Decades of investigation have sought the identification of melanoma antigens associated with immune-mediated tumor destruction. These efforts arose from a need for effective therapies as limited options for patients with advanced disease exist. Compelling evidence showed that melanoma stimulates endogenous immune responses that play a critical role in combating this disease and such can be manipulated in some individuals to result in clinically significant effects. The progression of melanoma from in situ to metastatic disease has been well described in clinical as well as pathologic terms. The molecular mechanisms responsible for this development, however, are only beginning to be appreciated. Technological advances in profiling cancer have brought attention to new genetic alterations involved with melanoma. These occur and are influenced by complex interactions with the host. As the result of these interactions, the changes that result with melanoma progression are likely selected, with the immune system playing a prominent role in this process.
In the current issue of Clinical Cancer Research, Barrow et al. (1) examined the expression pattern of two categories of melanoma antigens, the differentiation antigens represented by gp100, Melan-A, and tyrosinase and the cancer-testis antigens MAGE-A1, MAGE A4, and NY-ESO-1 in a large series of primary and metastatic samples. They discovered that the differentiation antigens were strongly expressed in the vast majority of both primary and metastatic melanomas. In comparison, two of three of the cancer-testis antigens revealed greater expression in metastatic deposits. Specifically, MAGE-A1 was more prevalent in ulcerated primaries, a significant adverse prognostic sign as exemplified by its important addition to the 2002 American Joint Committee on Cancer melanoma staging system.
Previous attempts to correlate antigen expression patterns and tumor progression in melanoma have resulted in varying conclusions (2–5). Consistently reported is higher expression of the cancer-testis antigens in metastatic deposits compared with primary lesions. Expression patterns, however, were previously determined by differing methods that included both PCR and immunohistochemical staining. The significance of the current report is the large numbers of samples analyzed for protein expression by immunohistochemical staining and the ability to contrast expression patterns with disease progression in the same individual. These results may now be applied within the context of our current understanding to improve critical insights involving melanoma progression.
Most primary melanoma begins with a radial growth phase represented by the horizontal extension of abnormal cells along the epidermis with minimal invasion of the papillary dermis (Fig. 1). During the radial growth phase, a dermal lymphocytic infiltrate resulting in partial tumor destruction is commonly seen on histologic examination (6) and is one criterion used to distinguish melanoma from a benign nevus. Following this initial radial growth phase, the vertical growth phase proceeds with penetration of malignant cells to deeper levels of skin accompanied by further potential to metastasize (Fig. 1). In contrast to the radial growth phase, a brisk lymphocytic infiltrate during the vertical growth phase occurs relatively infrequently (10-20% of cases), but when present is tightly correlated with prolonged survival and a reduced incidence of developing metastatic disease (7, 8). Furthermore, the presence of CD4, CD8, CD68 macrophage, and HLA-DR cells independently offers a favorable outcome. Even when melanoma metastasizes to local regional lymph nodes, a lymphocytic infiltrate may occasionally be witnessed that is also highly associated with improved survival (9). In rare cases of disseminated melanoma undergoing spontaneous regression, pathologic examination strongly implicates immunomediated tumor destruction with disease sites diffusely infiltrated by lymphocytes, plasma cells, and macrophages (10).
Identified through autologous T cell– and antibody-based approaches, human melanoma antigens can be grouped into several classes (Table 1). The melanocyte lineage proteins, or normal differentiation antigens, function to produce melanin pigment present in both melanoma and normal melanocytes (11–14). Those antigens pose important questions regarding immune tolerance and cancer, specifically whether the epidermis where normal melanocytes are found provides a site of immune privilege. The cancer-testis antigens are highly expressed in normal tissues during development, but in adults expression is limited to the testis and placenta (15–19). Interestingly, they are also found aberrantly expressed in a variety of cancers. Because they are principally expressed in the testis, the cancer-testis antigens may also exist in a site of immune privilege further supported by the blood-testis barrier. Other antigen classes comprise subtle mutations of normal proteins (20, 21) or intronic sequences expressed as a result of cellular transformation (22–24). Finally, antibody-based strategies identified a variety of antigens, including the melanoma gangliosides (25–31). Patient sera used to screen melanoma cDNA expression libraries initially identified a novel 37 kDa protein D-1, MAGE-1, tyrosinase, and SSX-2 (32–35). This serologic-based cloning strategy has identified hundreds of antigens, representing a wide array of targets.
Although melanoma cells express multiple antigenic targets that result in innate immune responses, this is insufficient to prevent disease progression in patients that develop clinically significant disease. With the cloning of MAGE-1, the first gene product identified to be the target of T cells, antigen-specific immunization has been investigated (36). The most common antigens tested are MAGE 1, MAGE 3, MART-1, tyrosinase, gp100, and NY-ESO-1. Vaccination with MAGE-1 and MAGE-3 HLA-A1-restricted peptides (37) resulted in ∼30% responses, but MAGE-3-specific T cells were not detected in patients. NY-ESO-1 peptides with binding affinity for HLA-A2 have also been tested (38), revealing T-cell responses associated with disease stabilization or regression in a subset of patients. Newly defined class I and class II peptide epitopes for NY-ESO-1 have led to additional clinical trials using a variety of vaccination strategies.
Several epitopes of the melanocyte differentiation antigens found to be CTL targets were initially tested in vaccination trials and resulted in objective tumor responses in some patients (39). The most frequent differentiation antigens targeted in HLA-A2-specific vaccination strategies have been MART-1, tyrosinase, and gp100 (40). These efforts included the use of both native peptide motifs as well as peptide sequences modified for high-affinity binding to HLA-A2. These melanoma antigens have been loaded directly onto dendritic cells ex vivo from hematogenous progenitors followed by inoculation into skin, blood, or lymph node of melanoma patients (39–41). A final antigen-specific treatment approach involves the generation and expansion of tumor-reactive T cells ex vivo followed by their infusion into unmanipulated of lymphopenic hosts. This showed disease stabilization or responses in a number of patients, but limited ability to assess antitumor T-cell responses. Recently, HLA-A2-specific peptides have been combined with an antibody antagonist to cytotoxic T lymphocyte antigen 4 (CTLA-4) to augment specific T-cell effector activity and impede regulatory T-cell function.
The transformation from benign melanocytes to metastatic melanoma is the result of a compilation of genetic alterations crucial to cell division, differentiation, antiapoptosis, invasion, angiogenesis, and sustenance in a microenvironment distant from the point of origin of the cell. The complexity of genetic alterations involved in melanoma development is just beginning to be appreciated. Analyses of family kindreds via loss of heterozygosity (42) discovered tumor suppressor genes involving the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus, encoding the proteins p16 INK4A and p14 ARF by alternative reading frames. This is believed to be one of the earliest events in melanoma development. Other familial genetic alterations include xeroderma pigmentosa, retinoblastoma, RECQL2, BRCA2, and TP53 (43).
In addition to mutations in relevant tumor suppressor genes, activation of members of cell signaling pathways have also proved important (44). Activating NRAS mutations believed to drive the progression from radial growth phase to vertical growth phase lead to activation of both the RAF and phosphatidylinositol 3-kinase (PI3K) pathways (Fig. 2). Subsequent identification of genetic alterations in members of both of these pathways further confirms their importance. BRAF mutations are found in a significant proportion of both primary melanomas and benign nevi, ranging in incidence from 30% to 70%, strongly suggesting that BRAF plays a key role in the process of melanocyte transformation. In the PI3K pathway, the frequent loss of function of phosphatase and tensin homologue (PTEN) is believed to be involved in late phases of melanoma progression (Fig. 2; ref. 43), whereas Akt is constitutively activated in >50% of melanomas, with expression directly related to progression and inversely to survival. These signaling pathways converge on the microphthalamia-associated transcription factor (MITF) promotor that leads to transcription of a number of genes under its control, including the melanosomal differentiation antigens and INK4A (45). MITF recently has been found to be amplified in metastatic melanoma and seems to work in conjunction with BRAF and p16 in melanoma progression (46).
