Abstract
Background Melanoma antigen encoding gene A3 (MAGE-A3) gene, also called cancer/testis (CT) antigen, is a member of MAGE multigene family, which is located on the long arm of the X chromosome, and its expression can be caused by promoter region demethylation. The MAGE-A3 proteins' functions are unknown, but they were found to play a role in cell cycle progression, transcriptional regulation, and drug resistance. The aims of this study were to determine the expression of the MAGE-A3 gene in an Egyptian cohort of de novo acute myeloid leukemia (AML) patients and to define its role in the development of AML and its correlation with clinical presentation, laboratory data, as well as treatment outcome.
Patients and Methods This study included 40 de novo AML patients as well as 30 age- and sex-matched normal healthy subjects as a control group. They were all subjected to reverse transcription-polymerase chain reaction assay for the detection of MAGE-A3 gene expression.
Results Our study revealed that 23 AML patients (57.5%) expressed the MAGE-A3 gene, whereas none of the control group subjects (0%) expressed this gene. It was found that the expression of MAGE-A3 gene was associated with an increased risk of AML (odds ratio = 2.763; 95% confidence interval, 1.890-8.041). Regarding treatment outcome, a highly statistical significant difference was found between MAGE-A3-positive and -negative AML patients with P < 0.001, as the MAGE-A3-positive AML patients had a higher incidence of unfavorable treatment outcome, whereas the MAGE-A3-negative patients had a higher incidence of favorable outcome. This clarifies that the MAGE-A3 gene expression was found to have a significant impact on the risk of treatment failure (odds ratio = 3.591; 95% confidence interval, 1.273-10.462).
Conclusions The expression of MAGE-A3 gene may have a clinical relevance and important role as a risk factor in the development of AML. It may be considered as a prognostic marker and may be useful as a predictive test for treatment outcome in AML.
Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder characterized by a block in differentiation of hematopoiesis, resulting in an overgrowth of a clonal population of immature cells or blast cells of myeloid lineage that is frequently associated with genetic instability characterized by a diversity of chromosomal and molecular changes.1Acute myeloid leukemia is a complex disease with a wide range of clinical, morphologic, biologic, cytogenetic, molecular, and immunophenotypic features.2With this multitude of disease-associated variables, it is not surprising that response to treatment differs considerably among patients.3Although significant progress has been made in the management of these disorders, most patients with leukemia who fail frontline therapies or who relapse after an initial response, generally have a poor prognosis and eventually will relapse from minimal residual disease (MRD) and die of progressive disease. As the relative ineffectiveness and toxicities of traditional cytotoxic therapies become more appreciated, the search for therapeutic advances is increasingly focused on affecting the critical steps involved in the development and mutation of malignant clones.4Also, a better understanding of leukemic cells is needed to identify new prognostic markers and to choose adapted therapeutic strategies.5
Several leukemia-associated antigens (LAAs) have been identified in patients with AML as melanoma antigen-encoding genes (MAGE), BAGE, receptor for hyaluronic acid-mediated motility, PRAME, FLT3-ITD, Wilms tumor antigen 1, human telomerase reverse transcription, survivin, and proteinase 3.6The expression of these LAAs might play a critical role in leukemogenesis and the persistence of MRD in AML and therefore might be associated with poor clinical outcome in AML.7
Leukemia-associated antigens recognized by autologous T lymphocytes are encoded by genes, including those of the MAGE gene family, which includes MAGE-A, -B, -C, -D, -E, -F, -G, and -H subfamilies, which encodes peptides recognized by autologous cytotoxic T lymphocytes in a major histocompatibility complex class I-restricted fashion.8The MAGE-A, MAGE-B, and MAGE-C subfamilies, which are encoded on the X-chromosome are now called class I MAGE antigens. The most common are the MAGE-A genes, which are located on the chromosome Xq28, whereas MAGE-B genes, also called DAM-6, are located on chromosome Xp21, whereas MAGE-C genes are located on chromosome Xp26-27.6
Melanoma antigen encoding gene A3 (MAGE-A3) gene, also called cancer/testis (CT) antigen, is a member of MAGE multigene family, which is located on the long arm of the X chromosome and is composed of 3 exons, and the large open reading frame is entirely located in the third exon.9Functions of MAGE-A3 proteins are unknown, but they were found to play a role in cell cycle progression, transcriptional regulation, and drug resistance.10
MAGE-A3 gene is silent in normal healthy adult tissues with the exception of placenta and testis where it seems to be expressed in male germline cells that do not bear HLA class I molecules.11It is widely expressed in cancers of several histologic types such as melanomas, carcinomas of lung, head and neck, liver, urinary bladder and colorectal, ovarian tumors, seminomas, neuroblastomas, and osteosarcomas.12Also, MAGE-A3 is expressed in hematological malignancies but in a lower frequency than solid tumors.13MAGE-A3 is considered to play a pivotal role in oncogenesis and in cancer progression, and its overexpression in adult leukemia potentially contributes to leukemogenesis and development of AML by permitting proliferation and prolonging the survival of malignant cells.14
Leukemia-associated antigens such as proteins encoded by melanoma antigen-encoding genes including MAGE-A3, which is an immunogenic antigen in leukemias and solid tumors because it induces specific T-cell immune responses in tumor patients, might represent a potential target structure for specific cellular immunotherapies and antibody therapies of leukemia.8
The aim of this work was to study the expression of MAGE-A3 among de novo AML patients using reverse transcription-polymerase chain reaction (RT-PCR) assay and to define and determine its role in the development of AML and its correlation with the clinical presentation, laboratory data, as well as treatment outcome.
PATIENTS AND METHODS
Patients
The present study included 40 patients with de novo AML, their ages ranged between 12 and 63 years, with a mean (SD) of 33.36 (13.43) years and a median of 32 years. They were 26 male (65%) and 14 female (35%). Patients were studied before chemotherapy. All patients were diagnosed and recruited among cases referred to El-Kasr El-Aini Teaching Hospital, Cairo University, Cairo, Egypt. Thirty age- and sex-matched normal healthy individuals were also included as a control group.
The diagnosis of leukemia was based on complete history taking, clinical examination for organomegaly and lymphadenopathy, and laboratory investigations for diagnosis of AML including complete blood cell count, cytochemistry, and immunophenotyping of leukemic blast cells and special laboratory investigations (for patients and controls) to detect MAGE-A3 expression using RT-PCR technique according to the method described by Martínez et al.14
Methods
Sample Collection
Three milliliters of venous blood was collected by sterile venipuncture under complete aseptic conditions on 1.5 mg/mL potassium ethylene diamine tetra-acetic acid sterile vacutainer from patients and control subjects and divided as follows: 1 mL of venous blood for performing complete blood picture and 2 mL for the study of MAGE-A3 expression by RT-PCR assay.
Detection of MAGE-A3 Expression by RT-PCR
Separation of Mononuclear Cells
Mononuclear cells layer was separated under complete aseptic conditions using Ficoll Hypaque (Biochrom AG, Berlin) density gradient centrifugation.
RNA Extraction
Genomic RNA was extracted from cells by using TRIzol one-step RNA isolation reagent (Life Technologies, Paisley, UK) according to the manufacturer's protocol.
PCR for Amplification of MAGE-A3 Gene
One microgram of genomic RNA was used to synthesize complementary DNA by M-MuLV reverse transcription enzyme (Life Technologies) according to the manufacturer's protocol. Then, a mixture 25-μL reaction consisted of 2.5 μL of genomic DNA, 12.5 μL of PCR master mix (Qiagen, Crawley, UK), 2 μL of each primer, and 6 μL of DW was prepared.
MAGE-A3 Gene
The following primers (Qiagen) were used: forward primer 5′-TGGAGGACCAGAGGCCCCC-3′ and reverse primer 5′-GGACGATTATCAGGAGGCCTGC-3′.
The following cycles were used: initial heat activation step at 94°C for 10 minutes and 35 cycles of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, extension at 72°C for 4 minutes and then final extension at 72°C for 10 minutes.
β-Actin
The following primers (Qiagen) were used: forward primer 5′-TCATGTTTGAGACCTTCAA-3′ and reverse primer 5′-GTCTTTGCGGATGTCCACG-3′.
The following cycles were used: initial heat activation step at 94°C for 5 minutes and 35 cycles of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, extension at 68°C for 2 minutes, and then final extension at 68°C for 7 minutes.
Detection of PCR Products
The amplified products samples were then run in parallel on 2% agarose gel using gel electrophoresis (electro-4, Thermal Hybaid; Promega, Madison, WI), stained with ethidium bromide and visualized on a UV transilluminator (wavelength, 312nm) to detect the presence or absence of DNA bands. A DNA molecular weight marker (Fermentas AM, Cairo, Egypt) (100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 bp) was used. For MAGE-A3 and β-actin, 815- and 500-bp fragments were amplified, respectively, as shown in Figures 1 and 2.
Declaration of Ethics
A written informed consent was obtained from all patients according to the Helsinki guidelines of research ethics.
Statistical Analysis
Data were analyzed using SPSS Windows Statistical Package Version 15 (SPSS Inc., Chicago, IL). Numerical data were expressed as a mean, SD, and range as appropriate. Qualitative data were expressed as frequency and percentage. The χ2 test or the Fisher exact test was used to examine the relation between qualitative variables. Odds ratio (OR) and 95% confidence interval (CI) were calculated for risk estimation. For quantitative data, the Student t test for independent samples was used in comparing 2 groups normally distributed and the Mann-Whitney test was used in comparing 2 groups not normally distributed. The relation between numerical variables was tested using Pearson product-moment correlation coefficient. P < 0.05 was considered significant, and P < 0.01 was considered highly significant.
RESULTS
Characteristics of patient and control groups are summarized in Table 1.
The statistical comparison of MAGE-A3 gene expression in AML patients and control group subjects was studied. The comparison revealed a highly significant difference between the 2 groups, with a much higher expression of MAGE-A3 gene among AML patients than among control group subjects because 23 (57.5%) of 40 AML patients had a positive MAGE-A3 gene expression, although none of the control group (0%) had a positive expression (P < 0.001).
The correlation of MAGE-A3 gene expression with age, sex, clinical, and laboratory data in AML patients revealed a highly significant negative correlation between MAGE-A3 expression and hemoglobin level and platelet count (r = −0.712, P < 0.001 and r = −0.705, P < 0.001, respectively). On the other hand, a highly significant positive correlation was found with total leukocyte count, peripheral blood, and bone marrow blast cells' percentage (r = 0.530, P < 0.001; r = 0.691, P < 0.001; and r = 0.526, P < 0.001, respectively), but a significant positive correlation was found with lymphadenopathy (r = 0.478, P = 0.02). Meanwhile, there was insignificant correlation found between MAGE-A3 expression and age, sex, and other clinical data including hepatomegaly, FAB subtypes, and other laboratory data including immunophenotyping and cytogenetic abnormalities with P > 0.05.
Results of the statistical comparison between MAGE-A3-positive and -negative AML patients regarding age, sex, clinical, and laboratory data are shown in Table 2. There was a highly significant difference between the 2 groups of AML patients regarding laboratory data including the hemoglobin level, total leukocyte count, platelet count, peripheral blood (PB), and bone marrow (BM) blast cells' percentage with P < 0.001, although there was a significant difference regarding clinical data including lymphadenopathy and immunophenotypic markers including CD14, glycophorin A, and CD36 (P = 0.01, P = 0.04, and P = 0.04, respectively). On the other hand, there was an insignificant difference regarding age, sex, hepatomegaly, FAB subtypes, and immunophenotypic markers including HLA-DR, CD41, CD42, CD61, CD34, and cytogenetic abnormalities (P > 0.05).
Table 3 displays the statistical comparison between MAGE-A3 gene expression and mortality regarding laboratory data. The comparison revealed a significant difference between MAGE-A3-positive alive and dead AML patients regarding the hemoglobin level, total leukocyte count, platelet count, PB, and BM blast cells' percentage (P = 0.02, P = 0.01, P = 0.03, P = 0.01, and P = 0.01, respectively).
A statistical comparison between AML patients and the control group regarding the risk of developing AML was studied. MAGE-A3 gene expression was associated with a significantly increased risk of AML (OR = 2.763; 95% CI, 1.890-8.041).
The statistical comparison between MAGE-A3-positive and -negative AML patients regarding treatment outcome revealed a highly significant difference (P < 0.001), where the MAGE-A3-positive AML patients (78%) had a higher incidence of unfavorable outcome because 12 (52%) of 23 patients were in relapse and 6 (26%) of 23 patients died; on the other hand, the MAGE-A3-negative AML patients (76%) had a higher incidence of favorable outcome because 13 (76%) of 17 patients were in remission. This suggests that MAGE-A3 gene expression has a significant impact on the risk of treatment failure (OR = 3.591; 95% CI, 1.273-10.462).
DISCUSSION
Acute myeloid leukemia is a malignant clonal disorder of immature cells in the hemopoietic system, which dominates bone marrow activity and leads to marrow failure. The malignant cells replace the bone marrow, circulate in the blood, and may accumulate in other tissues, including the lymph nodes, liver, and spleen.15The malignant clone is frequently associated with genetic instability and several LAAs, which have a role in leukemogenesis.7
The cancer testes antigens are a group of proteins originally defined by their normal expression in testes and their aberrant expression in melanomas hematopoietic malignancies and other cancers. The first cancer testes antigens discovered were members of the MAGE family of proteins, including the MAGE-A, MAGE-B, and MAGE-C subfamilies, which are encoded on the X chromosome and which are now called class I MAGE antigens.12They are a group of highly homologous proteins whose expression is suppressed in all normal tissues except developing sperm and are coregulated in gametogenesis and in tumors.10
The expression of MAGE gene can be caused by promoter region demethylation and is widespread in malignancies.16Despite their widespread expression and that MAGE proteins have long been recognized as tumor-specific targets, however, the functions of most class I MAGE molecules have not been determined, but it was found to play a role in cell cycle progression and transcriptional regulation, and it is not known whether their expression is a functionally irrelevant byproduct of cellular transformation or could actually contribute to the development of malignancies.12Data have accumulated suggesting that the expression of MAGE proteins by malignant cells may contribute to leukemogenesis, advanced disease, or resistance to chemotherapy by permitting proliferation and prolonging the survival of malignant cells.17
In the present study, the MAGE-A3-positive expression was found exclusively in AML patients and was not detected in any of the healthy control group subjects; contrary to its lack of expression in control subjects (0%), 23 patients (57.5%) of the studied AML patients expressed MAGE-A3 gene. Comparison between AML patients and healthy control subjects revealed a highly significant difference and the MAGE-A3 gene expression was associated with a significantly increased risk of AML (OR = 2.763; 95% CI, 1.890-8.041). Results of the present study were nearly similar to the results of other researchers who reported the expression of MAGE-A3 in 47% of their AML patients and did not find any MAGE-A3 expression in healthy control subjects and stated a significant difference between the 2 groups and documented that MAGE-A3 expression was a risk factor in developing AML.9On the other hand, other investigators who studied the MAGE-A3 expression in 32 de novo AML patients reported a lower percent than this study did, which might be explained by the small sample size because the frequency was 31% for their patients and none of the control subjects had MAGE-A3 expression and MAGE-A3 expression was associated with an increase in the risk and susceptibility of AML development.14These results are in accordance with those recorded by recent studies, which stated a significant difference between the 2 groups regarding total leukocyte count, platelet count, PB, and BM blast cells because MAGE-A3 plays a role in increasing leukemic cell proliferation and survival in the bone marrow, and also, migration of leukemic cells from the bone marrow to the peripheral blood so MAGE-A3 expression were associated with a higher count of circulating AML blast cells, while they could not find a significant difference regarding age, sex, clinical, and other laboratory data.
The correlation of MAGE-A3 gene expression with age, sex, clinical, and laboratory data in AML patients showed a highly significant negative correlation between MAGE-A3 expression and hemoglobin level and platelet count (r = −0.712 and r = −0.705, respectively). On the other hand, a highly significant positive correlation was found with total leukocyte count, PB, and BM blast cells' percentage (r = 0.530, r = 0.491, and r = 0.526, respectively), but a significantly positive correlation was found with lymphadenopathy (r = 0.387). Nevertheless, an insignificant correlation was found between MAGE-A3 expression and age, sex, and other clinical data including hepatomegaly, FAB subtypes, and other laboratory data including immunophenotyping and cytogenetic abnormalities. This comes in agreement with previous studies, which also elicited a highly significant correlation between MAGE-A3 expression and total leukocyte count, PB, and BM blast cells' percentage and reported that higher total leukocyte count, PB, and BM blast cells' percentage occur with MAGE-A3 expression, although they could not elicit a correlation with age, sex, clinical, and other laboratory data.6,14
In the current study, comparison between MAGE-A3-positive and -negative AML patients regarding age, sex, clinical, and laboratory data revealed a highly significant lower hemoglobin level and platelet count and higher total leukocyte count, PB, and BM blast cells' percentage in patients with positive MAGE-A3 expression than in patients with negative expression. Also, patients with positive MAGE-A3 expression were significantly more prone to have lymphadenopathy and express CD14, CD36, and glycophorin A. On the other hand, there was an insignificant difference regarding age, sex, hepatomegaly, FAB subtypes, other immunophenotypic markers including HLA-DR, CD41, CD42, CD61, and CD34, and cytogenetic abnormalities.
These results are in accordance with those recorded by recent studies, which stated a significant difference between the 2 groups regarding total leukocyte count, platelet count, PB, and BM blast cells because MAGE-A3 plays a role in increasing leukemic cell proliferation and survival in the bone marrow. Also, migration of leukemic cells from the bone marrow to the peripheral blood and MAGE-A3 expression were associated with a higher count of circulating AML blast cells, although they could not find a significant difference regarding age, sex, clinical, and other laboratory data.6,9Most CD14 +ve AML patients showed a significant positive expression of MAGE-A3. This agrees with previous studies that reported that the expression of CD14 might play a role in the tendency of AML samples with monocytoid differentiation (AML; M4-M5) toward increased expression of MAGE-A3.14Also, an insignificant difference was found regarding FAB subtypes because MAGE-A3-positive AML patients did not show certain FAB subtypes preference, but slightly they tended to have M4-M5 FAB subtypes. These results are in concordance with those of other researchers who reported that MAGE-A3 expression was independent of FAB classification.6
Our findings showed a significant difference between MAGE-A3 expression and mortality regarding laboratory data, in which significantly higher total leukocyte count, PB, and BM blast cells' percentage and lower hemoglobin level and platelet count were found in MAGE-A3-positive dead AML patients than in MAGE-A3-positive alive patients, this means that MAGE-A3 gene expression is a bad prognostic marker related to total leukocyte count, PB, and BM blast cells' percentage because it increases blast cell proliferation and survival in the bone marrow and also migration of blast cells from the bone marrow to the peripheral blood, leading to higher total leukocyte count and blast cells' percentage, although it is an independent marker of hemoglobin level and platelet count. Our results are in agreement with those recorded by a recently reported study.9
Lastly, a highly significant difference was found between MAGE-A3-positive and -negative AML patients regarding treatment outcome, where the MAGE-A3-positive AML patients had a higher incidence of unfavorable treatment outcome (relapse and death), whereas the MAGE-A3-negative AML patients had a higher incidence of favorable outcome (remission). This suggests that MAGE-A3 gene expression has a significant impact on the risk of treatment failure (OR = 3.591; 95% CI, 1.273-10.462).
This comes consistently with other studies that found that the expression of MAGE-A3 influences the outcome of AML therapy in the study of de novo AML patients and concluded that MAGE-A3 expression was significantly related to response to therapy and prognosis of AML because AML patients with a positive MAGE-A3 expression had a lower complete remission rate, indicating that MAGE-A3 can be used as a poor prognostic factor in AML and reported that monitoring of the MAGE-A3 expression may be a useful means for the detection of MRD in AML and the detection of the MAGE-A3 expression may be useful in selecting individual chemotherapy regimens for patients with AML.9Also, in agreement with our study, other researchers reported a correlation between MAGE-A3 expression on AML cells and poor outcome and stated that the MAGE-A3 expression showed a significant unfavorable outcome among AML patients and has a significant impact on the risk of treatment failure because it induces specific T-cell responses and is involved in crucial mechanisms for cell growth of leukemic cells; hence, MAGE-A3 represents a novel target for the development of effective treatment of AML. Therefore, compounds that target MAGE-A3 could disrupt leukemic cells' proliferation and survival and make leukemic cells more accessible to conventional therapy.8,16MAGE might provide tools for immunotherapy for leukemia because identification of immunogenic LAAs as target structures is mandatory for the specific immunotherapy for leukemia.14The expression of MAGE-A3 suggests that immunotherapeutic treatments targeting this antigen may prove useful in the treatment of AML as early as disease presentation and eliminate the MRD after chemotherapy.13
In conclusion, the expression of MAGE-A3 has a role in leukemogenesis and in the pathophysiology, development, disease progression, and response to chemotherapy for AML. Its expression may have clinical relevance and important role as a prognostic factor in AML. It may be useful as a predictive marker for treatment outcome in AML patients and could be of interest in the future for new targeted therapies. Thus, determining the expression of MAGE-A3 is important for the prediction of both the risk of developing AML and the treatment outcome.
ACKNOWLEDGMENT
The author thanks the staff of El-Kasr El-Aini Hospital, Cairo University, Cairo, Egypt, for helping in performing this study and also patients for their willing participation in this research.