Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.

Like many epithelial tumors, head and neck squamous cell carcinoma (HNSCC) contains a heterogeneous population of cancer cells. We developed an immunodeficient mouse model to test the tumorigenic potential of different populations of cancer cells derived from primary, unmanipulated human HNSCC samples. We show that a minority population of CD44(+) cancer cells, which typically comprise <10% of the cells in a HNSCC tumor, but not the CD44(-) cancer cells, gave rise to new tumors in vivo. Immunohistochemistry revealed that the CD44(+) cancer cells have a primitive cellular morphology and costain with the basal cell marker Cytokeratin 5/14, whereas the CD44(-) cancer cells resemble differentiated squamous epithelium and express the differentiation marker Involucrin. The tumors that arose from purified CD44(+) cells reproduced the original tumor heterogeneity and could be serially passaged, thus demonstrating the two defining properties of stem cells: ability to self-renew and to differentiate. Furthermore, the tumorigenic CD44(+) cells differentially express the BMI1 gene, at both the RNA and protein levels. By immunohistochemical analysis, the CD44(+) cells in the tumor express high levels of nuclear BMI1, and are arrayed in characteristic tumor microdomains. BMI1 has been demonstrated to play a role in self-renewal in other stem cell types and to be involved in tumorigenesis. Taken together, these data demonstrate that cells within the CD44(+) population of human HNSCC possess the unique properties of cancer stem cells in functional assays for cancer stem cell self-renewal and differentiation and form unique histological microdomains that may aid in cancer diagnosis.

MAGE, BAGE and GAGE gene expression in human rhabdomyosarcomas.

MAGE, BAGE and GAGE genes encode tumor-associated antigens that are presented by HLA class I molecules and recognized by CD8(+) cytolytic T lymphocytes. These antigens are currently regarded as promising targets for active, specific tumor immunotherapy because MAGE, BAGE and GAGE genes are expressed in many human cancers of different histotype and are silent in normal tissues, with the exception of spermatogonia and placental cells. MAGE, BAGE and GAGE gene expression has been extensively studied in different tumors of adults but is largely unknown in many forms of pediatric solid cancer. Using RT-PCR, we analyzed MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, BAGE, GAGE-1,-2 or -8 and GAGE-3,-4,-5,-6 or -7b gene expression in 31 samples of pediatric rhabdomyosarcoma, the most frequent form of malignant soft tissue tumor in children. MAGE genes were expressed in a substantial proportion of patients (MAGE-1, 38%; MAGE-2, 51%; MAGE-3, 35%; MAGE-4, 22%; MAGE-6, 35%), while expression of BAGE (6%); GAGE-1, GAGE-2 and GAGE-8 (9%); and GAGE-3, GAGE-4, GAGE-5, GAGE-6 and GAGE-7B (16%) was less frequent. Overall, 58% of tumors expressed at least 1 gene, and 35% expressed 3 or more genes simultaneously. Our data suggest that a subset of rhabdomyosarcoma patients could be eligible for active, specific immunotherapy directed against MAGE, BAGE and GAGE antigens.

MAGE, BAGE, and GAGE gene expression in patients with esophageal squamous cell carcinoma and adenocarcinoma of the gastric cardia.

BACKGROUND: The MAGE, BAGE, and GAGE gene families code for distinct, tumor specific antigens that are recognized by cytotoxic T lymphocytes in the context of HLA molecules. The purpose of this study was to analyze MAGE, BAGE, and GAGE gene expression in the two major histologic types of esophageal carcinoma, squamous carcinoma (ESCc) and adenocarcinoma (CAc), and to correlate their expression patterns with the principal prognostic parameters and long term survival. METHODS: Gene expression was analyzed in surgical samples from 24 patients with ESCc and 24 patients with CAc by reverse transcriptase-polymerase chain reaction amplification (RT-PCR). None of the patients had received preoperative chemotherapy or radiotherapy, and all were followed until death or for a minimum of 4 years. RESULTS: Sixteen ESCc samples (67%) and 9 CAc samples (37.5%) expressed at least one of the genes under study. The expression of each MAGE gene in the two histologic types was not significantly different, with the exception of MAGE-4, which was expressed more in ESCc samples than in CAc samples. BAGE and GAGE expression was rather low and, in every case, was associated with the expression of at least one MAGE gene. CONCLUSIONS: In the group as a whole, and in both ESCc and CAc subgroups, no significant correlation emerged between the expression of any gene and prognostic parameters, such as pathologic tumor, lymph node, or disease stage. Nevertheless, BAGE or GAGE expression was related significantly to a poor prognosis, whereas the expression of MAGE genes (in the absence of BAGE and GAGE expression) was related significantly to a good prognosis.

Dendritic cells acquire the MAGE-3 human tumor antigen from apoptotic cells and induce a class I-restricted T cell response.

In an attempt to transduce monocyte-derived dendritic cells (DCs) with a retroviral vector coding for an intracytoplasmic tumor antigen (TAA), we were confronted by the evident dissociation between the ability of the treated DCs to induce a TAA-specific response, and the presence of integrated vector proviral DNA. The TAA, i.e., MAGE-3, was acquired by DCs and presented to immune effectors, thanks to the property of DCs to uptake the apoptotic bodies released by the irradiated vector-producing cells. Indeed, we observed that upon irradiation vector-producing cells underwent apoptotic cell death, monitored by annexin V and propidium iodide staining, and were phagocytosed by DCs. Lymphocytes obtained from a patient affected by a MAGE-3(+) melanoma, were stimulated in vitro with autologous DCs previously exposed to irradiated MAGE-3-expressing cells. This procedure led to the induction of MAGE-3-specific cytotoxic effectors, directed against a yet unknown MAGE-3 epitope presented by HLA-A*B5201 molecules. These data demonstrate that DCs can present engulfed human TAAs, thus providing strategies for cancer vaccination.

Identification of a promiscuous T-cell epitope encoded by multiple members of the MAGE family.

One of the major limitations of tumor-specific vaccination is the generation of antigen-loss variants that are able to escape the immune response elicited by a monoantigenic peptide epitope. Here, we report the identification of a new HLA-B*3701-restricted epitope shared by four different members of the MAGE family. Peripheral blood lymphocytes isolated from a melanoma patient were stimulated in vitro with the autologous HLA-negative melanoma line transfected with autologous HLA B*3701 molecule. This protocol led to the induction of tumor-specific, B*3701-restricted CTLs specific for a peptide epitope encoded by codons 127-136 of the gene MAGE-1. The same epitope is also encoded by the homologous region of three other members of the MAGE family, MAGE-2, -3, and -6. Consistent with the notion that the peptide encoded by MAGE-1 codons 127-136 is, indeed, processed from the proteins encoded by all four MAGE family members, the CTLs were able to specifically recognize Cos-7 cells cotransfected with HLA-B*3701 and any of these MAGE genes. Moreover, the CTLs also recognized a MAGE-6-positive melanoma line transfected with the B*3701 molecule. These findings allow the inclusion of a new set of tumor patients into clinical cancer vaccination trials. Furthermore, they suggest that some promiscuous peptide epitopes shared by different members of the MAGE family might be less prone to escape the immune response by generation of MAGE antigen loss variants.

The DAM gene family encodes a new group of tumor-specific antigens recognized by human leukocyte antigen A2-restricted cytotoxic T lymphocytes.

The DAM family of genes has a high degree of homology with MAGE, both in nucleotide sequence and in neoplastic tissue-specific expression. This study describes, for the first time, the identification of CTLs specific for a peptide epitope encoded by DAM genes. A human leukocyte antigen (HLA)-A2-restricted CTL clone was raised against a peptide, D10/6-271, encoded by codons 271-279 in the DAM cDNA. The corresponding peptide in the MAGE-3 sequence, M3-271, has been shown previously to be a natural T-cell epitope for HLA-A2-restricted CTLs recognizing the MAGE-3 protein. The D10/6-271-specific CTL clone required approximately 3 nM exogenous peptide for half-maximal lysis of target cells and was able to specifically recognize endogenous DAM antigen on HLA-A2+ melanoma cells infected with a vaccinia vector recombinant for gene DAM-6. These data suggest that DAM genes might encode a new group of tumor-specific antigens useful for the design of specific antitumor vaccines.

High homogeneity of MAGE, BAGE, GAGE, tyrosinase and Melan-A/MART-1 gene expression in clusters of multiple simultaneous metastases of human melanoma: implications for protocol design of therapeutic antigen-specific vaccination strategies.

Human melanoma cells express several antigens which are recognized by autologous and specific CTL clones in association with HLA-class-I molecules. Many of these antigens represent suitable targets for tumor immunotherapy, since their expression in human melanoma cells is common and highly specific. In order to achieve real clinical success with therapeutic vaccination strategies, one important requirement is the expression of the target antigen by all the tumor lesions of a patient. We have studied this issue by assessing, through an RT-PCR approach, the expression of MAGE-1, MAGE-2, MAGE-3, BAGE, GAGE-1/2, Tyrosinase and Melan-A/MART-1 genes in 17 clusters of simultaneous in-transit or regional lymph-node metastases collected from 15 stage-III and 1 stage-IV (AJCC/UICC pTNM system) melanoma patients. In 14 out of 17 clusters of simultaneous metastatic lesions (82%), the homogeneity in the pattern of gene expression within the cluster was complete. Heterogeneity within the same cluster was observed in only 3 out of 17 clusters (18%) and represented only minor features. Our data reveal that, in AJCC-stage-III melanoma patients, different but simultaneous metastatic lesions express the same pattern of antigen-coding genes. These observations have 2 main clinical implications: (i) the antigenic characterization of one single and easily accessible lesion allows identification of optimal targets for an active antigen-specific immunotherapy treatment; (ii) almost all the metastatic lesions are expected to be hit by the immune response eventually induced against the tumor antigen. Moreover, these data suggest that active specific immunotherapy directed against MAGE-1, MAGE-3, BAGE, GAGE-1/2, Melan-A/MART-1 and Tyrosinase antigens could be exploited as an adjuvant treatment to surgery in high-risk AJCC-stage-III-melanoma patients.