overall survival (OS) for all patients included in the study and disease-free survival (DFS) only for patients achieving complete response, and the og rank test was used to compare survival curves. Univariate and multivariate Cox proportional hazards models were fitted to assess the effect of patient and tumor characteristics on OS and disease-free survival. Comparison between qualitative variables was performed using the χ2 -test.
| Results|| |
The clinical and laboratory data are presented in [Table 1] and [Table 2], consequently. The total CXCR4 was detected in 31 patients (62%) by FC and pCXCR4 was detected in 18 patients (36%) by immunocytochemistry.
All B-ALL patients with positive expression of pCXCR4 by immune-cytochemistry were also positive for CXCR4 by FC. There was a statistically significant correlation between CXCR4 expression detected by FC and pCXCR4 expression detected by immune-cytochemistry (r2 = 0.058 and P = 0.01).
Both the total CXCR4 and pCXCR4 showed no significant association either with the degree of maturity of B-ALL on the basis of immunophenotyping ([Table 3]) or with extramedullary involvement.
|Table 3 The expression of total and phosphorylated CXCR4 in different immunophenotypic groups|
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Complete response was achieved in 41 patients, of whom 93% had negative pCXCR4 expression and 27% had positive expression. The complete response rate was significantly higher (P = 0.01) in patients with negative pCXCR4 expression compared with patients with positive expression (93.7 and 61.1%, consequently) as shown in [Table 4].
In the Univariate Cox proportional hazards model ([Table 5]), poor OS was significantly associated with pCXCR4 expression (P = 0.03), low serum albumin (P = 0.04), high B-2 microglobulin (P = 0.02), high serum lactate dehydrogenase (P = 0.03), the presence of a Philadelphia chromosome (P = 0.01), and late complete response (P = 0.01), whereas total CXCR4 was not significantly associated with OS (P = 0.12).
|Table 5 The univariate cox proportional hazards model to assess the association between the covariates and the overall survival|
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In the multivariate Cox proportional hazards model ([Table 6]), poor OS was significantly associated with the presence of a Philadelphia chromosome (P = 0.01), late complete response (P = 0.01), and pCXCR4 expression (P = 0.024).
|Table 6 The multivariate cox proportional hazards model to assess the association between the covariates and the overall survival|
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In the univariate Cox proportional hazards model, a shorter disease-free survival was significantly associated with the presence of a Philadelphia chromosome (P = 0.026), late complete response (P = 0.032), and pCXCR4 (P = 0.021) ([Table 5])
In the multivariate Cox proportional hazards model, a shorter disease-free survival was significantly associated with the presence of a Philadelphia chromosome (P = 0.033) and pCXCR4 expression (P = 0.027), whereas late complete response lost significance.
The median OS of patients with positive expression of pCXCR4 was 18 months (95% confidence interval; 15.7-20.3), whereas the median OS of patients with negative expression was not reached. The OS of patients who had -ve expression of pCXCR4 was significantly higher than that of patients with positive expression (log rank test; P = 0.01) [Figure 1].
|Figure 1 Histograms from an apparent healthy individual (upper) and a patient (lower) show the expression of CXCR4 that revealed high fluorescent intensity of CXCR4 expression in the patient compared to the control.|
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The median disease-free survival of patients with positive expression of pCXCR4 was 16 months (95% confidence interval; 8.1-24.8), whereas that of patients with negative expression was not reached. The disease-free survival of patients who had negative expression of pCXCR4 was significantly higher than that of patients with positive expression (log rank test; P = 0.02) [Figure 3].
| Discussion|| |
Chemokines regulate chemotactic responses of cells that are essential for organogenesis and immunity through the orchestration of cell movement from one location to another  . Cancer cells exploit these regulatory mechanisms to stimulate their own growth, invasion, and metastasis  .
The chemokine receptor CXCR4, which is expressed in as many as 23 different tumor types, is a particularly exciting new target  . Most models of CXCR4 function in cancer focus on its potential role as a mediator of motility, invasiveness, and metastatic behavior  . Juarez et al.  demonstrated that CXCR4 mediates the homing of B-ALL cells to the bone marrow.
The regulation of phosphorylation and internalization has significant effects on CXCR4-mediated cellular responses; therefore, some authors believed that the descriptions of total CXCR4 expression alone are inadequate  . The results of the current study demonstrating a prognostic impact only for PCXCR4 expression in adult patients with B-ALL support this notion.
The expression of the chemokine receptor was found to be sinusoidal, and the highest in the pre-B transition to mature B-cell stage. Crazzolara et al.  noted high expression in the mature B-immunophenotype and low expression in pre-B ALL. In our study, we found no significant correlation between either the total CXCR4 or PCXCR4 and the degree of maturity of B-ALL on the basis of immunophenotyping and immunocytochemistry.
This finding is supported by the study of Konoplev et al.  who reported similar results. However, other studies noted that mature B immunophenotype had the highest expression of CXCR4, whereas preB ALL had the lowest expression  .
Crazzolara et al.  suggested that high CXCR4 expression predicted more prominent extramedullary involvement in B-ALL, as measured primarily by assessing the liver and spleen size. However, in our study and the study of Konoplev et al.  , we did not appreciate any difference in pCXCR4 expression or CXCR4 expression in patients with or without extramedullary involvement.
We found that pCXCR4 expression was associated with a shorter disease-free survival and OS. Schneider et al.  reported that patients with B-ALL who had high CXCR4 expression had a shorter disease-free survival. Others reported that they did not detect an association between pCXCR4 or CXCR4 expression and a shorter disease-free survival; however, pCXCR4 expression was associated with a shorter OS  . We cannot explain the discrepancies between our results and those of others on the basis of the results of the current study. Potential explanations include our small patient population and differences in the patient age (adults vs. children).
| Conclusion|| |
We conclude that adult B-ALL patients with positive expression of pCXCR4 have an inferior response to chemotherapy, disease-free survival, and OS compared with patients with negative expression. This observation is potentially important clinically and therapeutically, because anti-CXCR4 agents currently are available and can be added to current chemotherapy protocols designed for patients with B-ALL ([Figure 1], [Figure 2], [Figure 3]).
|Figure 2 The immunocytochemistry studies of pCXCR4 expressions reveal negative control (left) and positive ALL case (right).|
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|Figure 3 Overall (upper) and Disease Free Survival (lower) with positive and negative pCXCR4 in ALL patients were shown to be highly signifi cant in patients with-ve pCXCR4 expression than patients with +ve pCXCR4 expression (Log Rank test; P = 0.01 and P = 0.02 respectively).|
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| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gökbuget N, Hoelzer D. Treatment of adult acute lymphoblastic leukemia. Semin Hematol
Tsukada N, Burger JA, Zvaifler NJ, Kipps TJ. Distinctive features of 'nurselike' cells that differentiate in the context of chronic lymphocytic leukemia. Blood
Ghia P, Chiorazzi N, Stamatopoulos K. Microenvironmental influences in chronic lymphocytic leukaemia: the role of antigen stimulation. J Intern Med
Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell'Aquila M, Kipps TJ .Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood
Vindrieux D, Escobar P, Lazennec G. Emerging roles of chemokines in prostate cancer. Endocr Relat Cancer
Spoo AC, Lübbert M, Wierda WG, Burger JA. CXCR4 is a prognostic marker in acute myelogenous leukemia. Blood
Konoplev S, Jorgensen JL, Thomas DA, Lin E, Burger J, Kantarjian HM, et al
. Phosphorylated CXCR4 is associated with poor survival in adults with B-acute lymphoblastic leukemia. Cancer
Kantarjian HM, O'Brien S, Smith TL, Cortes J, Giles FJ, Beran M, et al.
Results of treatment with hyper-CVAD, a dose-intensive regimen, in adult acute lymphocytic leukemia. J Clin Oncol
Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A, van't Veer MB. Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia
Onciu M, Schlette E, Medeiros LJ, Abruzzo LV, Keating M, Lai R. Cytogenetic findings in mantle cell lymphoma cases with a high level of peripheral blood involvement have a distinct pattern of abnormalities. Am J Clin Pathol
Perper RJ, Zee TW, Mickelson MM. Purification of lymphocytes and platelets by gradient centrifugation. J Lab Clin Med
Weir DM. Hand book of experimental immunology
. 3rd ed. Blackwell Scientific Publications: Oxford; 1986. 15.
Bisgaard K, Pluzek K. Use of polymer conjugate in immunocytochemistry comparative study of a traditional staining method utilizing polymer conjugate. Budapest, Hungary: I Cong Intl Acad Pathol and 12th world cong acad environ path; 1996. 20-25
Juarez J, Dela Pena A, Baraz R, Hewson J, Khoo M, Cisterne A, et al
. CXCR4 antagonists mobilize childhood acute lymphoblastic leukemia cells into the peripheral blood and inhibit engraftment. Leukemia
Balkwill F. Cancer and the chemokine network. Nat Rev Cancer
Balkwill F. The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol
Zlotnik A. Chemokines in neoplastic progression. Semin Cancer Biol
Juarez JG, Thien M, Dela Pena A, Baraz R, Bradstock KF, Bendall LJ. CXCR4 mediates the homing of B cell progenitor acute lymphoblastic leukaemia cells to the bone marrow via activation of p38MAPK. Br J Haematol
Crazzolara R, Kreczy A, Mann G, Heitger A, Eibl G, Fink FM, et al
. High expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood acute lymphoblastic leukaemia. Br J Haematol
Schneider P, Vasse M, Al Bayati A, Lenormand B, Vannier JP. Is high expression of the chemokine receptor CXCR-4 of predictive value for early relapse in childhood acute lymphoblastic leukaemia? Br J Haematol
[Figure 1], [Figure 3], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]