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The antigenicity of melanomas is known better than that of all other human tumors. Melanoma antigens recognized by T lymphocytes fall into four groups. They can be encoded by genes that are mutated in the tumor cells, by the cancer-germline genes which are not expressed in non-tumor cells that bear HLA molecules, by melanocyte differentiation genes, and by genes that are overexpressed in tumor cells. Only the antigens of the first two groups can be considered as melanoma-specific and therefore can be used safely in active or passive immunizations

T cell-based cancer immunotherapy relies on advancements made over the last 20 years on the molecular mechanisms underlying the antigenicity of tumors. This review focuses on human tumor antigens recognized by T lymphocytes, particularly the reasons why some are tumor-specific but others are not, and on the immunologic adjuvants used in clinical trials on therapeutic vaccination with defined tumor antigens.

We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1 beta. This effect is NF-kappa B and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1 beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1 alpha or IL-1 beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that similar to 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

BACKGROUND: Cancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1. SIGNIFICANCE: These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.

Several genes with specific expression in germ cells show aberrant activation in different types of tumors. These genes, termed cancer-germline (CG) genes, encode tumor-specific antigens, which represent potential targets for therapeutic vaccination against cancer. The germline-specific gene BORIS (Brother Of the Regulator of Imprinted Sites), which encodes an 11-zinc-fingers transcriptional regulator, was recently qualified as a new CG gene, as it was found to be activated in a variety of tumor samples. Moreover, it was suggested that BORIS might be responsible for the activation of most other CG genes, including gene MAGE-A1, in tumors. In the present study, we evaluated the frequency of BORIS activation in melanoma by quantitative RT-PCR. BORIS activation was detected in 27% (n = 63) melanoma tissue samples. Surprisingly, many melanoma samples expressed MAGE-A1 and other CG genes in the absence of BORIS activation, suggesting that BORIS is not an obligate factor for activation of these genes in melanoma. Consistently, forced expression of BORIS in melanoma cell lines did not induce expression of MAGE-A1. Our results indicate that BORIS may serve as a useful target for immunotherapy of melanoma. However, it appears that BORIS is neither necessary nor sufficient for the activation of other CG genes.

Cancer-germline genes (CGGs) code for immunogenic antigens that are present in various human tumors and can be targeted by immunotherapy. Their expression has been studied in a wide range of human tumors in adults. We measured the expression of 12 CGGs in pediatric brain tumors, to identify targets for therapeutic cancer vaccines. Real Time PCR was used to quantify the expression of genes MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, NY-ESO-1 and GAGE-1,2,8 in 50 pediatric brain tumors of different histological subtypes. Protein expression was examined with immunohistochemistry. Fifty-five percent of the medulloblastomas (n = 11), 86% of the ependymomas (n = 7), 40% of the choroid plexus tumors (n = 5) and 67% of astrocytic tumors (n = 27) expressed one or more CGGs. Immunohistochemical analysis confirmed qPCR results. With exception of a minority of tumors, the overall level of CGG expression in pediatric brain tumors was low. We observed a high expression of at least one CGG in 32% of the samples. CGG-encoded antigens are therefore suitable targets in a very selected group of pediatric patients with a brain tumor. Interestingly, glioblastomas from adult patients expressed CGGs more often and at significantly higher levels compared to pediatric glioblastomas. This observation is in line with the notion that pediatric and adult glioblastomas develop along different genetic pathways.

Gene MAGE-A3 encodes tumor-specific antigenic peptides recognized by T cells on many tumors. MAGE-A3 peptides presented by HLA class I molecules have been identified using CD8 lymphocytes stimulated with cells that either expressed gene MAGE-A3 or were pulsed with candidate peptides. One antigen identified with the latter method is peptide MAGE-A3(195-203) IMPKAGLLI, presented by HLA-A24 molecules. It has been used to vaccinate advanced cancer patients. Here, we have used HLA/peptide tetramers to detect T cells recognizing this peptide. Their frequency was estimated to be 2 x 10(-8) of the blood CD8 cells in non-cancerous HLA-A24(+) individuals, which is tenfold lower than the reported frequencies of T cells against other MAGE peptides. In the blood of a patient vaccinated with MAGE-A3, the estimated frequency was 5 x 10(-7). Anti-MAGE-3.A24 cytolytic T cell clones were derived, that lysed peptide-pulsed cells with half-maximal effect at the low concentration of 500 pM. However, these CTL did not recognize a panel of HLA-A24(+) tumor cells that expressed MAGE-A3 at levels similar to those found in HLA-A1(+) tumor cells recognized by anti-MAGE-3.A1 CTLs. Furthermore, 293-EBNA cells transfected with MAGE-A3 and HLA-A24 constructs were hardly recognized by the anti-MAGE-3.A24 CTL clones. These results suggest that peptide MAGE-A3(195-203) is poorly processed and is not an appropriate target for cancer immunotherapy.

BACKGROUND: Recently, periostin (POSTN), a gene encoding a protein with similarity to the fasciclin family and involved in cell survival and angiogenesis, has emerged as a promising marker for tumor progression in various types of human cancers. There is some controversy regarding both POSTN expression levels and the nature of periostin-producing cells within tumors. In this study, we used quantitative RT-PCR to assess periostin gene expression in normal tissues, primary cell cultures, tumor tissues and tumor cell lines. RESULTS: Periostin expression levels are highly variable in both normal tissues and tumors and strong POSTN overexpression is mostly detected in tumors from pancreas and liver. POSTN is not expressed in blood cancers. In melanoma samples, average periostin expression is not increased in primary tumors whereas POSTN overexpression was detected in about 60% of melanoma metastatic tumors in the liver or lymph nodes. Identification of the cellular source of periostin production in melanoma metastases -cancer cells or stroma- was assessed by comparing periostin expression in 23 newly-established melanoma cell lines and matched tumors. In contrast to the reduction by more than 99% of COL6A3 stromal marker mRNA in all cell lines, significant POSTN transcription was maintained in some melanoma cell lines, suggesting that both stromal cells and melanoma cells express periostin. The high level of periostin expression in primary cultures of skin fibroblasts suggests that fibroblasts may contribute for a large part to periostin production in melanoma-associated stroma. On the other hand, periostin expression in melanoma cells is probably acquired during the tumorigenic process as 1) normal melanocytes do not express POSTN and 2) melanoma cells from distinct metastases of the same patient were associated with very different levels of periostin expression. CONCLUSION: Our comparative analysis suggests that, although periostin overexpression is clearly detected in

A panel of cytolytic T lymphocyte (CTL) clones was isolated from metastases and blood samples of a melanoma patient vaccinated with MAGE-3.A1-pulsed autologous dendritic cells. We report here the identification of a new antigen encoded by the MAGE-C2 cancer-germline gene. This antigen is recognized by some of these CTL on HLA-B*4403. The sequence of the peptide is SESIKKKVL. It is processed in various melanoma cell lines expressing MAGE-C2 and HLA-B*4403. Because of the expression pattern of gene MAGE-C2, this new antigen is strictly tumor-specific and could therefore be used for peptide-based antitumoral vaccination.

Cancer-germline genes (CGGs) code for immunogenic antigens that are present on various human tumors but not on normal tissues. The importance of CGGs in cancer immunotherapy has led to detailed studies of their expression in a range of human tumors. We measured the levels of expression of 12 CGGs in various pediatric solid tumors to identify targets for therapeutic cancer vaccines. Quantitative real-time PCR (qPCR) was used to measure the expression of 8 MAGE genes and of genes LAGE-2/NY-ESO-1 and GAGE-1, 2, 8 in 9 osteosarcomas, 10 neuroblastomas, 12 rhabdomyosarcomas and 18 Ewing's sarcomas. Nine tumors were also examined by immunohistochemistry with monoclonal antibodies specific for the MAGE-A1, MAGE-A4 and NY-ESO-1 proteins. All osteosarcoma and 80% of neuroblastoma samples expressed several CGGs at high levels. Six of 12 rhabdomyosarcomas and 11 of 18 Ewing's sarcomas expressed at least one CGG. Immunohistochemistry data correlated well with qPCR results and showed a homogeneous protein distribution pattern in most positive tumors. No correlation was found between the levels of CGG expression in the tumors and clinicopathological parameters of the patients. Pediatric solid tumors express several CGGs, which encode antigens that could be targeted in therapeutic vaccination trials. Several CGGs of the MAGE, GAGE and LAGE families are coexpressed in a large proportion of osteosarcoma and neuroblastoma samples. Some rhabdomyosarcomas express several of these genes at high levels. Ewing's sarcomas have an overall low CGG expression. (c) 2006 Wiley-Liss, Inc.

The purpose of this phase 1/2 study was to evaluate toxicity, tumor evolution and immunologic response following administration of a fixed dose of a recombinant MAGE-3 protein by subcutaneous and intradermal routes in the absence of immunologic adjuvant. Thirty-two patients with detectable metastatic melanoma expressing gene MAGE-3 were included and 30 received at least one injection with a fixed dose of a ProtD-MAGE-3 fusion protein. The immunization schedule included 6 intradermal and subcutaneous injections at 3-week intervals. Afterward, patients without major tumor progression who required other treatments received additional vaccinations at increasing time intervals. The vaccine was generally well tolerated. Among the 26 patients who received at least 4 vaccinations, we observed 1 partial response and 4 mixed responses. For these 5 responding patients, time to progression varied from 3.5 to 51+ months. An anti-MAGE-3 CD4 T-lymphocyte response was detected in 1 out of the 5 responding patients. The majority of patients had no anti-MAGE-3 antibody response. The clinical and immunologic responses generated by the vaccine are rather limited. Nevertheless, given the potential antitumor efficacy and the very mild toxicity of vaccinations, further studies combining MAGE proteins and/or peptides with potent immunologic adjuvants are warranted, not only in metastatic melanoma, but also in the adjuvant setting. (c) 2005 Wiley-Liss, Inc.

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells ( DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

After vaccination of melanoma patients with MAGE antigens, we observed that even in the few patients showing tumor regression, the frequency of anti-vaccine T cells in the blood was often either undetectable or <10(-5) of CD8 T cells. This frequency being arguably too low for these cells to be sole effectors of rejection, we reexamined the contribution of T cells recognizing other tumor antigens. The presence of such antitumor T cells in melanoma patients has been widely reported. To begin assessing their contribution to vaccine-induced rejection, we evaluated their blood frequency in five vaccinated patients. The antitumor cytotoxic T lymphocyte (CTL) precursors ranged from 10(-4) to 3 x 10(-3), which is 10-10,000 times higher than the anti-vaccine CTL in the same patient. High frequencies were also observed before vaccination. In a patient showing nearly complete regression after vaccination with a MAGE-3 antigen, we observed a remarkably focused antitumoral response. A majority of CTL precursors (CTLp's) recognized antigens encoded by MAGE-C2, another cancer-germline gene. Others recognized gp100 antigens. CTLp's recognizing MAGE-C2 and gp100 antigens were already present before vaccination, but new clonotypes appeared afterwards. These results suggest that a spontaneous antitumor T cell response, which has become ineffective, can be reawakened by vaccination and contribute to tumor rejection. This notion is reinforced by the frequencies of anti-vaccine and antitumor CTLs observed inside metastases, as presented by Lurquin et al. (Lurquin, C., B. Lethe, V. Corbiere, I. Theate, N. van Baren, P.G. Coulie, and T. Boon. 2004. J. Exp. Med. 201:249-257).

SCOPUS: ar.j, info:eu-repo/semantics/published

The genes MAGE-1, -2, -3 and -4 are expressed in tumors of different histological types, but not in normal tissues, with the exception of testis and placenta. Short peptides derived from MAGE-1 and MAGE-3 gene products are recognized by cytolytic T lymphocytes when presented by HLA-class-I molecules, and represent potential targets for specific immunotherapy. We have determined whether esophageal carcinoma patients should be eligible for MAGE-peptide-based vaccine therapies. The expression og genes MAGE-1,-2,-3 and -4 in tumor samples was assessed by reverse-transcription and polymerase-chain-reaction amplification. Out of the 49 esophageal squa-mous-cell carcinomas studied, 53% expressed MAGE-1, 49% MAGE-2, 47% MAGE-3 and 71% MAGE-4. Eighty-four percent of the tumors expressed one or more of the four MAGE genes. Owing to the high incidence of MAGE gene expression in esophageal squamous-cell carcinoma, a large proportion of patients could be suitable candidates for immune therapies involving tumor-specific antigens encoded by MAGE genes.

Human genes MAGE-1 and MAGE-3 code for antigens that are recognized on melanoma cells by autologous cytolytic T lymphocytes. These antigens may constitute useful targets for specific anti-tumor immunization of cancer patients, since genes MAGE-1 and MAGE-3 are expressed in a number of tumors of different histological types, but are not expressed in normal adult tissues other than testis. This also applies to genes MAGE-2 and MAGE-4, which are closely related to MAGE-1 and MAGE-3. We have analyzed the expression of these 4 MAGE genes in cutaneous melanoma. Sixteen of 100 primary tumors vs. 69 (48%) of 145 metastases from individual patients expressed MAGE-1. Similar differences in the frequency of gene expression between primary and metastatic tumor samples were observed for MAGE-2, MAGE-3, and MAGE-4. MAGE expression in primary tumors was correlated with tumor thickness: there was a significantly increased frequency in the expression of MAGE-1, -2 and -3 in tumors of greater thickness. Benign and dysplastic nevi, as well as in site melanomas, did not express any of the 4 MAGE genes. (C) 1995 Wiley-Liss, Inc.

An optimal anticancer vaccine probably requires the cooperation of both CD4(+) Th cells and CD8(+) CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome-associated membrane protein-1 (LAMP1) and DC-LAMP. Human DCs were electroporated before or after maturation with mRNA encoding unmodified enhanced green fluorescent protein (eGFP) or eGFP linked to various targeting signals. The lysosomal degradation inhibitor chloroquine was added, and eGFP expression was evaluated at different time points after electroporation. DCs were also electroporated with unmodified MAGE-A3 or MAGE-A3 linked to the targeting signals, and the presentation of MAGE-A3-derived epitopes in the context of HLA class I and class II molecules was investigated. Our data suggest that proteins linked to the different targeting signals are targeted to the lysosomes and are indeed presented in the context of HLA class I and class II molecules, but with different efficiencies. Proteins linked to the LAMP1 or DC-LAMP signal are more efficiently presented than proteins linked to the invariant chain-targeting signal. Furthermore, DCs electroporated after maturation are more efficient in Ag presentation than DCs electroporated before maturation.