Role of Melanoma-Associated Antigens

Chapter 35 / Immunogenic Melanoma-Associated Antigens 619 35 Role of Melanoma-Associated Antigens

Rishab K. Gupta, Ana M. McElrath-Garza, and Donald L. Morton

CONTENTS INTRODUCTION EVIDENCE FOR IMMUNITY TO HUMAN MELANOMA HOST IMMUNE RESPONSE AND DEVELOPMENT OF MALIGNANT MELANOMA RECOGNITION OF MAA EXPRESSED BY HUMAN MELANOMA CELLS ROLE OF IMMUNOGENIC MAA IN MELANOMA PROGRESSION OR INHIBITION CONCLUSIONS AND PERSPECTIVES REFERENCES

Summary The dynamic interaction between malignant melanoma cells and the host’s immune system has a profound influence on the development and progression of disease. Numerous studies have documented the therapeutic potential of immunological manipulation in patients with melanoma, and many tumor- associated antigens have been studied as possible targets of melanoma-specific immunotherapy. This chapter outlines the most significant melanoma-associated antigens, highlighting those with the greatest potential for influencing future treatment modalities. Specific antigens defined by T- and B-cells are discussed, and how these antigens are recognized, processed, and manipulated by the host’s immune system is explained. The role of gangliosides in distinguishing different stages of melanocyte differentiation is described, and a 90-kDa tumor-associated glycoprotein antigen which has considerable promise as a marker of melanoma tumor burden is introduced. Finally, this chapter reviews technological advances in assays used to detect various melanoma-associated antigens. Key Words: Melanoma; melanoma-associated antigens; tumor-associated antigens; immunotherapy; TA90.

INTRODUCTION The malignant transformation of melanocytes to melanoma can be characterized by changes in cytology, morphology, proliferative index, biochemistry, and gene expression. Activation of genes in transformed cells leads to overexpression and/or unique expression of molecules, such as tumor-associated antigens. Some of these molecules may be present in normal cells but sequestered from the immune system. In transformed

From: From Melanocytes to Melanoma: The Progression to Malignancy Edited by: V. J. Hearing and S. P. L. Leong © Humana Press Inc., Totowa, NJ

619 620 From Melanocytes to Melanoma cells, the antigens are expressed on nonsequestered sites. Tumor-associated antigens are considered immunogenic when the host recognizes them as nonself and generates an immune response. This chapter reviews the different melanoma-associated antigens (MAA) (e.g., melanoma antigen gene [MAGE]-1, NY-ESO-1, and TA90) and their roles in immune surveillance and the development/progression of melanoma. More than 40 yr ago, anecdotal reports of an increased incidence of melanoma regression in immunosuppressed patients, such as those with congenital immunodeficiency disorders, suggested an important immune component in the development and control of disease (1–6). The more recent discovery of tissue-specific and common tumor- associated melanoma antigens further supports the key role for the immune system in the development and progression of melanoma. Additional evidence for immune modulation by MAA includes the prognostic significance of lymphocytes and other tumor-cell infiltrates (7–9), the delayed recurrence of rapidly progressive disease after successful treatment of the primary tumor (10–12), the presence of tumor cells in lymphatics, peripheral blood, and operative wounds without subsequent development of metastases (13–20), and the elimination of circulating tumor cells before the establishment of metastatic colonies (21). Ongoing investigations to identify clinically relevant target antigens and enhance the production of corresponding autoantibodies should lead to improvements in immunotherapy for melanoma.

EVIDENCE FOR IMMUNITY TO HUMAN MELANOMA Spontaneous regression of primary and metastatic melanoma, although rare, has been well documented (1–6). Published reports have correlated regression with minor viral and bacterial infections, fever of unknown origin, changes in hormonal balance, and congenital immunodeficiency disorders. Development of metastases that are histologi- cally and functionally identical to the primary tumor has been reported 10 to 20 yr after successful treatment of the primary tumor. Delayed recurrence suggests a clinically effective antitumor response during the disease-free interval. Although biological, physi- ological, and endocrine factors could contribute to spontaneous regression and long- term remission, clinical evidence implicates humoral and cellular immunological mechanisms (22,23).

HOST IMMUNE RESPONSE AND DEVELOPMENT OF MALIGNANT MELANOMA Because malignant melanoma is one of the most immunogenic solid tumors, it has served as a prototype for investigations of active immunotherapy against cancer (24–27). Determining the mechanisms of tumor escape from immunosurveillance is necessary to formulate effective immunotherapy against tumor cells (28). Despite the highly immunogenic nature of melanoma cells, as evidenced by the infiltration of tumor antigen- specific T-cells, most tumors are not completely destroyed by the host and can lead to progression of disease. An effective immune response against the host’s melanoma cells requires immune recognition of target peptides in the context of major histocompatibil- ity complex (MHC) molecules (29–31). This is particularly true for T-cell mediated responses. In addition, co-stimulatory signals are necessary for cell-mediated immunity (32,33). The absence of proper MHC or co-stimulatory molecule expression can prevent an effective antitumor immune response (34). Thus, a melanoma cell that expresses Chapter 35 / Immunogenic Melanoma-Associated Antigens 621 immunogenic MAA may not induce a T-cell mediated antimelanoma response without proper co-stimulation, even if the MAA are recognized by the T-cells. Furthermore, many responses defined as antitumor effector mechanisms can become ineffective or protumorigenic under certain conditions. For example, immune selection pressure could result in outgrowth of resistant tumor variants.

RECOGNITION OF MAA EXPRESSED BY HUMAN MELANOMA CELLS MAA have been detected by in vitro and in vivo cellular reactions and by in vitro serological tests. T- and B-cells that recognize and reject autologous cells have been widely used to identify MAA (35). In serological approaches, both sera from cancer patients and antisera produced in xenogeneic hosts were used as the source of antibody. Antigens Defined by T-Cells Genetic (36), biochemical (37), and immunological (38) approaches have been used to identify MAA recognized by cytolytic T-lymphocytes. MAGE-1 (39) was the first tumor-associated antigen to be identified by a genetic approach. This antigen belongs to a family of at least 12 closely related genes (40) expressed in variable proportions by melanoma cells (41). Expression levels of MAGE antigens, as assessed by polymerase chain reaction technology, vary considerably among tissue samples and cell lines. MAGE-2 and MAGE-3 are the most common. MAGE-1 has been detected by specific monoclonal antibodies (MAbs) in clinical tumor specimens. The pattern of reactivity observed was heterogeneous within individual specimens (42). When MAGE-1 is expressed, MAGE-2 or MAGE-3 is also expressed, making it feasible to use MAGE-1 and MAGE-3 for therapeutic purposes and reduce the risk of tumor escape caused by emergence of antigen-loss variants (41). Benign and dysplastic nevi, as well as in situ melanomas, do not express MAGE genes (43–45). MAGE-1, -2, -3 and -4 are more often expressed by metastatic melanoma than by primary tumors. This difference illustrates the selective activation of certain genes during malignant transformation and progression. Immune responses to the products of these genes may serve as markers of disease status as well as components of a therapeutic antimelanoma response. Melanoblasts, which are precursors of melanocytes, are presumed to have a pheno- typic pattern similar to that of early melanoma cells. MAA such as Melan-A/melanoma antigen recognized by T-cells (MART)-1 (46–48), tyrosinase (49,50), Pme117/gp100 (37,51), gp75/tyrosine-related protein (TRP)-1 (52), and AIM-2 (53) are expressed by normally differentiated melanocytes and by melanoma cells. These antigens are melanoma-specific; they are not expressed by other tumor cells. Houghton et al. (54) reported that melanoma cells in early stages of differentiation do not express antigens that are present during the later stages (such as tyrosinase). Using a real-time polymerase chain reaction method, Johansson et al. (55) found that tyrosinase, TRP-1, TRP-2, and MART-1/ Melan-A expression varied from undetectable to highly detectable levels in the different cell lines at different time intervals. Peptides derived from these MAA have been used as targets for immunotherapy in several clinical trials. Although a significant proportion of patients with disease limited to the dermis, subcutaneous tissue, and/or lymphatics developed an immune response, only a minority of patients with metastatic disease had regression of the tumor. Studies by Slingluff et al. (56) suggest that, if a tumor does not express melanocytic differentia- 622 From Melanocytes to Melanoma tion proteins, immunotherapy should be directed against unique tumor antigens, as well as more frequently encountered melanocytic antigens. Antigens Defined by B-Cell Factors Some MAA have been localized on the cell surface, whereas others are found in the cell cytoplasm (57,58). The presence of antinucleolar antibodies in the sera of melanoma patients has also been reported (59). Before the advent of MAbs, numerous studies used patient sera to demonstrate the existence of tumor antigens in melanoma. Use of serological techniques, such as antibody-mediated cytotoxicity, immune adherence, immunofluorescence, mixed hemadsorption, complement fixation, radioimmunoassay, and enzyme immunoassay demonstrated the immunological reactivity between these antigens and sera from melanoma patients. Based on the humoral crossreactivity and absorption studies, MAA can be grouped into four categories: fetal antigens, common MAA, group-specific antigens with variable expression, and individually specific antigens. Membrane-rich fractions prepared from melanoma tumors showed wide crossreactivity against sera from patients with malignancies of various histological types. However, absorption of the sera with fetal tissue homogenates revealed that the crossreactivity was caused by fetal antigens. Similar wide crossreactivity was observed with the partially purified spent culture medium of a melanoma cell line, suggesting the expression of these antigens by fetal cells and melanoma cells. Within the last decade, serological expression cloning has been used to identify tumor antigens that elicit a strong antibody response in cancer patients (60). The genes SSX2 (61), NY-ESO-1 (62), and SYCP-1 (63) were identified during serological expression cloning analyses of human cancer. These genes, as well as others such as MAGE, BAGE, and GAGE, have been named “cancer/testis (CT) antigens” (60) because they are expressed predominantly in normal testis and in cancer cells. CT genes are considered ideal target antigens for a cancer vaccine because of their immunogenicity and lack of expression in normal tissues. Of the various CT antigens, NY-ESO-1 has been the most extensively studied as a potential component of cancer vaccines (64). There is a clear distinction between CT genes identified by messenger RNA expression analysis and those identified through immunological methods. The former genes have immunogenic potential, whereas the latter are immunogenic in cancer patients (63). Forty-four CT antigen families have been reported. With the exception of a few genes, such as those of the MAGE family, there is no general evolutionary linkage between CT genes. In addition, protein products of only 19 CT gene families have been demonstrated to elicit an immune response in cancer. The responses were strickly humoral in 13 cases, strictly cellular in 3 cases, and cellular plus humoral in 3 cases (63). Some of the antigenic diversity of human malignant melanoma cells could reflect differences in serological assay techniques, antibody sources (patient sera), and target antigen purity. Conditions for in vitro culture are not the same as those for in vivo tumor growth; the selective pressure of culture conditions may change antigenic expression on or in the cultured melanoma cells. In our experience, the expression level of human MAA fluctuates markedly with passage from one generation to another. Incorporation of exogenous components from growth medium into the membrane of cultured cells is well documented (65), and may influence the results of serological assays. This artifact has been minimized by preparing the target antigen from cells cultured in fetal calf serum-free medium or other medium that is free of potentially immunogenic supplements (66). Chapter 35 / Immunogenic Melanoma-Associated Antigens 623

Hybridoma technology has allowed development of MAbs that recognize tumor- associated antigens expressed in variable levels on autologous cells, melanoma cells, and tumor cells of other histological types. Consistent identification of these antigens— despite varied serological techniques and nonstandardized reagents—confirms existence of the four groups of tumor-associated antigens on melanoma cells. These immunogenic antigens are recognized by allogeneic antibodies and their expression significantly influences the development and progression of melanoma (67). Gangliosides as Melanoma Antigens Gangliosides are carbohydrates that have been used to distinguish different stages of melanocyte/melanoma differentiation (68). These acidic glycolipids contain a hydro- phobic ceramide moiety and are expressed by both normal melanocytes and melanoma cells. GM3, GD3, GM2, GD2, and O-acetyl GD3 are found on the cell membrane of melanocytic cells. Expression of some gangliosides is significantly (>1000-fold, in the case of GD3) upregulated after malignant transformation of melanocytes (69). Regula- tion of ganglioside expression could trigger neoplastic transformation of melanocytes and progression of transformed cells. The diversity of ganglioside expression in neoplastically transformed cells is governed by individual genotypic differences. Changes in ganglioside profile of neoplastically transformed melanocytes correlate with changes in the proliferation, migration, and metastasis of malignant cells (70). Although this chapter is limited to the immunogenic antigens of melanoma, both immunogenic and nonimmunogenic gangliosides play a role in transformation of melanocytes and progression of the transformed cells. The presence of autoantibodies to gangliosides has been correlated with an improved prognosis in melanoma patients (71–73). Sialyl Lewis(a) is not immunogenic in melanoma, but its expression has been correlated with melanoma progression (74). Antigens Defined by Antibody Phage Display Library Method A phage-Fab library derived from the B-cells of a melanoma patient in remission after immunotherapy has been used to identify and isolate a 23-kDa glycoprotein (75). The p23 antigen is on the surface and in the cytoplasm of melanoma cells, and it is expressed at high levels by cultured melanoma cell lines, vertical growth-phase primary melanoma, and metastatic melanoma. The antigen is not expressed in radial growth-phase primary melanoma, nevi, and normal skin. By identifying potentially stage-specific MAA and determining the mechanisms involved in neoplastic progression of transformed melanocytes, the antibody phage display library method (76,77) may have therapeutic and diagnostic potential (78).

ROLE OF IMMUNOGENIC MAA IN MELANOMA PROGRESSION OR INHIBITION Reactions observed in vitro between sera and autologous melanoma cells might also occur in vivo. These reactions, which involve both tumor and host functions, could promote or prevent tumor growth and progression (79). Efforts are underway to increase the quality and magnitude of MAA-specific immune responses to a clinically effective level (80). However, the possibility of antigen loss from tumor cells resulting from immunoselection could result in immune escape and subsequent tumor progression (81–84). 624 From Melanocytes to Melanoma

Several regulatory pathways and molecules may be involved in melanoma antigen silencing, tumor cell differentiation, and the outcome of antitumor immune responses (85–87). Certain melanoma antigens may be modulated by cytokines. In addition, cell and matrix adhesion molecules, growth factors, proteases, gangliosides, and MHC class I and II molecules contribute to the course of malignant melanoma. Antigens expressed on melanoma cells but not on mature melanocytes may be used as markers for the degree of cell differentiation; dedifferentiation of melanoma cells decreases their antigenic similarity to normal melanocytes (88). Different genes responsible for expression of melanoma antigens have been identified in various stages of the disease and may be used as tumor markers for recurrence or progression. Modulation of melanoma antigens has been suggested as a mechanism for tumor progression in the presence of cytolytic T-lymphocytes. This might explain the common finding of Melan-A/MART-1-specific tumor-infiltrating lymphocytes in clinically pro- gressing melanomas, and it might represent a possible pathway for therapeutic interven- tion (89). The antigen-derived T-cell response evoked by early melanoma becomes attenuated with disease progression. A variety of cell adhesion molecules on the surface of melanoma cells may regulate cellular cytotoxicity. The use of MAA, antiantigen autoantibody levels, and immune complexes (IC) has shown promise for detecting disease and for planning and monitoring treatment. Many reports indicate that appearance of antibodies to MAA is associated with improved prognosis. These circulating antimelanoma antibodies can bind to their corresponding antigens and form IC. A clinically interesting example is the hypopigmentation associated with binding of antimelanoma antibodies to normal melanocytes. Serum from patients with vitiligo contains a high titer of naturally occurring antimelanoma antibodies and reportedly induced regression of melanoma metastases in mice. Although the association with prognosis is not clear, the appearance of hypopigmentation in patients with melanoma serves as evidence for the activity of antimelanoma antibodies (90). Prognostic Role of Anti-TA90 Immunity in Melanoma Initial studies used allogeneic antibodies to identify a high molecular weight complex in the urine of patients with metastatic melanoma (91,92). This complex comprises multiple subunits, including an immunogenic 90-kDa glycoprotein called TA90 (93). TA90 is a heat-stable antigen with an isoelectric point of 6.1. It is expressed by about 75% of human solid tumors. Immunochemical analysis, including antibody-blocking studies, revealed that TA90 is distinct from known tumor markers such as carcino- embryonic antigen, prostate-specific antigen, CA15-3, D-fetoprotein, and other oncofetal antigens (94). Most cancer patients whose tumors express TA90 have endogenous anti-TA90 antibodies of immunoglobulin (Ig)-M and IgG isotypes. In patients with early- or intermediate-stage melanoma, the immune response to TA90 has been correlated with occult nodal disease and survival (95). In addition, evidence suggests that the endogenous immune response to TA90 might determine the postoperative outcome of patients undergoing surgical therapy for metastatic melanoma (96). Despite the fact that tumor cells have been detected in the blood of patients with metastatic melanoma, many of these patients have enjoyed prolonged survival or cure after surgical resection. This suggests that enhancement of a specific antitumor immune response might prolong survival. In fact, melanoma patients receiving a polyvalent specific active immunothera- Chapter 35 / Immunogenic Melanoma-Associated Antigens 625 peutic (Canvaxin, CancerVax Corp, Carlsbad, CA) that contains TA90 exhibit increased humoral (IgM and IgG) and delayed-type hypersensitivity responses (97,98). In a series of investigations in which patients received Canvaxin after surgical resection of melanoma, elevated humoral and delayed-type hypersensitivity responses to Canvaxin were associated with prolonged survival (99–101). These observations were the basis for an immunological model to predict the survival of patients who received Canvaxin for regional metastatic melanoma (98). In general, the incidence and level of antibodies to TA90 or other MAA are higher when melanoma is localized than when it is disseminated (102). Surgery and immunotherapy with autologous or allogeneic vaccines will affect levels of circulating antibody in melanoma patients. Prognostic Role of TA90-Specific Immune Complex in Melanoma When the original high molecular weight glycoprotein complex was purified and used to develop a murine MAb (AD1-40F4) of IgM isotype (103), Western blot analysis revealed that AD1-40F4 recognized a 90-kDa band in urine and serum of cancer patients (93). This epitope is different from the epitope recognized by the autoantibody in the serum of melanoma patients (104). AD1-40F4 was used to develop a TA90-IC assay that discriminated between normal and melanoma sera (105). This assay has a sensitivity of 83% in detecting nodal metastases in melanoma patients undergoing lymphadenectomy (94). To test the hypothesis that the postoperative serum level of TA90-IC could have a significant correlation with recurrence and survival in patients with thick primary melanomas, postoperative TA90-IC status was correlated with disease-free survival (DFS) and overall survival (OS). Standard prognostic factors for melanoma were then compared with TA90-IC. The sensitivity and specificity of TA90-IC for predicting recurrence were 70% and 85%, respectively. Five-year DFS and OS rates were significantly higher when TA90-IC was negative. At a median follow-up of 25 mo, multivariate analysis identified postoperative TA90-IC status as the only independent predictor of DFS (106). Therefore, a positive postoperative TA90-IC level suggests the presence of micrometastases that may become clinically significant. Because standard prognostic factors, including precise staging of the regional lymph nodes, cannot accurately determine which early-stage melanomas will metastasize, and because TA90-IC status in thick primary melanoma showed a correlation with survival, TA90-IC was also investigated as a prognostic marker for patients with thin primary melanoma. Patients with 1.01- to 2.00-mm primary melanomas and tumor-negative regional lymph nodes were divided into two groups (95). Group 1 comprised 50 patients who died of metastases within 7 yr after complete surgical treatment; group 2 comprised 50 patients who were matched with group 1 by six standard prognostic factors (including tumor thickness, Clark’s level, and presence of ulceration), but who lived at least 10 yr without recurrence. Excluded from study were patients whose stage I melanoma had spread to the regional nodes (stage III disease), either at the time of lymphadenectomy or at any time before the development of distant metastases. This eliminated the possibility of studying patients with occult lymph node metastases or locoregional relapse. In addition, this study excluded any patient who received any form of postoperative adjuvant therapy; this eliminated the possible influence of adjuvant therapy on clinical outcomes. All patients in both groups underwent staging of the regional lymph nodes by 626 From Melanocytes to Melanoma complete lymph node dissection or, more recently, by lymphatic mapping and selective dissection of the sentinel lymph node. Postoperative sera from these patients were ana- lyzed for TA90-IC. Standard microscopic examination determined the status of all excised lymph nodes. The incidence of TA90-IC positivity was 82% in group 1 and 18% in group 2 (p < 0.001). Thus, positive TA90-IC level correlated with distant metastasis when melanoma was low- or intermediate risk by standard prognostic factors. TA90-IC has been identified in the sera of numerous melanoma patients. The correlation between TA90-IC and overall survival suggests that subclinical metastasis can be detected before surgical treatment of early-stage melanoma. A subsequent study used postoperative sera to determine TA90-IC levels in patients with melanoma and evaluate their relationship to recurrence and survival (107). Multiple archived serum samples were prospectively collected during postoperative surveillance of 166 patients with American Joint Committee on Cancer stage I, II, or III melanoma. TA90-IC results were correlated with disease recurrence and survival data. The TA90-IC status in the early postoperative period was highly predictive of survival. Five-year OS was 64% for TA90-IC-negative patients and 36% for TA90-IC-positive patients (p = 0.0001). Median OS was 40 mo for TA90-IC-positive groups and 160 mo for the negative groups. TA90-IC-positive patients had a 5-yr DFS of only 24%, compared with 74% for TA90-IC-negative patients (p = 0.0001). Median DFS was 18 mo and 160 mo in TA90-IC-positive and TA90-IC-negative groups, respectively. In a study of 125 stage IV melanoma patients undergoing complete resection of distant metastases, postoperative TA90-IC positivity was again the most important prognostic variable for DFS (108). Thus, there is ample evidence to suggest that a positive TA90-IC assay strongly correlates with recurrence after surgical resection of American Joint Committee on Cancer stage II, III, and IV melanoma.

CONCLUSIONS AND PERSPECTIVES Tumor immunology represents a complex interplay of many different factors that affect tumor cell growth. Results of various investigations of immune responses against melanoma antigens indicate that both cellular and humoral immune responses to MAA are critical to the development and control of progressive disease (109). Clinical reports of spontaneous regression of melanoma and the improved outcomes seen in patients receiving experimental vaccines are promising indications of the significant impact that immunotherapy may have on the future treatment of melanoma. However, development of recurrence and the progression of treated disease suggest that melanoma cells may escape immune destruction by losing or downregulating expression of MAA targets. Continued studies of MAA-based interactions may allow the development of antigen- targeted immunotherapy that prevents the malignant transformation of melanocytes to melanoma.

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