|Year : 2013 | Volume
| Issue : 1 | Page : 1-6
Clinical relevance of thrombospondin receptor (CD36) expression in Egyptian de novo adult acute myeloid leukemia
Sherin M. Abd El-Aziz1, Dalia A. Salem1, Manal A. Salah-Eldin2
1 Department of Clinical Pathology, Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt
2 Department of Medical Oncology, Oncology Center, Mansoura Faculty of Medicine, Mansoura, Egypt
|Date of Submission||11-Sep-2012|
|Date of Acceptance||01-Oct-2012|
|Date of Web Publication||20-Jun-2014|
Sherin M. Abd El-Aziz
Department of Clinical Pathology, Mansoura Faculty of Medicine, 35516 Mansoura
Source of Support: None, Conflict of Interest: None
Acute myeloid leukemias (AMLs) are a heterogeneous group of disorders that often present with different morphological, immunophenotypic, and cytogenetic patterns. Identification of these characteristics may be useful for a better prognostic evaluation and for a more appropriate therapeutic approach. CD36 is a transmembrane, highly glycosylated, glycoprotein commonly expressed on blasts in acute monocytic leukemia, megakaryoblastic leukemia, and erythroleukemia.
Patients and methods
We evaluated CD36 surface expression in 97 newly diagnosed AML patients, and the results were correlated with the morphology, immunophenotype, cytogenetic pattern, and clinical outcome.
CD36 antigen was recorded in 48 of 97 patients (49.5%) and particularly in those with M5 and M6 FAB subtypes. Moreover, CD36 expression was significantly associated with the expression of CD11b (P=0.001) and CD14 (P=0.0001), unfavorable cytogenetic abnormalities (P=0.001), shorter overall survival (P>0.0001), and leukemia-free survival (P=0.03).
On the basis of the results of the study, it can be concluded that CD36 expression in AML patients may identify a subgroup with a poor prognosis, and may thus be a valuable adjunct to be added to the current prognostic factors.
Keywords: acute myeloid leukemia, CD36, immunophenotyping
|How to cite this article:|
Abd El-Aziz SM, Salem DA, Salah-Eldin MA. Clinical relevance of thrombospondin receptor (CD36) expression in Egyptian de novo adult acute myeloid leukemia. Egypt J Haematol 2013;38:1-6
|How to cite this URL:|
Abd El-Aziz SM, Salem DA, Salah-Eldin MA. Clinical relevance of thrombospondin receptor (CD36) expression in Egyptian de novo adult acute myeloid leukemia. Egypt J Haematol [serial online] 2013 [cited 2020 Feb 18];38:1-6. Available from: http://www.ehj.eg.net/text.asp?2013/38/1/1/128295
| Introduction|| |
Acute myeloid leukemia (AML) is a complicated, heterogeneous disease involving the presence of a clonal expansion of neoplastic myeloid cells with variable degrees of differentiation. Interactions of hemopoietic progenitor cells, endothelial cells, and stromal cells play a role in the migration or homing of these cells and in the regulation of hemopoiesis and are mediated by several specific cell surface receptors 1,2. AML often presents with different morphological, immunophenotypic, and cytogenetic patterns 3. Identification of these characteristics may be useful for a better prognostic evaluation and for a more appropriate therapeutic approach. Various chromosomal aberrations have been identified in AML patients and are uniquely associated with distinct clinical entities and prognostic relevance. Cytogenetics in AML is widely accepted as one of the major prognostic factors in all age groups and was the initial factor considered in the reclassification of AML by the WHO 4,5.
Immunophenotyping of AML has controversial implications with respect to prognosis. Many associations have been described between individual antigen expression on myeloid blasts and prognosis; however, few are consistent. Immunophenotypic markers that, in various studies, have been implicated as predictive of adverse outcomes include CD7, CD9, CD11b, CD13, CD14, CD15, CD33, CD34, CD38, CD56, CD117, myeloperoxidase (MPO), and terminal deoxynucleotidyl transferase 6–14; however, other studies have yielded conflicting results. The lack of methodological standardization, the size and type of patient population studied, single versus multi-institutional series, and other factors have all contributed toward the controversy.
CD36 is a transmembrane, highly glycosylated, glycoprotein expressed by monocytes, macrophages, platelets, microvascular endothelial cells, and adipose tissues. It recognizes and interacts with a large variety of ligands, including collagen type I and IV, thrombospondin, lipids, and plasmodium falciparum-infected erythrocytes. It is involved in tumor angiogenesis, phagocytosis of apoptotic cells, and foam cell formation by uptake of oxidized low-density lipoproteins. CD36 is commonly expressed on blasts in acute monocytic leukemia, megakaryoblastic leukemia, and erythroleukemia 15–17.
It has been reported previously that the presence of the CD36 antigen on blast cells in AML patients could be used to identify a subgroup of patients with an adverse prognosis irrespective of molecular or cytogenetic abnormalities 18.
To better clarify the prognostic role of CD36 expression in AML cells, in the present study, we evaluated the presence of this antigen on leukemic cells of 97 newly diagnosed adult patients with AML and the results were correlated with the presence of other antigens with prognostic significance, cytogenetic pattern, and clinical outcome.
| Patients and methods|| |
A total of 103 adult patients with untreated AML, excluding patients with acute promyelocytic leukemia and secondary AML, were enrolled in this study from March 2009 to February 2011. Six patients (5.8%) received only supportive care and were thus excluded from this analysis. The median age of the remaining 97 patients was 39 years (range: 19–66 years), with a male/female ratio of 1.8 : 1. The clinical and laboratory findings of the patients are listed in [Table 1]. After informed consent and institution ethical committee approval were obtained, peripheral blood and/or bone marrow samples were taken at diagnosis for morphological examination, enzyme cytochemical staining, flow cytometric immunophenotyping, conventional karyotyping, and fluorescent in-situ hybridization (FISH) analyses. The diagnosis and classification of the AML patients were based on clinical presentation, morphology, immunophenotyping, and cytogenetic analysis 19.
|Table 1: Demographic, clinical, and laboratory findings in the adult acute myeloid leukemia patients studied|
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Flow cytometric analysis
All EDTA-anticoagulated peripheral blood/bone marrow samples were immediately transported to the FC laboratory. For surface antigen staining, RBCs were lysed using ammonium chloride lysing solution (8 g ammonium chloride, 1 g EDTA, and 0.1 g dihydrogen potassium phosphate in 1 l distilled water to yield ×10 lysing solution). The cells were stained with different combinations of fluorescently labeled monoclonal antibodies (mAbs) according to the manufacturer’s recommendations (DakoCytomation, Glostrup, Copenhagen, Denmark and Beckman Coulter, Hialeah, Florida, USA). For the detection of cytoplasmic and nuclear antigens, the Intraprep Permealization Kit (Beckman Coulter) was used before the addition of mAb to allow its access into the cytoplasm according to the manufacturer’s recommendations. The mAbs were used in different combinations of fluorochromes, namely, fluorescein isothiocyanate, phycoerythrin, and phycoerythrin-cyanine 5. Triple staining of the blast cells with cCD3/cCD79a/MPO was carried out as a primary step to confirm their myeloid origin. After that, different combinations of mAb against the following antigens were used: CD34, human leukocyte antigen (HLA-DR), CD117, CD13, CD33, CD14, glycophorin A, CD41, CD61, CD36, and CD11b. The immunophenotyping was performed on an EPICS-XL flow cytometer (Coulter, Miami, Florida, USA). The cells were analyzed with the most appropriate blast gate using the combination of forward and side scatters. If the percentage of an antigen was more than 20%, the leukemic sample was considered to be positive for that surface marker 20.
Conventional karyotyping and fluorescent in-situ hybridization analysis
The results of conventional karyotyping were retrieved from patients’ files, whereas FISH analysis was carried out in our laboratory. The target DNA sequence in the chromosome to be analyzed is denatured and hybridized to a single-strand fluorophore-labeled complementary nucleic acid sequence (probe) using the Vysis Hybrite machine (Olympus Life Science), which is then detected using fluorescent microscopy (Olympus BX61 microscope; Olympus Life Science, Hamburg, Germany). The results of FISH analysis were reported using the International System for Human Cytogenetic Nomenclature (ISCN-1995) 21–23. Fluorophore-labeled locus-specific identifier probes for AML1/ETO fusion, MLL break apart, and CBFβ-MYH11 fusion were used for the detection of the t(8;21)(q22;q22), rearrangement of the MLL gene at chromosome 11 band q23 and inversion in chromosome (16)(p13q22), respectively (Cytocell, Cambridge, UK).
Patients received intravenous standard induction treatment including a combination of anthracycline (doxorubicine 50 mg/m2/day for 3 days) and cytosine arabinoside (100–200 mg/m2/day for 7 days) adapted according to age. Postremission therapy for individuals younger than 55 years of age was priority based and adapted according to cytogenetic risk. In intermediate-risk and high-risk patients, an allogeneic transplantation with a family donor had the highest priority if they had an HLA-matched sibling donor. All other patients received postremission therapy with four courses of high-dose cytosine arabinoside (2–3 g/m2) every 12 h for 3 days. Patients older than 55 years of age received only one to two postremission therapy course(s) of high-dose cytosine arabinoside after complete remission. Some patients (6/103), because of their advanced age or their bad performance status, received only supportive treatment and were excluded from this study.
Data were analyzed on a personal computer running SPSS for windows (statistical package for social science, version 17; SPSS Inc., Chicago, Illinois, USA). For descriptive statistics of the qualitative variables, the frequency distribution procedure was run with the calculation of the number of patients and percentages. For descriptive statistics of the quantitative variables, the mean and SD were used to describe the central tendency and dispersion. The χ2-test was used to compare the differences between the groups. The correlation among CD36 expression and other antigens was estimated using Spearmen’s correlation coefficient. Overall survival (OS) was measured from the date of enrollment until date of death, and leukemia-free survival (LFS) for patients who achieved complete remission (CR) was measured from the date of CR to relapse or death. OS and LFS were plotted using the Kaplan–Meier method. Comparison of the survival was carried out using the log-rank test. A univariate Cox proportional hazards model was used to evaluate the possible associations between OS, LFS, and each risk factor singly. Variables identified as statistically significant in univariate analysis were subsequently included in a stepwise multivariate Cox proportional hazards model. Differences were considered to be statistically significant when the P value was 0.05.
| Results|| |
Antigenic expression in acute myeloid leukemia patients
According to the Frensh-American-British (FAB) criteria, the most frequent subtype observed in our patients was M4 (43.3%), followed by M2 (24.7%). Immunophenotypic results showed that all our patients expressed MPO, whereas none expressed cCD79a or cCD3. The most frequently expressed antigens on myeloblasts were CD33 (95.9%), HLA-DR (87.6%), and CD13 (82.5%) [Table 1]. CD34 and CD117 were mostly expressed on myeloblasts of the M1-FAB subtype (82.8 and 89.4%, respectively). However, HLA-DR was expressed frequently on M2 myeloblasts (87.2%). CD13 and CD33 were most commonly expressed in the M4-FAB subtype (88.1 and 97.8%, respectively). The most frequently expressed antigens in AML-M5 were CD14, CD36, and CD11b (100, 81.3, and 68.8%, respectively). In addition, CD36 was expressed in 100% (4/4) of M6 patients.
Correlation of CD36 expression with immunophenotype and cytogenetic results
Immunophenotyping analysis showed that the CD36 antigen was positive in 48 of 97 patients (49.5%). It was most frequently expressed in M6-FAB (100%), M5-FAB (81.3%), and M4-FAB (50%), whereas it was less commonly expressed in M2-FAB and M1-FAB (37.5 and 18.2%, respectively). We did not find any significant correlation between CD36 and other myeloid markers such as CD33 or CD13 expression. However, CD36 expression was correlated negatively with some immature markers, namely, CD34 and CD117 (P=0.0001 and 0.03, respectively). Despite the low number of patients, an increased expression of CD11b and CD14 was found in 72.9 and 77.1%, respectively of CD36+ patients and only in 26.5 and 34.7%, respectively of CD36− patients, suggesting a strict correlation between CD36, CD11b, and CD14 positivity (P=0.0001 and 0.001, respectively).
Cytogenetic analysis was evaluable only in 76 of 97 patients, 40 of whom were CD36+ and 36 were CD36−. Fourteen patients (18.4%) were categorized as low risk [t(8;21) or inv 16], 39 (51.3%) as intermediate risk (normal karyotyping), and 23 (30.3%) with unfavorable cytogenetics (11q23 abnormality and complex karyotyping) [Table 1]. On comparing the distribution of unfavorable cytogenetics between patients either CD36+ or CD36−, it was found that unfavorable cytogenetics was detected in 17 of 40 CD36+ patients and only in six of 36 CD36− patients, with a highly significant correlation (P=0.001) [Table 2].
|Table 2: Correlation of CD36 expression with demographic, clinical, laboratory findings, and patient outcome|
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Correlation of CD36 with remission rate and outcome
To address the hypothesis of whether CD36 expression could represent an adverse prognostic factor in AML, its presence was correlated with clinical response. Response was evaluated after induction therapy in 91 of 97 patients who received the same treatment. Six AML patients, equally distributed in both the CD36+ and the CD36− groups, who died during induction (i.e. death during the first period after induction therapy before hematological recovery) were considered as failures in the response analysis and were regularly included in the survival analysis.
The overall CR rate in our series of patients was 60.4%. CR was achieved in 32 of 46 (69.5%) CD36− patients and in 23 of 45 (51.1%) evaluable CD36+ patients [Table 2]. Although a larger number of responders could be recorded among CD36− patients versus CD36+ patients, the difference was not significant (P=0.073).
Survival analysis was carried out and, as shown in [Figure 1] and [Figure 2], the 2-year OS and LFS rates were shorter in CD36+ than in CD36− patients. The OS rates were 37.5% for CD36+ patients and 44.9% for CD 36− patients (P=0.001), and the LFS rates were 33.3 and 40.6% for CD36+ and CD 36− patients, respectively (P=0.03).
Prognostic factors in studied acute myeloid leukemia patients
We used the Cox proportional hazards model to determine associations between LFS, OS, and the various risk factors available to us in this group of AML patients. The expression of CD36, CD11b, and CD34 on blast cells, WBCs count, and adverse cytogenetics were all significant factors in determining LFS and OS [Table 3]. When positive prognostic factors in univariate analysis were considered in a multivariate analysis, the expression of CD36, CD34, CD11b, and adverse cytogenetics retained their statistical significance as independent negative prognostic factors for the detection of OS and LFS.
|Table 3: Univariante and multivariante analysis for overall survival and leukemia-free survival|
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| Discussion|| |
AML represents a heterogeneous complex disorder on the basis of diverse cytogenetics and molecular alterations with a huge influence on prognosis and therapeutic decisions. Approaches to improve AML therapy have often focused on the stratification of patients for the intensity of postinduction treatment according to defined risk categories mainly on the basis of genetic criteria. However, about half of the patients have no detectable specific chromosomal abnormalities, and additional prognostic factors are required to optimize the use of presently available therapies.
Flow cytometric immunophenotyping plays a well-established role as a diagnostic modality in acute leukemia, particularly as a tool for assigning lineage and facilitating further pathologic classifications 24. Although several authors have investigated the prognostic implications of flow cytometric immunophenotyping in AML, no clear consensus has emerged on its role in predicting treatment response, relapse, or OS.
In this study, we examined the expression and prognostic value of CD36 in 97 de novo AML patients at the initial diagnosis. A positive expression of CD36 was found in 49.5% of patients, which was higher than the level recorded in previous studies, which varied between 37 and 41.5% 18,25. This variation may be because of limited number of cases and the higher prevalence of M4/M5-FAB subtypes that express more CD36 in our study.
In our patient cohort, low proportions of CD36 were detected in AML-M2 and AML-M1, whereas the highest proportions of CD36 were detected in M5 and M6 FAB subtypes. This had already been reported by other groups and could be explained at least in part by the differential expression of CD36 during the course of myeloid differentiation 18,25–27. Plesa et al. 28 used both CD36 and CD34 as key points in the differentiation of myelomonocytic progenitor cells into either monoblasts or myeloblasts. In both cases, the expression of CD34 specific for myelomonocytic progenitor cells decreased. Differentiation into monoblasts was accompanied by an increase in CD36 expression, whereas this was not the case for differentiation into myeloblasts. This finding could be attributed to the fact that the CD36 antigen is constitutively expressed in normal monocyte and monocyte-derived cells.
In agreement with previous reports 25, 29, our results indicate that the expression of CD36 was correlated with antigens frequently associated with a negative prognostic significance such as CD11b and CD14 expression levels. When the expression patterns of specific antigens in AML were explored by Webber et al. 27, monocytic markers were found to be moderately frequent, including CD11b (41%), CD36 (34%), and CD14 (16%). Moreover, CD36 was found to be expressed in both mature and immature monocytic cells, making it a useful marker of monocytic differentiation in immature monocytes that lack CD14 expression 30.
An important point that needs to be considered is the correlation between CD36 positivity and cytogenetic abnormalities. In this study, CD36 was more commonly expressed in the unfavorable cytogenetic group, which is in agreement with a previous study that reported a significant correlation between CD36 expression and the high-risk cytogenetics characterizing a prognostically unfavorable group of AML patients 31.
In terms of clinical outcome, our findings support previous reports ascribing a poor prognosis to AML patients expressing CD36 18. In our patient population, the expression of CD36 on leukemic cells was confirmed by multivariate analysis as an independent prognostic factor for the prediction of a group of patients with a significant decrease in OS and LFS. Although significance was not reached, there was a tendency toward a lower response rate to chemotherapy in the group of patients with a positive CD36 expression compared with CD36− patients.
| Conclusion|| |
The understanding of the importance of CD36 expression in AML is relatively recent, and not all laboratories routinely include this marker in the diagnostic characterization panel. We believe that the results presented here, although preliminary, should foster accurate research of CD36 expression in AML at diagnosis, which would allow the analysis of its prognostic significance in larger cohorts.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]