The need for effective adjuvant therapies and evidence that the immune system plays a prominent role in the development and progression of melanoma (Curiel-Lewandrowski & Demierre, 2000) have led to the investigation of several methods to stimulate the immune system to treat melanoma or prevent its recurrence. While to date, no large, randomized trial has demonstrated an overall survival advantage to the use of a melanoma vaccine, investigations have continued, in part because vaccines have the potential of not only significant clinical efficacy, but minimal morbidity. Furthermore, the 2004 guidelines for melanoma from the National Comprehensive Cancer Network (NCCN) recommend that patients with melanomas ≥ 4 mm be offered melanoma vaccine clinical trials.
The immune system is known to function via two interdependent types of immune responses known as innate immunity and adaptive immunity. In innate immunity, an immediate immune response is triggered that signals immune cells to attack and contain patho gens/tumor cell antigens until a long-term and more specific response can be generated. Adaptive immunity is a highly specific, long-lasting response to a particular pathogen or antigen. Adaptive immunity will fight the existing pathogen/antigen and enables the immune system to recognize and respond more rapidly to a subsequent encounter with the same antigen (immune memory). The dendritic cells of the immune system are an important link between innate and adaptive immunity. Dendritic cells can capture antigens from surrounding tissue, and, if activated, present antigens to other cells of the adaptive immune system, including T cells and B cells. In the adaptive immune response, B cells produce antigen-specific proteins, called antibodies, to mount an antigen-specific response. T cells can become either killer T cells, called cytotoxic T cells (CTLs), which can directly kill foreign or aberrant cells, or mature into T helper cells, which secrete proteins that stimulate other cells to participate in the immune response.
Current vaccine strategies aim to increase specific cellular and humoral responses (T and B-cell responses), enhance dendritic cells' capacity to present antigens to T cells, thus promoting immune response, and promote resistance to local immunosuppressive factors secreted in melanoma. Indeed melanoma is a "smart" tumor, often able to "avoid," or suppress the immune system (Demierre, Swetter, & Sondak, in press).
Cumulative data of studies on BCG, levamisole, transfer factor, C. Parvum, have suggested that nonspecific stimulation of the immune system does not significantly affect the clinical course of melanoma (Demierre et al., 2005). Thus, the focus has been on active specific immunotherapy (Curiel-Lewand rowski & Demierre, 2000). Active specific immunotherapeutic agents, generally vaccines, are designed to elicit a host immune response to known or unknown tumor-associated antigens (examples of well known melanoma-associated antigens include tyrosinase, Mart-1/Melan-A, gp75, gp100, MAGE).
Using the principle that melanoma-associated antigens are shared among a large number of patients, the development of an allogeneic vaccine, generally prepared from cultured cell lines, should stimulate an anti-tumor immune response. There has been documented evidence that this type of vaccination could induce immune responses to several melanoma antigens (Morton et al., 1992; Takahashi, Johnson, Nishi naka, Morton, & Irie, 1999). All ogeneic vaccines are readily available, can be standardized, preserved, and distributed in a manner similar to any other therapeutic agent, and thus have been more readily available for evaluation in large prospective, randomized trials. Two allogeneic vaccines were evaluated in large-scale, randomized trials as an adjuvant therapy for melanoma. Both incorporated an immunologic adjuvant to incite sufficient local immune response to promote sensitization to tumor-associated antigens.
Canvaxin® . Canvaxin is an allogeneic vaccine comprising three viable irradiated mela noma cell lines (whole cells are used). The cell lines were chosen for their high content of immunogenic mela noma and tumor-associated antigens, and contain at least 11 known tumor-associated antigens such as MAGE-1, MAGE-3, tyrosinase, gp100, gp75, and Mart-1/Melan-A (Morton & Barth, 1996). This vaccine enhances the immune response to melanoma, and this response correlates with outcome (Di Fronzo et al., 2002; Hsueh, Gupta, Qi, & Morton, 1998; Hsueh, Essner, Foshag, Ye, & Morton, 2002; Morton, 2001; Takahashi et al., 1999). Multi center randomized controlled trials compared Canvaxin plus BCG to placebo plus BCG in both stage III and stage IV melanoma patients status post-surgical resection. The phase III trial for stage III melanoma patients reached accrual and closed in September 2004. The phase III trial for stage IV patients was closed April 2005 prior to complete accrual.
Melacine®. As opposed to using whole cells, Melacine consists of a lysate of two homogenized melanoma cell lines that are combined with the adjuvant DETOX ("detoxified Freund's adjuvant," comprising mono phosphoryl lipid A and a purified mycobacterial cell-wall skeleton) (Mitchell, 1998; Mitchell, Harel, & Groshen, 1992). Based on studies of Melacine in stage IV mela noma patients, Melacine was approved in Canada in May 2000 for treating advanced melanoma.
The Southwest Oncology Group completed a large (689 patients) randomized trial evaluating Melacine in the adjuvant setting (SWOG 9035). Patients with intermediate thickness (1.5-4.0 mm), node-negative melanoma were randomized to either Melacine or observation after surgery. Overall, there was no significant advantage of the vaccine compared to observation (Sondak et al., 2002). However, prospective identification of patients HLA typing showed that for the vaccine arm, A2+ and/or C3+ vaccinated patients (178 patients out of 294 total vaccine arm patients [61%]) had an 82% 4-year disease-free survival (p=0.001 compared to observation arm or A2/C3 vaccine arm patients) (Sosman et al., 2002a). Moreover, the 5-year estimate indicated an improved survival for the subset of Melacine patients with HLA-A2 or C3 (p=0.003) (Sosman et al., 2002b). Melacine has also been evaluated in combination with interferon. This study closed in 2003 and results are eagerly awaited.
Another approach has consisted of using the patient's own tumor to create a vaccine, postulating that whatever relevant antigens do exist would be represented on that individual's tumor cells. Approaches have included both autologous cellular vaccines and dendritic cell vaccines. Autologous tumor vaccines require the surgical resection of a sample of the patient's melanoma, which can be irradiated or mechanically lysed, and given back to the patient to promote immune recognition. There have been several attempts and the approach by Berd, Sato, Maguire, Kairys, and Mastrangelo (2004) appeared most promising. Due to logistical difficulties, a phase III trial was halted, illustrating the challenges of conducting large trials with autologous vaccines (Demierre et al., 2005). There is continued interest in this approach and another phase III trial may be planned.
While both allogeneic and autologous melanoma vaccines provide numerous potential antigens, the most important are not known and monitoring the immune response has been challenging. The use of defined antigens has permitted the measurement of immune responses specific to those antigens.
Ganglioside Vaccines. Ganglio sides (for example, GM2), present on melanoma cells and some non-neoplastic cells (Hersey et al., 1991), have a moiety expressed on the cell surface that is available for antibody recognition. Gangliosides are effective targets for active immunotherapy (Livingston et al., 1989; Tai et al., 1985; Takahashi, Chang, Morton, & Irie, 1995). To increase the humoral response to GM2 vaccination, investigators conjugated the GM2 to the xenogeneic protein keyhole limpet hemocyanin (KLH) and included the saponin-derived adjuvant QS-21 (Chapman et al., 2000), resulting in a vaccine called GMK. A recent intergroup trial phase III (ECOG 1694/S9512/C509801) studied this vaccine in patients with resected stage IIB-III melanoma, comparing it to high-dose interferon. The trial demonstrated a significant difference in favor of interferon-alfa versus the vaccine in terms of both relapse-free and overall survival (Kirkwood et al., 2001). There appeared to be no evidence of any adverse effect of GMK on RFS or OS, with a trend toward improved outcome for patients with positive IgM and IgG titers suggesting that GMK may have provided some clinical benefit to responding patients. This vaccine is being further explored in a phase III EORTC trial in stage II melanoma patients (those with intermediate thickness mela noma, node negative) comparing the same vaccine, for 2 years, to observation after surgery. This vaccine can be combined with high-dose interferon without any adverse impact on the level of induction of anti-ganglioside antibodies by the vaccine (Kirkwood et al., 2001).
Peptide Vaccines. While ganglioside vaccines aim to stimulate a humoral (B-cell) response, peptide vaccines are intended to stimulate T-cell based responses to tumor-specific antigens ex pressed on the surface of cells through a major histocompatibility complex (MHC) class-I restricted process (Eisen bach, Bar-Haim, & El-Shami, 2000). The majority of peptide antigens are, on their own, only weakly immunogenic, and so they are typically delivered to the patient along with an immune adjuvant. These adjuvants, such as BCG or "incomplete Freund's adjuvant," are meant to induce inflammation and initiate the immune process (Weber, 2000). Cytokines, such as IL-12 (Lee et al., 2001) or GM-CSF (Weber et al., 2003) have also been given simultaneously with the vaccines to further stimulate the immune response. A drawback of using single antigens has been the increased possibility that melanoma cells could escape recognition through antigenic modulation (selective down-regulation of antigen expression to escape immune destruction). Since not all individuals have the same tumor antigen expression profile, having multiple antigens and adding cytokines could further stimulate the immune system. One intergroup phase III trial (E4697) is currently evaluating a multiple antigen peptide vaccine (Mart-1®, tyrosinase, Gp100) alone or administered with GM-CSF compared to placebo or GM-CSF alone in high-risk stage III (for example, gross extranodal extension, satellites, in-transit lesions) and resected stage IV patients. Patients' HLA-A2 status must be known prior to randomization, since T cell recognition of an antigen depends on the presentation of that antigen on a specific MHC molecule, the human lymphocyte antigen (HLA) type class I. Only in the context of HLA-A2 can a given peptide induce an immune response.
A recent approach consists of using heat shock protein-peptide complexes (HSPPC) as vaccines. Heat shock proteins are stress proteins that act as "chaperones" for antigenic proteins, alerting the immune system to eliminate disease. These peptide complexes extracted from melanoma cells can stimulate antigen-specific CD8+ T cells from melanoma patients' peripheral blood mononuclear cells (Castelli et al., 2001). Since preliminary data have indicated feasibility, minimal toxicity, clinical responses in 18% of patients along with tumor-specific T-cell responses in 50% to 60% of subjects (Belli et al., 2002), a phase III study was initiated. This study compares a heat shock peptide vaccine (HSPPC-96) derived from autologous tumor to standard therapies (IL-2 and/or dacarb azine/temozolomide based therapy and/or complete tumor resection) in stage IV melanoma patients (see Table 2 ). This study closed in September 2004 as it had reached accrual.
An exciting strategy has been to develop agonists of TLR9, a toll-like receptor found in a subset of dendritic and B cells. TLR9 recognizes a specific pattern of nucleotides in the DNA, known as CpG DNA, that is common in bacteria and viruses, but uncommon in human DNA. Using synthetic CpG DNA sequences that mimic (copies) those found in pathogens, synthetic CpG se quences are capable of binding to and activating TLR9, thus becoming DNA agonists (Klinman, 2004). When administered, TLR9 agonists initiate a cascade of cellular signals that result in a highly specific and targeted immune response. TLR9 agonists initiate both an innate and an adaptive immune response, generating cytotoxic T cells (CTLs) and disease-specific (pathogen or tumor) antibodies. In addition, via activation of dendritic cells, TLR9 agonists fight against the development of immune tolerance to pathogens and cancers. CPG 7909 is a single-strand oligodeoxynucleotide (a TLR9 agonist), that has been optimized for potent modulation of innate immunity and subsequent adaptive immune functions. CPG 7909 injection is being studied as a targeted cancer immunotherapeutic and as an adjuvant to melanoma chemotherapy. A phase II study has been completed in patients with advanced melanoma and a phase III will begin in patients with unresectable stage IIIb/c or stage IV melanoma (see Table 2 ).
The most recent approach has been the development of a monoclonal antibody that targets the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4). This CTLA4 limits the therapeutic potency of cancer vaccines by decreasing T-cell function (Cham bers, Kuhns, Egen, & Allison, 2001), which is critical in melanoma immunity. Blocking CTLA4 with a monoclonal antibody against CTLA4 showed increased tumor immunity in previously vaccinated stage IV melanoma (Hodi et al., 2003). To date, side effects related to the use of the monoclonal antibody against CTLA4 include skin eruptions and itching in one-third of patients. As a result, a randomized phase III double blind trial will evaluate the effectiveness of anti-CTLA4 monoclonal antibody (MDX-010) alone or in combination with another vaccine MDX-1379 (a peptide vaccine made up of two peptides, pieces from the melanoma protein gp100). Because these peptides bind to HLA-A2 which is recognized by T-cells, only patients with stage III or IV previously treated melanoma (with unresectable disease) who are HLA-A*0201 positive will be eligible (see Table 2 ).
The future for melanoma vaccines is promising. Our growing understanding of important factors in melanoma immune response is permitting us to develop more specific approaches. While melanoma vaccines are traditionally first evaluated in the metastatic setting, their relative lower toxicity makes them ideal candidates to eventually evaluate in earlier stages of melanoma (for example the EORTC phase III of GMK-KLH/QS-21 vaccine in node negative intermediate thickness melanoma AJCC [stage II]). Nurses will play an increasingly important role in clinical trials of patients receiving melanoma vaccines (see Table 3 ).
Dermatology Nursing. 2005;17(4):287-295. © 2005 Jannetti Publications, Inc.
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