In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4+ T cells from the blood of a healthy donor. CD4+ T cells strongly recognized MAGE-3281–295 and, to a lesser extent, MAGE-3141–155 and MAGE-3146–160. Moreover, CD4+ T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4+ T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3–positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4+ T cells recognized MAGE-3281–295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4+ T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4+ T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4+ T cell epitopes.
MAGE-3; CD4+ epitopes; melanoma; tumor vaccines; adoptive immunotherapy
MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues except testis and placenta. MAGE-A antigens and their epitope peptides have been used in tumor immunotherapy trials. MAGE-A4 antigen is extensively expressed in various histological types of tumors, so it represents an attractive target for tumor immunotherapy. In this study, we predicted HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes of MAGE-A4, followed by peptide/HLA-A*0201 affinity and complex stability assays. Of selected four peptides (designated P1, P2, P3, and P4), P1 (MAGE-A4286-294, KVLEHVVRV) and P3 (MAGE-A4272-280, FLWGPRALA) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/Kb transgenic mice. And the induced CTLs could lyse target cells in an HLA-A*0201-restricted fashion, demonstrating that the two peptides are HLA-A*0201-restricted CTL epitopes and could serve as targets for therapeutic antitumoral vaccination.
MAGE-A4 has been considered as an attractive cancer-testis (CT) antigen for tumour immunotherapy. It has been well accepted that T-helper type 1 (Th1) cell-dominant immunity is critical for the successful induction of antitumour immunity in a tumour-bearing host. The adoptive Th1 cell therapy has been shown to be an attractive strategy for inducing tumour eradication in mouse systems. However, Th1-cell therapy using human tumour-specific Th1 cells, which were expanded from peripheral blood mononuclear cells (PBMCs) in a clinically useful protocol, has never been performed. Here, we first identified MAGE-A4-derived promiscuous helper epitope, peptide (MAGE-A4 280–299), bound to both HLA-DPB1*0501 and DRB1*1403. Using the peptide, we established a suitable protocol for the propagation of MAGE-A4-specific Th1 cells in vitro. Culture of CD4+ T cells with IFN-γ-treated PBMC-derived adherent cells in the presence of helper epitope peptide resulted in a great expansion of MAGE-A4-reactive Th cells producing IFN-γ , but not IL-4. Moreover, it was shown that ligation of MAGE-A4-reactive Th1 cells with the cognate peptide caused the production of IFN-γ and IL-2. Thus, our identified MAGE-A4 helper epitope peptide will become a good tool for the propagation of tumour-specific Th1 cells applicable to adoptive immunotherapy of human cancer.
helper epitope; promiscuous peptide; tumour-specific CD4+ T cells; adoptive Th1-cell therapy
Cancer immunotherapy has become one of the most important therapeutic approaches to cancer in the past two decades. Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). Antigen-specific CTLs induced by MAGE-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumor. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma. It is highly homologous to the MAGE-A gene subfamily, particularly to MAGE-3 (93%). MAGE-n-derived peptide QLVFGIEVV is a novel HLA-A2.1-restricted CTL epitope that induces MAGE-n-specific CTLs in vitro. Identification of these CTL epitopes may lead to clinical applications of these peptides as cancer vaccines for patients with MAGE-n+/HLA-A2+ tumors. In the present study, HLA-A/A24-restricted CTL epitopes of antigen MAGE-n were predicted using the NetCTL1.2 Server on the web, COMB >0.85. The results showed that the NetCTL1.2 Server prediction method improved prediction efficacy and accuracy. Additionally, 8 HLA-A2- and 9 HLA-A24-restricted CTL epitope candidates (nonamers) derived from the tumor antigen MAGE-n were predicted. These nonamers, following identification via experimentation, may contribute to the development of potential antigen peptide tumor vaccines.
MAGE-n; HLA-A2/A24; cytotoxic T-lymphocyte epitope; prediction
MAGE-type genes are expressed by many tumors of different histological types and not by normal cells, except for male germline cells, which do not express major histocompatibility complex (MHC) molecules. Therefore, the antigens encoded by MAGE-type genes are strictly tumor specific and common to many tumors. We describe here the identification of the first MAGE-encoded epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules to CD4+ T lymphocytes. Monocyte-derived dendritic cells were loaded with a MAGE-3 recombinant protein and used to stimulate autologous CD4+ T cells. We isolated CD4+ T cell clones that recognized two different MAGE-3 epitopes, MAGE-3114–127 and MAGE-3121–134, both presented by the HLA-DR13 molecule, which is expressed in 20% of Caucasians. The second epitope is also encoded by MAGE-1, -2, and -6. Our procedure should be applicable to other proteins for the identification of new tumor-specific antigens presented by HLA class II molecules. The knowledge of such antigens will be useful for evaluation of the immune response of cancer patients immunized with proteins or with recombinant viruses carrying entire genes coding for tumor antigens. The use of antigenic peptides presented by class II in addition to peptides presented by class I may also improve the efficacy of therapeutic antitumor vaccination.
human; invariant chain; peptide; tumor; histocompatibility leukocyte antigen class II
BACKGROUND: A number of tumors express antigens that are recognized by specific cytotoxic T cells. The normal host immune responses, however, are not usually sufficient to cause tumor rejection. Using appropriate immunization strategies, tumor-specific antigens may serve as targets against which tumor-destructive immune responses can be generated. MAGE-1 and MAGE-3 are two clinically relevant antigens expressed in many human melanomas and other tumors, but not in normal tissues, except testis. Here, we have investigated whether DNA and cellular vaccines against MAGE-1 and MAGE-3 can induce antigen-specific anti-tumor immunity and cause rejection of MAGE-expressing tumors. MATERIALS AND METHODS: Mice were immunized against MAGE-1 and MAGE-3 by subcutaneous injection of genetically modified embryonic fibroblasts or intramuscular injection of purified DNA. Mice were injected with lethal doses of B16 melanoma cells expressing the corresponding MAGE antigens or the unrelated protein SIV tat, and tumor development and survival were monitored. RESULTS: Intramuscular expression of MAGE-1 and MAGE-3 by plasmid DNA injection and subcutaneous immunization with syngeneic mouse embryonic fibroblasts transduced with recombinant retroviruses to express these antigens induced specific immunity against tumors expressing MAGE-1 and MAGE-3. Both CD4+ and CD8+ T cells were required for anti-tumor immunity. Coexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) or B7-1 significantly increased anti-tumor immunity in an antigen-specific manner and resulted in a considerable proportion of mice surviving lethal tumor challenge. CONCLUSIONS: Our results suggest that genetic and cellular vaccines against MAGE and other tumor antigens may be useful for the therapy of tumors expressing specific markers, and that GM-CSF and B7-1 are potent stimulators for the induction of antigen-specific tumor immunity.
Patients with Hodgkin’s Lymphoma (HL) relapsing after hematopoietic stem cell transplant (HSCT) have limited options for long-term cure. We have shown that infused cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV+ HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients an alternative is to target the cancer/testis antigen MAGE-A4 present in EBV antigen-negative HL tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors.
We explored the feasibility of combining adoptive T cell therapy with epigenetic modification of tumor antigen expression. We further characterized MAGE-A4-specific T cell phenotype and function, and examined the effects of the epigenetic-modifying drug decitabine on these T cells.
Cytotoxic T cells were generated specifically recognizing MAGE-A4 expressed by autologous HL targets and tumor cell lines. Decitabine – previously shown to increase tumor antigen expression in Hodgkin’s Lymphoma – did not compromise MAGE-A4 specific T cell phenotype and function. In patients treated with decitabine, expanded MAGE-A4 specific T cells had a broader anti tumor T cell repertoire consistent with increased antigen stimulation in vivo.
Adoptive transfer of MAGE-A4 specific T cells, combined with epigenetic-modifying drugs to increase expression of the protein may improve treatment of relapsed Hodgkin’s Lymphoma.
T cell immunotherapy; relapsed Hodgkin’s Lymphoma; MAGE-A4; decitabine; epigenetic modification of tumors; cancer testis antigens
The androgen receptor (AR) is a key transcriptional regulator and therapeutic target in prostate cancer. During androgen deprivation therapy to treat metastatic prostate cancer, surviving cells acquire increased AR signaling through a variety of mechanisms, one of which is enhanced interactions with AR coactivators. One recently identified AR-specific coregulator expressed only in human and nonhuman primates is the melanoma antigen gene protein-A11 (MAGE-11). MAGE-11 increases AR transcriptional activity through direct interactions with AR and other coactivators, and its levels increase during prostate cancer progression to castration-recurrent growth. The MAGE-11 gene is located at Xq28 on the human X chromosome as part of an X-linked MAGE gene family of cancer–testis antigens. MAGE-11 stabilizes AR when androgen levels are low, and functions in a transcriptional hub to promote AR-mediated gene activation. The evolutionary development and organization of the MAGE-11 gene within the cancer–testis antigen family suggests that MAGE-11 provides a gain-of-function to AR among primates in both normal physiology and cancer, and may serve as a therapeutic target in the treatment of advanced prostate cancer.
androgen receptor; cancer–testis antigens; MAGE-11; MAGE-A11; N/C interaction; X chromosome; X-linked genes
The evolutionary mode of a multi-gene family can change over time, depending on the functional differentiation and local genomic environment of family members. In this study, we demonstrate such a change in the melanoma antigen (MAGE) gene family on the mammalian X chromosome. The MAGE gene family is composed of ten subfamilies that can be categorized into two types. Type I genes are of relatively recent origin, and they encode epitopes for human leukocyte antigen (HLA) in cancer cells. Type II genes are relatively ancient and some of their products are known to be involved in apoptosis or cell proliferation. The evolutionary history of the MAGE gene family can be divided into four phases. In phase I, a single-copy state of an ancestral gene and the evolutionarily conserved mode had lasted until the emergence of eutherian mammals. In phase II, eight subfamily ancestors, with the exception for MAGE-C and MAGE-D subfamilies, were formed via retrotransposition independently. This would coincide with a transposition burst of LINE elements at the eutherian radiation. However, MAGE-C was generated by gene duplication of MAGE-A. Phase III is characterized by extensive gene duplication within each subfamily and in particular the formation of palindromes in the MAGE-A subfamily, which occurred in an ancestor of the Catarrhini. Phase IV is characterized by the decay of a palindrome in most Catarrhini, with the exception of humans. Although the palindrome is truncated by frequent deletions in apes and Old World monkeys, it is retained in humans. Here, we argue that this human-specific retention stems from negative selection acting on MAGE-A genes encoding epitopes of cancer cells, which preserves their ability to bind to highly divergent HLA molecules. These findings are interpreted with consideration of the biological factors shaping recent human MAGE-A genes.
To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer.
mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR) in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1.
MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20). In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41) and 36.6% (15/41), while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41) and 14.6% (6/41). In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7) and 14.3% (1/7). The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (P < 0.05). Gene expression rate was not correlated with menopause or lymph node metastasis. Positive expression of MAGE-1 and MAGE-3 was positively correlated with tumor differentiation and the clinical stage of the ovarian cancer. In addition, the positive expression rate of BAGE was significantly higher in ovarian cancer patients with ascites (P < 0.05). The mRNA expression profiles of MAGE, GAGE and BAGE in ovarian carcinoma cell lines SKOV3, A2780 and COC1 varied, but there was at least one gene expressed in each cell line.
Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer. These genes can be used as one of the important indicators for early diagnosis, efficacy evaluation and prognostic determination of ovarian cancer.
MAGE-A1 belongs to a family of 12 genes that are active in various types of tumors and silent in normal tissues except in male germ-line cells. The MAGE-encoded antigens recognized by T cells are highly tumor-specific targets for T cell-oriented cancer immunotherapy. The function of MAGE-A1 is currently unknown. To analyze it, we attempted to identify protein partners of MAGE-A1. Using yeast two-hybrid screening, we detected an interaction between MAGE-A1 and Ski Interacting Protein (SKIP). SKIP is a transcriptional regulator that connects DNA-binding proteins to proteins that either activate or repress transcription. We show that MAGE-A1 inhibits the activity of a SKIP-interacting transactivator, namely the intracellular part of Notch1. Deletion analysis indicated that this inhibition requires the binding of MAGE-A1 to SKIP. Moreover, MAGE-A1 was found to actively repress transcription by binding and recruiting histone deacetylase 1 (HDAC1). Our results indicate that by binding to SKIP and by recruiting HDACs, MAGE-A1 can act as a potent transcriptional repressor. MAGE-A1 could therefore participate in the setting of specific gene expression patterns for tumor cell growth or spermatogenesis.
Melanoma antigen family (MAGE) genes are widely expressed in various tumor types but silent in normal cells except germ-line cells lacking human leukocyte antigen (HLA) expression. Over 25 MAGE genes have been identified in different tissues, mostly located in Xq28 of human chromosome and some of them in chromosome 3 and 15, containing either single or multiple-exons. This in silico study predicted the genes on hTERT location and identified a distant relative of MAGE gene located on chromosome 5. The study identified a single exon coding ~850 residues polypeptide sharing ~30% homology with Macfa-MAGE E1 and hMAGE-E1. dbEST search of the predicted transcript matches 5′ and 3′ flanking ESTs. The predicted protein showed sequence homology within the MAGE homology domain 2 (MHD2). UCSC genome annotation of CpG Island around the coding region reveals that this gene could be silent by methylation. Affymetrix all-exon track indicates the gene could be expressed in different tissues particularly in cancer cells as they widely undergo a genome wide demethylation process.
telomerase; melanoma antigen; gene prediction; EST; ORF
Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, that codes for one of these antigens named MZ2-E. We show here that antigen MZ2-D, which is present on the same tumor, is encoded by another member of the MAGE gene family named MAGE-3. Like MAGE-1, MAGE-3 is composed of three exons and the large open reading frame is entirely located in the third exon. Its sequence shows 73% identity with MAGE-1. Like MZ2-E, antigen MZ2-D is presented by HLA-A1. The antigenic peptide of MZ2-D is a nonapeptide that is encoded by the sequence of MAGE-3 that is homologous to the MAGE-1 sequence coding for the MZ2-E peptide. Competition experiments using single Ala-substituted peptides indicated that amino acid residues Asp in position 3 and Tyr in position 9 were essential for binding of the MAGE-1 peptide to HLA-A1. Gene MAGE-3 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes. It is expressed in a larger proportion of melanoma samples than MAGE-1. MAGE-3 encoded antigens may therefore have a wide applicability for specific immunotherapy of melanoma patients.
MAGE-A genes are a subfamily of the melanoma antigen genes (MAGEs), whose expression is restricted to tumor cells of different origin and normal tissues of the human germline. Although the specific function of individual MAGE-A proteins is being currently explored, compelling evidence suggest their involvement in the regulation of different pathways during tumor progression. We have previously reported that MageA2 binds histone deacetylase (HDAC)3 and represses p53-dependent apoptosis in response to chemotherapeutic drugs. The promyelocytic leukemia (PML) tumor suppressor is a regulator of p53 acetylation and function in cellular senescence. Here, we demonstrate that MageA2 interferes with p53 acetylation at PML-nuclear bodies (NBs) and with PMLIV-dependent activation of p53. Moreover, a fraction of MageA2 colocalizes with PML-NBs through direct association with PML, and decreases PMLIV sumoylation through an HDAC-dependent mechanism. This reduction in PML post-translational modification promotes defects in PML-NBs formation. Remarkably, we show that in human fibroblasts expressing RasV12 oncogene, MageA2 expression decreases cellular senescence and increases proliferation. These results correlate with a reduction in NBs number and an impaired p53 response. All these data suggest that MageA2, in addition to its anti-apoptotic effect, could have a novel role in the early progression to malignancy by interfering with PML/p53 function, thereby blocking the senescence program, a critical barrier against cell transformation.
MAGE; PML; sumoylation; acetylation; p53; senescence
The melanoma antigen gene (MAGE) A1-A6 RT-PCR system was developed for the detection of lung cancer cells in the sputum. However, we identified MAGE expression in some patients with non-malignant lung diseases. To understand these patterns of MAGE expression, we performed MAGE A3 methylation-specific PCR (MSP) and p16 MSP. We collected 24 biopsy specimens of lung cancer tissue and performed MAGE A1-A6 RT-PCR, MAGE A3 MSP and p16 MSP. RNA and DNA were simultaneously extracted from induced sputum specimens of 133 patients with lung diseases and 30 random sputum specimens of healthy individuals and the 3 molecular analyses were performed. The patients were diagnosed as 65 cases of lung cancer and 68 of benign lung diseases. Positive rates of MAGE A1-A6 RT-PCR, MAGE A3 MSP and p16 MSP were as follows: in lung cancer tissue, 87.5, 58.3 and 70.8%; in the sputum of lung cancer patients, 50.8, 46.2 and 63.1%; benign lung diseases, 10.3, 30.9 and 39.7%; and healthy individuals, 3.3, 6.7 and 3.3%. Of the 40 MAGE-positive cases, 33 were diagnosed with lung cancer and 7 as having benign lung diseases. From the 7 cases of MAGE-positive benign lung diseases, 6 cases showed methylation abnormalities. The MAGE-positive group revealed significantly higher rates of methylation abnormalities. Of the 40 MAGE-positive cases, 39 cases were found to be lung cancer or benign lung diseases with abnormal methylation. Thus, MAGE expression in the sputum suggests the presence of lung cancer cells or pre-cancerous cells.
lung cancer; sputum; melanoma antigen gene RT-PCR; melanoma antigen gene A3; p16; methylation
The type I melanoma antigen gene (MAGE) proteins CT7 (MAGE-C1) and MAGE-A3 are commonly expressed in multiple myeloma (MM), and their expression correlates with increased plasma cell proliferation and poor clinical outcome. They belong to the cancer-testis antigen (CTAg) group of tumor-associated proteins, some of which elicit spontaneous immune responses in cancer patients. CT7 and MAGE-A3 are promising antigenic targets for therapeutic tumor vaccines in myeloma; therefore, it is critical to determine if they are immunogenic in MM patients. We analyzed cellular and humoral immune responses against CTAgs in patients with plasma cell dyscrasias: MM, monoclonal gammopathy of undetermined significance (MGUS), and Waldenström’s macroglobulinemia (WM). Bone marrow lymphocytes from two of four untreated MM patients exhibited CT7-specific cellular immune responses as measured by an autologous cellular immunity assay, the first such immune response to CT7 to be reported in cancer patients. Sera from 24 patients were screened by ELISA for humoral immune responses to CTAgs. Two patients with MM demonstrated positive titers, one for MAGE-A1 and the other for SSX1. These data demonstrate that CTAgs, particularly CT7, are immunogenic in MM patients and merit further exploration as targets of immunological therapy in MM.
human; multiple myeloma; CT antigens; cellular immunity; humoral immunity
The type I melanoma antigen gene (MAGE) proteins CT7
(MAGE-C1) and MAGE-A3 are commonly expressed in multiple myeloma (MM),
and their expression correlates with increased plasma cell proliferation
and poor clinical outcome. They belong to the cancer-testis antigen
(CTAg) group of tumor-associated proteins, some of which elicit
spontaneous immune responses in cancer patients. CT7 and MAGE-A3
are promising antigenic targets for therapeutic tumor vaccines in
myeloma; therefore, it is critical to determine if they are immunogenic
in MM patients. We analyzed cellular and humoral immune responses
against CTAgs in patients with plasma cell dyscrasias: MM, monoclonal
gammopathy of undetermined significance (MGUS), and Waldenström's
macroglobulinemia (WM). Bone marrow lymphocytes from two of four
untreated MM patients exhibited CT7-specific cellular immune responses
as measured by an autologous cellular immunity assay, the first
such immune response to CT7 to be reported in cancer patients. Sera
from 24 patients were screened by ELISA for humoral immune responses to
CTAgs. Two patients with MM demonstrated positive titers, one for
MAGE-A1 and the other for SSX1. These data demonstrate that CTAgs,
particularly CT7, are immunogenic in MM patients and merit further
exploration as targets of immunological therapy in MM.
myeloma; CT antigens; cellular immunity; humoral
The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aero-digestive tract (UADT) tumor cells and its association with T cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE specific immunotherapy. Using quantitative RT-PCR (QRT-PCR), we evaluated the expression of MAGE-3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA-A*0201+squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using western blot. HLA-A*0201:MAGE-3(271–279) specific cytotoxic T lymphocytes (MAGE-CTL) from SCCHN patients and healthy donors showed that MAGE-3/6 expression was highly associated with CTL recognition in vitro. Based on MAGE-3/6 expression we could identify 31 (47%) of the 65 UADT tumors which appeared to express MAGE-3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE-3 expression was responsible for CTL recognition, two MAGE-3/6 mRNAhigh SCCHN cell lines, PCI-13 and PCI-30, were subjected to MAGE-3/6 specific knockdown. RNAi–transfected cells showed that MAGE expression, and MAGE-CTL recognition, were significantly reduced. Furthermore, treatment of cells expressing low MAGE-3/6 mRNA with a demethylating agent, 5-aza-2'-deoxycytidine (DAC), increased the expression of MAGE-3/6 and CTL recognition. Thus, using QRT-PCR UADT cancers frequently express MAGE-3/6 at levels sufficient for CTL recognition, supporting the use of a QRT-PCR based assay for the selection of candidates likely to respond to MAGE-3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials.
MAGE; Head and neck cancer; T cells; QRT-PCR
The members of MAGE gene family are highly expressed in human hepatocellular carcinoma (HCC). In the present study, we tested the tumour-specific MAGE-1 and MAGE-3 transcripts in the peripheral blood of HCC patients by nested RT–PCR to detect the circulating tumour cells and evaluate their potential clinical implication. Of 30 HCC patients, the positive rate of MAGE-1 and MAGE-3 transcripts was 43.3% (13 out of 30) and 33.3% (10 out of 30) in PBMC samples, whilst the positive rate was 70% (21 out of 30) and 53.3% (16 out of 30) in the resected HCC tissue samples, respectively. The positivity for at least one MAGE gene transcript was 63.3% (19 out of 30) in PBMC samples of HCC patients and 83.3% (25 out of 30) in the resected HCC tissue samples. MAGE-1 and/or MAGE-3 mRNA were not detected in the PBMC of those patients from whom the resected HCC tissues were MAGE-1 or MAGE-3 mRNA negative, nor in the 25 PBMC samples from healthy donors. The detection of MAGE transcripts in PBMC was correlated with the advanced stages and tumour size of the HCC, being 82.4% (14 out of 17) in tumour stages III and IVa, 56.6% (five out of nine) in stage II, and null (nought out of four) in stage I. The serum α-FP in 33.3% (10 out of 30) of HCC patients was normal or slightly elevated (<40 ng ml−1). However, six of these 10 patients (α-FP <40 ng ml−1) were MAGE-1 and /or MAGE-3 mRNA positive in their PBMC. The follow-up survey of MAGE mRNA in PBMC was performed in 12 patients. Seven patients with persistent MAGE-1 and/or MAGE-3 mRNA positive or from negative turned to positive died because of metastasis and/or recurrence. In striking contrast, all four patients with MAGE-1 and/or MAGE-3 mRNA from positive turned to negative and one patient with persistent MAGE-3 transcript negative are alive after last test. Collectively, detection of MAGE transcripts with follow-up survey in PBMC is a feasible and reliable assay for the early prediction of the relapse and prognosis of the HCC patients.
British Journal of Cancer (2002) 86, 110–116. DOI: 10.1038/sj/bjc/6600016 www.bjcancer.com
© 2002 The Cancer Research Campaign
circulating tumour cells; hepatocellular carcinoma; MAGE transcripts; nested polymerase chain reaction; tumour-specific marker
This study was to investigate Melanoma-antigen gene (MAGE) expression by reverse transcription-nested polymerase chain reaction (RT-nested PCR) with the original common primers of MAGE-A1 to -A6 and analysis of correlation between its expression and the well-known clinical parameters in addition to evaluate the clinical feasibility of the common primers. Surgical tumor and corresponding nonneoplastic tissue samples from 38 patients with colorectal cancer were studied. To confirm the identities of RT-PCR products, direct sequencing was done after in vitro subcloning. No expression of MAGE was observed in the non-neoplastic colorectal mucosal tissues. Sixteen (42.1%) of 38 carcinomas expressed at least one of MAGE A-1 to -6. The expression of the MAGE genes was not related to age, sex, histological grades, the depth of invasion, metastasis to lymph nodes, vessel, neural, or perineural invasion. The identities with the corresponding mRNAs were confirmed in 6 cases for MAGE-A2 (15.8%), 6 cases for MAGE-A4 (15.8%), 2 cases for MAGE-A3 (5.3%), and one case for MAGE A-6 (2.6%). These results suggest that MAGE expressions, except those of MAGE-A2 and -A4, seem to have a limited role in the molecular pathogenesis of colon cancer. However, the common primer sets to detect of expressions for MAGE-A1 to -A6 simultaneously appear to be feasible to differentiate malignant from benign lesions in colorectal diseases.
Melanoma antigens (MAGE) are frequently expressed in lung cancer and are promising targets of anticancer immunotherapy. Our preliminary data suggested that MAGE may be expressed during early lung carcinogenesis, raising the possibility of targeting MAGE as a lung cancer prevention strategy. The purpose of this study was to investigate MAGE activation patterns in the airways of chronic smokers without lung cancer. MAGE-A1, -A3 and -B2 gene expression was determined in bronchial brush cells from chronic former smokers without lung cancer by reverse transcription-PCR (RT-PCR). The results were correlated with clinical parameters. The 123 subjects had a median age of 57 years, a median of 40 pack-years smoking history, and had quit smoking for at least one year prior to enrollment. Among the subjects, 31 (25%), 38 (31%), and 46 (37%) had detectable MAGE-A1, -A3 and -B2 expression, respectively, in their bronchial brush samples. Expression of MAGE-A1 and -B2 positively correlated with pack-years smoking history (P=0.03 and 0.03, respectively). The frequency of expression did not decrease despite a prolonged smoking cessation period. In conclusion, MAGE-A1, -A3 and -B2 genes are frequently expressed in the bronchial epithelial cells of chronic smokers without lung cancer, suggesting that chronic exposure to cigarette smoke activates these genes even before the malignant transformation of bronchial cells in susceptible individuals. Once activated, the expression persists despite long-term smoking cessation. These data support the targeting of MAGE as a novel lung cancer prevention strategy.
melanoma antigens; airway; smokers; lung cancer; prevention
Immunotherapy targeting MAGE-A3 in multiple myeloma (MM) could eradicate highly aggressive and proliferative clonal cell populations responsible for relapse. However, expression of many cancer-testis antigens, including MAGE-A3, can be heterogeneous, leading to the potential for tumor escape despite MAGE-A3-induced immunity. We hypothesized that a combination of the hypomethylating agent 5-azacitidine (5AC) and the histone deacetylase inhibitor (HDACi) MGCD0103 (MGC) could induce MAGE-A3 expression in MAGE-A3-negative MM, resulting in recognition and killing of MM cells by MAGE-A3-specific cytotoxic T lymphocytes (CTL).
Gene expression analyses of MAGE-A3 expression in primary MM patient samples at diagnosis and relapse were completed to identify populations that would benefit from MAGE-A3 immunotherapy. MM cell lines were treated with 5AC and MGC. Real-time polymerase chain reaction (PCR) and Western blotting were performed to assess MAGE-A3 RNA and protein levels, respectively. Chromium-release assays and interferon (IFN) secretion assays were employed to ascertain MAGE-A3 CTL specificity against treated targets.
Gene expression analysis revealed that MAGE-A3 is expressed in MM patients at diagnosis (25%) and at relapse (49%). We observed de novo expression of MAGE-A3 RNA and protein in MAGE-A3-negative cell lines treated with 5AC. MGC treatment alone did not induce expression but sequential 5AC/MGC treatment led to enhanced expression and augmented recognition by MAGE-A3-specific CTL, as assessed by 51Cr-release assays (P = 0.047) and enzyme-linked immunosorbent assay (ELISA) for IFN-γ secretion (P = 0.004).
MAGE-A3 is an attractive target for immunotherapy of MM and epigenetic modulation by 5AC, and MGC can induce MAGE-A3 expression and facilitate killing by MAGE-A3-specific CTL.
5-azacitidine; cancer-testis antigen; demethylation; epigenetics; histone deactylase inhibitor; hypomethylation; MAGE-A3; MGCD0103; multiple myeloma
We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti- MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.
Melanoma antigen D1 (MAGED1) is a member of the type II melanoma antigen (MAGE) family. The down-regulation of MAGED1 expression has been shown in breast carcinoma cell lines and in glioma stem cells and may play an important role in apoptosis and anti-tumorigenesis. However, there is no report on its clinical role in colorectal cancer (CRC).
We examined the expression of MAGED1 by qPCR in colorectal cancer tissues and their adjacent non-tumorous tissues taken from 6 cases and performed Western blotting and IHC analyses. In addition, we analyzed MAGED1 expression in 285 clinicopathologically characterized colorectal cancer patients.
MAGED1 expression was significantly down-regulated in colorectal cancer tissues compared with adjacent non-tumorous tissues and was associated with clinical stage (p < 0.001), T classification (p = 0.001), N classification (p < 0.001), M classification (p < 0.001) and pathologic differentiation (p = 0.002). Patients with lower MAGED1 expression had a shorter survival time than those with higher MAGED1 expression. Univariate and multivariate analyses indicated that MAGED1 expression was an independent prognostic factors (p < 0.001).
MAGED1 may serve as a novel prognostic biomarker of human colorectal cancer.
MAGED1; Colorectal cancer; Melanoma antigen and prognosis
The melanoma antigen gene (MAGE) and synovial sarcoma on X chromosome (SSX) gene families are silent in most normal adult tissues, but are expressed in a variety of malignant lesions. Therefore, detection of MAGE and SSX transcription may be useful for the diagnosis of head and neck cancers. The aim of this study is to detect MAGE and SSX gene transcripts of head and neck cancers using the MAGE 1-6 assay and the SSX 1-9 assay.
The transcripts of MAGE 1-6 and SSX 1-9 genes were detected by the MAGE 1-6 assay and the SSX 1-9 assay respectively, in cancer cell lines, cancer tissue, and induced sputum specimens from head and neck cancer patients.
The transcripts of MAGE 1-6 and SSX 1-9 genes were detected in 82.8% and 75.9% of head and neck cancer tissues (N=29) respectively, and 96.6% of cancer tissues expressed at least one of MAGE 1-6 or SSX 1-9 genes. In the induced sputum of head and neck cancer patients (N=18), the transcripts of MAGE 1-6 and SSX 1-9 genes were detected in 72.2% and 77.8%, respectively, and 94.4% of the sputum specimens were positive for either the MAGE 1-6 or the SSX 1-9 assay.
These results suggest that the combination of MAGE 1-6 and SSX 1-9 assays may be useful in the diagnosis of head and neck cancer.
MAGE and SSX gene; Head and neck cancer; RT-PCR