|Year : 2014 | Volume
| Issue : 2 | Page : 80-85
Prognostic utility of CD184 in acute myeloid leukemia
Mohamed A Attia1, Sahar M Hazzaa1, Ola A El-Shora1, Tamer A El-Bedewy2, Loi M El-Ahwal2
1 Clinical Pathology Department, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Internal Medicine Department, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||24-Dec-2013|
|Date of Acceptance||10-Mar-2014|
|Date of Web Publication||30-Aug-2014|
Mohamed A Attia
Clinical Pathology Department, Faculty of Medicine, Tanta University, PO Box 527, 31211 Tanta
Source of Support: None, Conflict of Interest: None
Background Chemokine induce directional migration of cells toward a gradient of chemotactic cytokines (chemotaxsis). CD184 is a chemokine receptor which regulates the localization of leukemic cells, and most leukemic cells respond to it, with increased adhesion, survival and proliferation.
Aim The work was conducted to evaluate the role of CD184 in newly diagnosed patients with acute myeloid leukemia.
Subject and method Eighty patients presented with de novo AML. Based upon the distribution of CD184 expression in our patients' population, we defined groups with low CD184 expression as group I (MFIR 4 to 8), intermediate CD184 expression as group II (MFIR 9 to 20), and high CD184 expression as group III (MFIR more than 21). Patients with acute myeloid leukemia were studied at first diagnosis and were previously untreated. After diagnosis, patients received chemotherapy and they were followed up for periods ranged of 24 months with special attention to clinical and laboratory markers of remission and relapse, taking care to estimate the date of first complete remission, date of relapse, death or last seen alive.
Results patients were divided according to CD184 expression (MFIR) into 3 groups; group (I) with low expression (MFIRs ≤9) had a mean of 5.04 ± 1.48, group (II) with intermediate expression (MFIRs between 9 to 20) had a mean of 10.03 ± 0.45, and group (III) with high expression (MFIRs ≥20) had a mean of 30.25 ± 6.17. In the present study, we found no relation between CD184 expression levels in AML patients and the age of patients. Also, no significant relation was found between CD184 expression levels and the presence or absence of hepatosplenomegally and lymphadenopathy (P > 0.05 ). As regards to haematological data and MFIR of CD184 expression in AML patients, the mean leukocytic count was significantly lower in group (I) than in group (II) and group (III), and leucocytic count was significantly related to CD184 expression in the three groups. Also, the percentage of blasts in peripheral blood and bone marrow was significantly related to CD184 expression with P value < 0.05 while no significant relation was found with haemoglobin or platelets (P > 0.05). As regards to prognosis and MFIR of CD184 expression, group (I) showed 70% remission, 20% relapse and 10% death, while group (II) showed 33.33% remission and 66.67% relapse and group (III) showed 100% deaths meaning that high and intermediate CD184 expression were associated with low rates of remission and high frequency of relapse and death. We can conclude that CD184 expression can represent a useful prognostic tool for AML patients.
Keywords: CD184, AML, flow cytometry
|How to cite this article:|
Attia MA, Hazzaa SM, El-Shora OA, El-Bedewy TA, El-Ahwal LM. Prognostic utility of CD184 in acute myeloid leukemia. Egypt J Haematol 2014;39:80-5
|How to cite this URL:|
Attia MA, Hazzaa SM, El-Shora OA, El-Bedewy TA, El-Ahwal LM. Prognostic utility of CD184 in acute myeloid leukemia. Egypt J Haematol [serial online] 2014 [cited 2020 Feb 18];39:80-5. Available from: http://www.ehj.eg.net/text.asp?2014/39/2/80/139772
| Introduction|| |
Acute myeloid leukemia (AML) is a heterogenous group of neoplasms arising from transformation of hematopoietic stem cells, usually with retained partial capacity of differentiation  . It is a quickly progressive hematological neoplasm in which there are too many immature blood-forming cells in the blood and bone marrow (BM), the cells being specifically those destined to give rise to the granulocytes or monocytes, both types of white blood cells that fight infections. In AML, these blasts do not mature and so become too numerous  . Chemokines represent a large family of cytokines  . Human chemokine system includes more than 40 chemokines and 18 chemokine receptors  . They induce directional migration of cells toward a gradient of chemotactic cytokines (Chemotaxis)  . CD184 is a chemokine receptor, previously known as LESTR (leukocyte expressed seven transmembrane receptor), which was discovered as a coreceptor for HIV entry into CD4+ T cells. This coreceptor was given the name Fusin because it aided HIV fusion, but now it is called CXCR4  and it became well known that this receptor is widely expressed on hematopoietic cells and is functional in leukemic blasts  . In addition, it became well established that it is the receptor of stromal cell-derived factor-1 a and b (SDF-1)  . SDF-1/CD184 axis regulates the localization of leukemic cells, and most of the leukemic cells respond to SDF-1 with increased adhesion, survival, and proliferation  . The homing of AML cells within the marrow microenvironment is a multistep process. First, adhesion molecules such as selectins and integrins, for example leukocyte function-associated antigen-1, very late activation antigen-4 (VLA-4), and VLA-5 integrins, support AML transient adhesion to extracellular matrix (stromal) ligand named fibronectin  . Then, chemokine SDF-1 modulates reversible adhesion into firm one  by activating CD184, which directs spontaneous migration (directional migration) of AML cells beneath the marrow stromal cells  . This is proved by the fact that AML cell migration is significantly inhibited by antibodies to CD184 and by CS1 peptide that blocks VLA-4 binding  . The study was conducted to evaluate the role of CD184 in newly diagnosed patients with AML.
| Patients and methods|| |
Peripheral blood (PB) or BM aspirates of 80 patients diagnosed with AML were obtained at the time of diagnosis and were examined for CD184 expression during routine flow cytometry work-up, generally within 2-4 h after the sample was drawn. The diagnosis of AML was established using standard morphology and cytochemistry according to the French-American-British (FAB) classification, immunophenotypic criteria, and cytogenetic evaluation. For immunodetection of CD184 on AML cells, a whole blood lysis method was used with directly labeled primary antibodies following the manufacturer's instructions (Becton Dickinson, Heidelberg, Germany). Briefly, 100 μl aliquots of PB or marrow aspirate containing EDTA were incubated for 25 min with 10 μl of monoclonal antibody conjugates. For CD184 detection, 10 μl of PE-conjugated anti-CD184 (12G5) was used. A control sample was incubated with the appropriate isotype control antibodies. After incubation, 1 ml of fluorescence-activated cell sorter (FACS) lysing solution was added for 20 min, and cells were washed twice and analyzed within 2 h on a FACS Calibur (Becton Dickinson). All antibodies and FACS lysis solution were purchased from Becton Dickinson. Flow cytometry data were analyzed using the cell Quest software. A total of 10 000 events were acquired and gating was performed using forward scatter and side scatter, and percent of positivity was calculated. Mean fluorescence intensity ratios (MFIRs) were calculated by dividing the mean fluorescence intensity for CD184 by the mean fluorescence of the respective nonspecific isotype control.
Eighty patients presented with de-novo AML. On the basis of the distribution of CD184 expression in our patients' population, we defined groups with low CD184 expression as group I (MFIR 4-8), with intermediate CD184 expression as group II (MFIR 9-20), and with high CD184 expression as group III (MFIR > 21).
All patients were attendant of the Hematology/Oncolgy Unit, Pediatric and Internal Medicine Department, Tanta University Hospitals.
Patients with AML were studied at first diagnosis and were previously untreated. The diagnosis of AML was assessed by morphological study and cytochemistry of BM samples according to the FAB classification of acute leukemia and by immunophenotyping.
After diagnosis, patients received chemotherapy and they were followed up for a period of 24 months with special attention to clinical and laboratory markers of remission and relapse, taking care to estimate the date of first complete remission (CR), date of relapse, death, or last seen alive.
Patients were judged to have achieved CR when BM aspirates showed trilineage regeneration with less than 5% blasts by morphologic and immunocytochemical analysis, in the presence of normal blood count that persisted for at least 1 month. All other patients were considered nonresponsive  .
Data were analyzed using SPSS version 16. Quantitative data were expressed in the form of mean ± SD and qualitative data were described in the form of number and percentage. Differences between groups were evaluated with ANOVA. The survival analysis was carried out according to Kaplan-Meier product limit estimates.
| Results|| |
Detection of CD184 by flow cytometry
We examined for CD184 surface expression on AML blasts. Representative samples of high and low CD184 expression by AML cells are displayed in [Figure 1].
|Figure 1: The mean fluorescence intensity of one patient (green) and its negative control (violet).|
Click here to view
Using flow cytometry, we observed a continuum in the levels of CD184 expressed by AML cells in the 80 samples, ranging from MFIR values of 4-35.6 [Table 2]. In 40 samples (50%), the CD34 AML cells displayed CD184 expression levels below the MFIR threshold of 8, 24 samples (30%) had an intermediate CD184 expression with MFIR values between 9 and 20, and 16 patients (20%) exceeded the MFIR threshold of 21. As such, we assigned three groups with low, intermediate, or high CD184 expression to these threshold levels.
Association between CD184 expression and clinical data
The characteristics of all AML patients are displayed in [Table 1]. In [Table 2], we displayed the patients' characteristics for each of the three different CD184 expression groups (low, intermediate, and high). We noticed higher white blood cell counts and a higher frequency of blast percent in PB and BM, and higher LDH in patients with higher CD184 expression (groups II and III) when compared with patients with low CD184 expression (group I).
|Table 2: Clinical and laboratory characteristics of the acute myeloid leukemia patients when categorized by CD184 expression according to low (group I), intermediate (group II), or high (group III) CD184 expression|
Click here to view
After receiving chemotherapy, all of the studied patients were put under observation for detection of response to therapy and disease outcome.
Patients with low CD184 expression (group I) were good responders to therapy (achieved and maintained CR) compared with those with intermediate and high CD184 expression (groups II and III) [Table 3]. Kaplan-Meier analysis of overall survival (OS) and disease-free survival as a prognostic test revealed a statistically significant difference in prognosis between the three groups with significantly higher OS [Figure 2] and disease-free survival [Figure 3] in group I when compared with groups II and III.
|Table 3: Relationship between CXCR4 expression in the patients group and their prognosis|
Click here to view
|Figure 2: Impact of CD184 expression on overall survival (OS) in acute myeloid leukemia (AML) patients. This graph displays Kaplan– Meier estimates of OS of AML patients on the basis of CD184 expression on the leukemia cells at the time of diagnosis. OS was calculated for patients with low CD184 expression as defined by mean fluorescence intensity ratios (MFIRs) between 4 and 8 (group, n = 10), intermediate CD184 expression with MFIRs between 9 and 20 (group II, n = 6), and high CD184 expression with MFIRs equal to or more than 21 (group III, n = 4). Higher CXCR4 MFIRs correlated with a reduced OS. Log rank = 42.47; P = 0.0000.|
Click here to view
|Figure 3: Prognostic impact of CD184 expression on acute myeloid leukemia cells by evaluating the disease-free survival for all included patients using the Kaplan– Meier procedure.|
Click here to view
| Discussion|| |
AML represents a group of clonal hematopoietic stem cell disorders in which both failure to differentiate and overproliferation result in the accumulation of blasts  . It is the most common leukemia affecting adults  . Experienced morphologists can classify AML by blast appearance on Romanowsky-stained smear based on nuclear and cytoplasmic features. Immunophenotyping has been added in undifferentiated patients in whom morphology and cytochemistry are unrevealing, and electron microscopy is suggested when megakaryoblastic leukemia is suspected. Moreover, cytogenetic and occasionally molecular genetic studies are used to fully characterize the leukemic blasts  .
A number of clinical and laboratory features evident at diagnosis have prognostic value for predicting the remission duration of patients treated for acute leukemia. The identification of these prognostic factors has provided a means of stratifying patients into different risk groups and tailoring treatment accordingly; yet, the outcome remains largely unpredictable for most of the patients irrespective of the initial seriousness of the disease  .
Chemokines have been classified into four groups (C, CC, CXC, CX3C) on the basis of the positioning of their cysteine residues  . The binding of chemokines to their G protein-linked receptors on target cells leads to signal transduction events  , which in turn lead to adhesion and most importantly directional migration  , controlling T-cell migration during immune and inflammatory responses that occurs in infection or tissue injury in which immune cells bearing chemokine receptors are drawn from far distances to the sites of insults, where chemokines are released  .
In hematopoiesis, stem cells and progenitor cells migrate to and from various organs and tissues under the directional guidance of chemokines  . In addition, a number of studies emphasized the idea that tumor cell migration and organ-specific metastasis are critically regulated by chemokines and their receptors  .
SDF-1 belongs to the CXC chemokine family, which is characterized by one intervening amino acid separating the first two N-terminal cysteines. It signals through a G protein-coupled receptor termed CD184, originally named LESTER or fusin. The chemokine receptor CD184 has been shown to be expressed on different leukocytes. SDF-1/CD184 contributes to the extravasation of leukocytes, which is important for lymphocyte trafficking  .
In the present study, patients were divided according to CD184 expression (MFIR) into three groups: group I with low expression (MFIRs ≤ 9) had a mean of 5.04 ± 1.48, group II with intermediate expression (MFIRs between 9 and 20) had a mean of 10.03 ± 0.45, and group III with high expression (MFIRs ≥ 20) had a mean of 30.25 ± 6.17.
In the present study, we found no relationship between CD184 expressions levels in AML patients and the age of patients. In addition, no significant relationship was found between CD184 expression levels and the presence or absence of hepatosplenomegaly and lymphadenopathy (P > 0.05). In accordance with the previous results, Michael et al.  confirmed that neither age nor hepatosplenomegaly and lymphadenopathy were related to the expression of CD184 in AML patients.
With respect to hematological data and MFIR of CD184 expression in AML patients, the mean leukocytic count was significantly lower in group I than in group II and group III, and leukocytic count was significantly related to CD184 expression in the three groups. In addition, the percentage of blasts in peripheral blood and BM was significantly related to CD184 expression with P value of less than 0.05, whereas no significant relationship was found with hemoglobin or platelets (P > 0.05).
In agreement with the present results, Anke et al.  reported a significant relationship between CD184 expression and leukocytic and blast count, with no relationship with the other traditional parameters.
With respect to prognosis and MFIR of CD184 expression, group I showed 70% remission, 20% relapse, and 10% death, whereas group II showed 33.33% remission and 66.67% relapse and group III showed 100% deaths meaning that high and intermediate CD184 expression were associated with low rates of remission and high frequency of relapse and death.
In agreement with the present results, Rombouts et al.  found a strong relationship between the surface expression of CD184 on AML cells and the OS. Patients who had low CD184 expression had a significantly longer OS than those who had intermediate or high expression. In addition, Konoplev et al.  confirmed the bad prognostic significance of CD184 expression in AML patients. In addition, Spoo et al.  described a decreased survival for the patients groups with high and intermediate expression.
A potent mechanism in the trafficking of leukemia cells is the interaction of the chemokine receptor CD184, which is variably expressed on leukemia cells, and its ligand SDF-1, which is constitutively produced by stromal cells in the BM  . This interaction contributes to marrow infiltration of leukemia blasts depending on the expression intensity of CD184 on malignant blasts  .
Broxmeyer et al.  hypothesized that the SDF-1/CD184 axis may also play essential roles in the increased survival, decreased proliferation and differentiation, and increased anchorage of human AML stem cells in the BM.
Exogenous SDF-1 moderately enhances in-vitro survival of AML cells that express CD184 on their surface in the absence of growth factors. This may give an advantage in the growth and dissemination of blast cells, especially in view of the fact that BM SDF-1 levels increase after irradiation and chemotherapy  .
Hope et al.  noticed a decreased proliferation and a partial block in differentiation of migrated AML cells within the stromal layer. This suggests that CD184 expression by AML cells favors the enrichment of a more primitive, noncycling subpopulation of AML cells within the stromal layer. These cells may be less susceptible to cytotoxic treatments  .
CD184 can represent novel therapeutic target in AML, as a previous study reported that CD184 antagonists could mobilize AML cells from the protective BM microenvironment and reverse chemotherapy resistance. The general feasibility and safety of such an approach has been demonstrated by studies in which CD184 antagonists were administered to patients or volunteers, although an increased toxicity to normal hematopoiesis due to simultaneous progenitor cell mobilization is a concern  .
We can conclude that CD184 expression can represent a useful prognostic tool for AML patients and its expression is easy to determine in routine flow cytometry analysis of AML samples at diagnosis.
| Acknowledgements|| |
| References|| |
|1.||Jaffe ES, Harris N, Stein H. (eds) World Health Organization classification of tumours: pathology and genetics of tumours of haematopoietic and lymphoid tissues. Lyon, France: IARC Press; 2001; pp 28-43. |
|2.||Cheson BD, Bennett JM, Kopecky KJ. Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol 2003; 21 :4642-4649. |
|3.||Sica A. Defective expression of the monocyte chemotactic protein-1 receptor CCR2in macrophages associated with human ovarian carcinoma. J Immunol 2000; 164 :733-738. |
|4.||Loetscher P, Moser B , Baggiolini M. Chemokines and their receptors in lymphocyte traffic and HIV infection. Adv Immunol 2000; 74 :127-180. |
|5.||Murdoch C, Finn A. CXCR4: chemokine receptor extraordinaire. Immunol Rev 2000; 177 :175-184. |
|6.||Crazzolara R, Kreczy A, Mann G. High expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood acute lymphoblastic leukaemia. Br J Haematol 2001; 115 :545-553. |
|7.||Bleul CC, Farzan M, Choe H. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 1996; 382 :829-833. |
|8.||Jaurez J, Bendall L. SDF-1 and CXCR4 in normal and malignant hematopoiesis. Histol Histopathol 2004; 19 :299-309. |
|9.||Peled A, Kollet O, Ponomaryoy T. The chemokine SDF-1 activates the integrins LFA-1. VLA-4 and VLA-5 on immature human CD34+cells: role in transendothelial / stromal migration and engrafment of NOD/SC1D mice. Blood 2000; 95 :3289-3296. |
|10.||Sieg DJ, Hauck CR, llic D. FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2000; 2 :249-256. |
|11.||Burger JA, Spoo A, Dwenger A. CXCR4 chemokine receptors (CD184) and alpha4beta1 integrins mediate spontaneous migration of human CD34_ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis). Br J Haematol 2003; 122 :579- 589. |
|12.||Konopleva M, Konoplev S, Hu W. Stromal cells prevent apoptosis of AML cells by up-regulation of anti-apoptotic proteins. Leukemia 2002; 16 :1713-1724. |
|13.||Nozomi N, Okabe -Kodo J, Michihiro N, et al. Plasma level of the differentiation inhibitiory factor nm-24H1 protein and their clinical implication in acute myelogenous leukemia. Blood 2000; 1 :3. |
|14.||Jemal A, Tiwari RC, Murray T. Cancer statistics 2004. CA Cancer J Clin 2004; 54 :8-29. |
|15.||Stewart AK, Schuh AC. Impact of understanding the molecular basis of haematological malignant disorders on clinical practice. Lancet 2000; 355 : 1447-1453. |
|16.||Payne AS, Cornelius LA. The role of chemokines in melanoma tumour growth and metastasis. J Invest Dermatol 2002; 118 :915-922. |
|17.||Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk between tumour cells and their microenvironment. Blood 2006; 107 :1761-1767. |
|18.||Kim J, Takeuchi H, Lam ST. Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk of recurrence and for poor survival. Journal of Clinical Oncology 2005; 23 :2744-2753. |
|19.||Mebius RE. Organogenesis of lymphoid tissues. Nat Rev Immunol 2003; 3 :292-303. |
|20.||Taichman RS, Cooper C, Keller ET. Use of the stromal cellderived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res 2002; 62 :1832-7. |
|21.||Burger M, Hartmann T, Krome M. Small peptide inhibitors of the CXCR4 (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCLL12 in chronic lymphocytic leukaemia B cells. Blood 2005; 106 :1824-1830. |
|22.||Michael L, Spoo AC, William G. CXCR4 is a prognostic marker in AML. Blood 2007; 109 :786-791. |
|23.||Spoo AC, Michael L, William G. CXCR4 is a prognostic marker in AML. Blood 2007; 109 :786-791. |
|24.||Rombouts EJ, Pavic B, Lowenberg B. Relation between CXCR-4 expression,Flt3 mutations, and unfavorable prognosis of adult acute myeloid leukemia. Blood 2004; 104 :550-557. |
|25.||Konoplev S, Rassidakis GZ, Estey E. Overexpression of CXCR4 predicts adverse survival in patients with unmutated FLT3 AML with normal karyotype. Cancer 2007; 109 :1152-1156. |
|26.||26 Spoo AC, Lubbert M, Wierda WC. CXCR4 is a prognostic marker in AML. Blood 2007; 109 :786-791. |
|27.||Corcione A, Arduino N, Ferretti E. Chemokine receptor expression and function in childhood acute lymphoblastic leukaemia of Blineage. Leuk Res 2006, 30 :365-372. |
|28.||Crazzolara R, Kreczy A, Mann G. High expression of the chemokine receptor CXCR4 predicts extra-meduulary organ infilteration in childhood acute lymphoblastic leukaemia. Br. J. Haematol 2002; 115 :545-553. |
|29.||Broxmeyer HE, Kohli L, Kim CH. Stromal cell-deried factor- 1/CXCL12 directly enhances survival/antiapoptosis of myeloid progenitor cells through CXCR4 and Galphai proteins and enhances engraftment of competitive, repopulating stem cells. J Leukoc Biol 2003; 73:630-638. |
|30.||30 Ponomaroov T, Peled A, Petit I. Increased expression of the chemokine SDF-1 following DNA damage: relevance for human stem cell function. J Clin Investig 2000; 106 :1331-1339. |
|31.||31. Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in selfrenewal capacity. Nat Immunol 2004; 5 :738-743. |
|32.||32. Broxmeyer HE, Orschell CM, Clapp DW. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 2005; 201 :1307-1318. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]