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Cancer/testis antigens (CTA) are tumor-associated antigens expressed during ontogenesis, in a number of solid tumors but not in normal tissues except testis. Most of these CTA are highly immunogenic, eliciting a humoral and cellular response in the patients with advanced cancer, and are useful for tumor-specific immunotherapy. Medullary thyroid carcinoma (MTC) is a neoplasm derived from the parafollicular cells of the thyroid and occurs in either a sporadic or a familial form. In the present study, we examined by RT-PCR the expression of a number of genes encoding CTA in 23 surgical samples of sporadic MTC. Among the 11 cDNA antigens examined, RAGE, MAGE-4, and GAGE 1-2, were not expressed in any of the tissues. SSX 2 was present only in one tissue, whereas BAGE, GAGE 1-6, MAGE-1, MAGE-2, MAGE-3, and SSX 1-5 were detected in two to five samples. NY-ESO-1 cDNA was the most frequent, being detected in 15 of 23 examined samples (65.2%). Six (26.1%) tissues did not express any CTA-specific mRNA, whereas 10 tumors expressed only one gene (43.5%), 3 (21.4%) expressed 2 genes, and 4 displayed a broad CTA gene expression. NY-ESO-1 expression in primary MTC tissues significantly correlated with tumor recurrence. The presence of specific anti-NY-ESO-1 antibodies was searched in the sera of MTC-affected patients examined by ELISA using recombinant NY-ESO-1 protein. A humoral response against this CTA was detected in 6 of 11 NY-ESO-1 expressing patients (54.5%), and in 1 of 6 patients with NY-ESO-1-negative tumor. No anti-NY-ESO-1 antibodies were detected in healthy subjects (n = 17). The presence of anti-NY-ESO-1 antibodies was searched also in the sera of MTC affected patients whose tissues were not available for CTA analysis. Anti-NY-ESO-1 antibodies were present in 15 of 42 sera (35.7%), demonstrating that MTC is a neoplasm frequently associated with humoral immune response to NY-ESO-1. Serological survey may be useful as a way to identify patients with humoral immune response to NY-ESO-1 that provide a new attractive target for vaccine-based immunotherapy of MTC.

Purpose: Limited therapeutic options are presently available for advanced renal cell carcinoma (RCC). This study was designed to define the clinical potential of the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) in human RCC, through its control of the expression of "therapeutic targets" of the cancer testis antigen (CTA) family, and of the tumor-associated antigen RAGE-1, in RCC cells., Experimental Design: Reverse transcription (RT)-PCR assays of a panel of RCC cells treated with 5-AZA-CdR, investigated the induction of the expression of several CTAs and of RAGE-1. Immunoprecipitation and Western blotting assessed whether the expression of CTA-specific mRNA induced by 5-AZA-CdR resulted in a translated protein of appropriate molecular weight. The functional activity of de novo expressed CTA was evaluated using (51)Cr release cytotoxicity assays of 5-AZA-CdR-treated HLA-A2-positive RCC cells using HLA-A2-restricted NY-ESO-1-specific CTLs., Results: Exposure to 5-AZA-CdR invariably induced the expression of the CTA MAGE-1, -2, -3, and -4, GAGE 1-6, and NY-ESO-1 in all of the RCC cells investigated. De novo expression of NY-ESO-1 was persistent, being still detectable 60 days after the end of treatment, and generated a functional protein efficiently recognized by HLA-A2-restricted NY-ESO-1-specific CTLs. 5-AZA-CdR also induced RAGE-1 expression in RAGE-1-negative RCC and sarcoma cells but not in neoplastic cells of different histology., Conclusions: This study provides the scientific rationale to establish new strategies of chemoimmunotherapy in RCC patients. The well-defined immunogenicity of the investigated CTAs and of RAGE-1 suggest that systemic administration of 5-AZA-CdR represents a promising strategy to enhance the constitutively poor immunogenic potential of RCC cells, and to propose that virtually all RCC patients receive active and/or adoptive CTA- or RAGE-1-based immunotherapy.

Cancer-testis antigens expressed by different-histotype transformed cells are suitable targets for tumor immunotherapy. However, their heterogeneous expression in neoplastic lesions limits the eligibility of patients for cancer-testis antigen-directed vaccination, and low levels of cancer-testis antigens' expression may impair immune recognition of malignant cells. Because of the primary clinical relevance of cancer-testis antigens' expression in neoplastic tissues, 68 unrelated or sequential metastatic lesions from 56 patients were used to characterize the molecular mechanisms regulating the presence and levels of expression of different cancer-testis antigens of the MAGE family (i.e., MAGE2, 3 and 4) in cutaneous melanoma. Polymerase chain reaction-based methylation analyses showed that methylation status of specific cytosine-guanine dinucleotides in the promoters of investigated cancer-testis antigens correlated with their heterogeneous expression within unrelated metastatic melanoma lesions, and with their homogeneous expression among sequential metastases from three patients with melanoma. Unlike methylated promoters, unmethylated promoters of MAGE2, 3 and 4 genes drove the expression of reporter gene-enhanced green fluorescent protein after transient transfection of cancer-testis antigen-positive Mel 142 melanoma cells. Furthermore, de novo expression of MAGE3 gene induced by the treatment of Mel 195 melanoma cells with the DNA hypomethylating agent 5-aza-2'-deoxycytidine was associated with a 6%-12% demethylation of selected cytosine-guanine dinucleotides in its promoter. Finally, 5-aza-2'-deoxycytidine induced a 16-fold increase of MAGE3 expression in Mel 313 melanoma cells expressing constitutively low levels of the antigen, but did not affect that of Mel 275 melanoma cells expressing high baseline levels of MAGE3. Overall, these findings identify promoter methylation as a shared mechanism directly regulating the expression of therapeutic cancer-testis antigens in metastatic melanomas, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemoimmunotherapeutic strategies in patients with melanoma.

Endoglin (CD105) is a cell membrane glycoprotein over-expressed on highly proliferating endothelial cells in culture, and on endothelial cells of angiogenetic blood vessels within benign and malignant tissues. CD105 binds several factors of the Transforming Growth Factor (TGF)-beta superfamily, and its over-expression modulates cellular responses to TGF-beta1. The complex of experimental findings accumulated in the last few years strongly indicate that CD105 is a powerful marker of angiogenesis, and that it might play a critical role in the pathogenesis of vascular diseases and in tumor progression. In this paper, we will review the structural, biological and functional features of CD105, as well as its distribution within normal and neoplastic tissues, emphasizing its foreseeable role as a molecular target for new diagnostic and bioimmunotherapeutic approaches in human malignancies., (Copyright 2001 Wiley-Liss, Inc.)

Conflicting evidences suggested that levels of HLA-A and -B antigens expressed on normal and neoplastic cells of given individuals are genetically predetermined, or, on the other hand, regulated by molecular mechanisms generating the down-regulated expression of HLA-B antigens frequently observed on melanoma cells. In our study, we quantitated, both at the protein and mRNA level, the amounts of HLA-A and -B antigens constitutively expressed on 23 primary cultures of metastatic melanomas and on autologous peripheral blood mononuclear cells (PBMC). Flow cytometric analyses identified a significantly (p < 0.01) lower expression of HLA-B antigens on melanoma cell cultures but not on autologous PBMC. Consistently, lower amounts of HLA-B antigens mRNA were detected by RNase protection assay exclusively in neoplastic cells. This unbalanced expression of HLA-A and -B antigens was readily reverted by interferon (IFN)-gamma but not by the DNA hypomethylating agent 5-aza-2'-deoxycytidine in 4 melanoma cell cultures investigated. Significantly (p < 0.05) lower levels of HLA-B antigens were also detected on cells from solid malignancies of different histotypes but not on neoplastic cells from hemopoietic neoplasms; levels of HLA-B antigens were rapidly up-regulated by IFN-gamma exclusively on non-hemopoietic transformed cells. Together, these data strongly argue against a genetic predetermination of the amounts of HLA-A and -B antigens expressed on normal and neoplastic cells of distinct melanoma patients and suggest that constitutively low levels of HLA-B antigens are a specific feature of non-hemopoietic transformed cells that is controlled by common regulatory mechanism(s) and that is possibly shared by non-hemopoietic normal cells., (Copyright 2001 Wiley-Liss, Inc.)

The clinical efficacy of therapeutic complement (C)-activating monoclonal antibodies (mAb) to melanoma-associated antigens can be impaired by the levels of expression of C-inhibitory molecules on neoplastic cells. Protectin (CD59) is a glycosylphosphatidylinositol (GPI)-anchored cell membrane glycoprotein, acting as terminal regulator of C cascade, which is heterogeneously expressed in melanomas and represents the main restriction factor of C-mediated lysis of melanoma cells. Thus, we investigated whether the overexpression of CD59 could influence the constitutive susceptibility of distinct melanoma cells to homologous C. Infection of CD59-positive Mel 100 and 70-W melanoma cells by a retroviral vector carrying the CD59 cDNA, significantly (P < 0.05) upregulated their constitutive expression of CD59, whereas it did not affect that of additional C-regulatory molecules. Transduced CD59 was entirely GPI-anchored and showed a molecular weight identical to native CD59. Additionally, higher amounts of soluble CD59 were detected in the conditioned media of CD59-transduced melanoma cells compared with parental cells. CD59-transduced melanoma cells, sensitized by the anti-GD3 disialoganglioside mAb R24, were significantly (P < 0.05) less susceptible to homologous C-lysis than were parental cells; this effect was fully reverted by the masking of CD59 with F(ab')(2) fragments of the anti-CD59 mAb YTH53.1. These results provide conclusive evidence demonstrating that absolute levels of CD59 expression regulate the susceptibility to homologous C of specific melanoma cells, and suggest an additional explanation for the poor clinical results obtained with C-activating mAb in the clinical setting., (Copyright 2000 Wiley-Liss, Inc.)

Over the past two decades, complement (C)-activating monoclonal antibodies (mAb), directed to specific tumor-associated antigens (TAA), have been extensively utilized for passive immunotherapy of solid tumors of different histology. However, the clinical outcome of this therapeutic approach has been substantially disappointing; antigenic heterogeneity of neoplastic cells and their limited accessibility by therapeutic mAb, have been provided as substantial explanations for the poor clinical results obtained. Nevertheless, in light of the recent advances in the knowledge of the mechanisms regulating C-activity, it begins to be evident that membrane and soluble C-inhibitory proteins play a key role in the protection of neoplastic cells from C-attack, providing additional insights on biological features of transformed cells that may hamper the clinical efficacy of humoral immunotherapy. Among C-regulatory proteins investigated, this review will focus on protectin (CD59) that represents the main restriction factor of C-susceptibility of neoplastic cells from solid malignancies. In view of the functional role of CD59, we will describe its tissue distribution and biological features in malignant neoplasms; major emphasis will be given to cutaneous melanoma, in which the C-regulatory role of CD59 has been extensively investigated, and clinical approaches of humoral immunotherapy have been implemented. According to the available data, the foreseeable strategies to improve the therapeutic efficacy of humoral immunotherapy of solid malignancies will be discussed.

Melanoma cells constitutively release intercellular adhesion molecule 1 (ICAM-1) as soluble ICAM-1 (sICAM-1), and its levels are elevated in melanoma patients and correlate with disease progression. However, this correlation is not absolute, suggesting that specific characteristics of neoplastic cells and/or ICAM-1-positive non-neoplastic cells may influence the amounts of circulating sICAM-1. In this study, we found a weak correlation (r = 0.55; r2 = 0.3) between sICAM-1 release by 40 metastatic melanomas (36 primary cultures and 4 cell lines), and ICAM-1 expression on neoplastic cells. In addition, melanoma-secreted interleukin-1alpha (IL-1alpha) (1/40) but not vascular endothelial growth factor (VEGF) (29/40), significantly (P < 0.05) up-regulated the shedding of sICAM-1 by human umbilical vein endothelial cells (HUVEC). This was completely abolished by IL-1alpha/beta neutralizing antibodies both at the protein and mRNA level. Altogether, our results suggest that (i) the extent of sICAM-1 release is distinctive for individual melanomas and can be independent of ICAM-1 expression; (ii) tumor endothelia may sustain levels of sICAM-1 in selected melanomas; (iii) melanoma-released VEGF does not affect ICAM-1 expression and sICAM-1 release by HUVEC. Melanoma-derived sICAM-1 inhibits cell-mediated cytotoxicity of melanoma cells; therefore, constitutive levels of sICAM-1 release and IL-1alpha secretion by individual melanomas can differentially influence tumor progression and the clinical effectiveness of cytotoxic-cell-based vaccines.

The immunogenic potential of melanoma cells and their recognition by the host's cytotoxic cells depends on the presence and on the level of expression of human leukocyte antigen (HLA) class I antigens, costimulatory molecules and melanoma-associated antigens (MAA), on neoplastic cells. In this study, we demonstrate that the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR), significantly (p < 0.05) enhanced the constitutive expression of HLA class I antigens, HLA-A1 and -A2 alleles, and of the costimulatory molecules intercellular adhesion molecule-1 and lymphocyte function-associated antigen-3, on a panel of 12 melanoma cells. This upregulation peaked at day 4, slowly decreased thereafter, and returned to baseline levels 32 days after the end of treatment. In addition, treatment with 5-AZA-CdR induced a persistent expression of MAGE-1 in Mel 275 melanoma cells; this was still detectable, by reverse transcriptase polymerase chain reaction, 60 days after the end of treatment. In contrast, 5-AZA-CdR did not affect the constitutive expression of the high molecular weight-MAA by the melanoma cells investigated. These observations, together with data obtained comparing the effect of 5-AZA-CdR with that of interferon-gamma, strongly suggest that 5-AZA-CdR may have prospective therapeutic implications in active and/or passive specific immunotherapy for human melanoma.