PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunother. Author manuscript; available in PMC Dec 7, 2012.
Published in final edited form as:
PMCID: PMC3517185
NIHMSID: NIHMS421447
Immunotherapy for Melanoma: Current Status and Perspectives
Doru T. Alexandrescu,* Thomas E. Ichim, Neil H. Riordan, Francesco M. Marincola, Anna Di Nardo,* Filamer D. Kabigting,* and Constantin A. Dasanu§
*Division of Dermatology, University of California at San Diego
Medistem Inc, San Diego, CA
Infectious Disease and Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center and Center for Human Immunology (CHI), National Institutes of Health, Bethesda, MD
§Department of Hematology-Oncology, Saint Francis Hospital and Medical Center, Hartford, CT
Reprints: Doru T. Alexandrescu, University of California at San Diego (UCSD), 9500 Gillman Drive, MC0971, La Jolla, CA 92093 (mddoru/at/hotmail.com; dtalexandrescu/at/ucsd.edu).
Immunotherapy is an important modality in the therapy of patients with malignant melanoma. As our knowledge about this disease continues to expand, so does the immunotherapeutic armamentarium. Nevertheless, successful preclinical models do not always translate into clinically meaningful results. The authors give a comprehensive analysis of most recent advances in the immune anti-melanoma therapy, including interleukins, interferons, other cytokines, adoptive immunotherapy, biochemotherapy, as well as the use of different vaccines. We also present the fundamental concepts behind various immune enhancement strategies, passive immunotherapy, as well as the use of immune adjuvants. This review brings into discussion the results of newer and older clinical trials, as well as potential limitations and drawbacks seen with the utilization of various immune therapies in malignant melanoma. Development of novel therapeutic approaches, along with optimization of existing therapies, continues to hold a great promise in the field of melanoma therapy research. Use of anti-CTLA4 and anti-PD1 antibodies, realization of the importance of co-stimulatory signals, which translated into the use of agonist CD40 monoclonal antibodies, as well as activation of innate immunity through enhanced expression of co-stimulatory molecules on the surface of dendritic cells by TLR agonists are only a few items on the list of recent advances in the treatment of melanoma. The need to engineer better immune interactions and to boost positive feedback loops appear crucial for the future of melanoma therapy, which ultimately resides in our understanding of the complexity of immune responses in this disease.
Keywords: malignant melanoma, immunotherapy, vaccines, cytokines, immunomodulation, dendritic cells
Most of the discoveries in human cancer immunology originate from studies of melanoma, a cancer shown to be among the most immunogenic of all tumors. In the past thirty years, much has been learned about the immunobiology of melanoma. As this knowledge continues to expand, so does the potential therapeutic role of immunotherapy in augmenting the antitumor immune responses against melanoma. A schematic representation of the antitumor immune responses generated in melanoma is presented in Figure 1.
FIGURE 1
FIGURE 1
Role of Dendritic Cells (DCs) and Mechanisms of Tumor-Mediated Immunosuppression (schematic). The activation of immature dendritic cells (iDCs) is followed by migration to lymphatic nodes, sites of transformation to mature dendritic cells. The uptake (more ...)
Melanoma was the first tumor model to reveal CD4 and CD8 cellular specificity to the tumor differentiation antigens gp100 and tyrosinase.1,2 The subsequent efforts to identify specific genes encoding tumor antigens and their corresponding epitopes yielded major progress in further understanding of the antitumoral immune responses. It became clear that genetic changes in cancer cells can lead to the build-up of new specific antigens, which are MHC-restricted and recognized by the CD4+ lymphocytes. MAGE-1 represented the first tumor antigen specifically recognized by the cytotoxic CD8+ lymphocytes.3 Initial studies on MAGE-1 supported the idea that the human immune system could respond to the tumor antigens, thus sparking a great deal of interest in identifying potential therapeutic targets and biomarkers predicting response to immunotherapy. These advances have contributed to the development of vaccines, biological agents such as inter-leukins and interferons, cellular therapies, and antibodies currently in use to treat melanoma. These therapies continue to be tested, either alone or in combination, in order to improve the largely disappointing tumor response rates (RRs) ranging only 5% to 10%.
The fact that successful preclinical studies do not always translate into clinically meaningful objective RRs in patients with melanoma has been a common theme. Although such therapies as vaccines are able to significantly induce tumor antigen-specific T-cells, it has only translated into marginal clinical responses, and often at the cost of severe or life-threatening autoimmune toxicities. The fact that specific cytotoxic T-cells are not capable of efficient tumor lysis led to the concept of tumor tolerance.4 It is now clear that various immunosuppressive elements in the tumor microenvironment limit the anti-tumor activity of induced anti-suppressor T-cells and other effector cells. Recent advances in the treatment of melanoma focus on targeting mechanisms of tumor immunosuppression, including cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed death-1 receptor (PD1). This review summarizes fundamental concepts and recent advances in our understanding and treatment of melanoma. Ongoing development of novel therapeutic approaches concurrent with optimization of existing therapies and identification of effective combination treatment regimens continue to hold much promise in the field of melanoma research.
A number of cytokines, including Interleukin-2 (IL-2), Interferon-a (IFN-α), alone or in combinations with IL-2, IL-12 and others have been tried with various degrees of success in the therapy of melanoma (Table 1).
TABLE 1
TABLE 1
Clinical Use of Cytokines in Melanoma
Interleukin-2 (IL-2)
The biological effects of IL-2 are complex. Relevant for cancer therapy is the enhancement of CTL and NK-cell lysis. In response to IL-2 stimulation, a mixture of NK and CD8+ cells acquire cytolytic properties, which lead to tumor cell killing in vitro, even in the absence of HLA-class I restriction. Complete responses (CR) were produced in 6% and partial responses (PR) in 16% to 20% of patients with metastatic disease that failed standard therapy that were treated with a high-dose (HD) regimen of 600,000 to 720,000 IU/kg i.v., repeated every 8 hours for 8 to 14 doses per cycle with two cycles spread by a week considered a treatment course.5,17 However, many patients are not able to tolerate more than 8 doses per cycle of treatment, with the second cycle typically consisting of fewer doses than the first. Although initial murine studies supported improved results with the addition of LAK cells to HD IL-2 therapy, subsequent clinical data in patients with metastatic disease showed an equivalent efficiency with the HD IL-2 alone.18,19 With a median response duration of 8.9 months and 44% of responders being alive at 6 years, HD IL-2 offered the possibility of cure for a small fraction of patients.20 An analysis of 374 metastatic melanoma patients receiving high-dose IL-2 suggested that patients having only cutaneous metastasis had a higher response as compared to however patients with diseases at other sites. Additionally, in the same analysis, it was found that development of vitiligo and thyroid dysfunction was associated with response.21 An elegant study by Sabatino et al22 used a multiplex antibody-targeted protein array platform to identify pretreatment elevation in VEGF and fibronectin as being indicators of poor response to IL-2 therapy.
The toxicity of IL-2 therapy prompted studies assessing feasibility of lowering the doses of IL-2. While this results in reduced adverse effects, it is associated with an inferior RR (frequently under 5%), duration and quality of responses.23 A frequently employed continuous infusion of IL-2 of 18 MU/m2/d for 5 days is characterized by a similar toxicity and response rate as HD IL-2, but a shorter duration of response.6 The current consensus is that although the high dose regimens are currently approved by the FDA, the issue of optimum dose appears to still be unresolved, primarily due to lack of randomized data controlled for prognostic factors across institutions.
Interferon-α (IFN-α), IL-2+IFN-α Combinations
IFN-α exerts anticancer activities through numerous mechanisms, including direct antiproliferative/apoptotic effects,24 increasing immunogenicity of tumors,25 suppression of angiogenesis,26,27 as well as modulating the innate and adaptive immune response.28 Response rates of 14% with rhIFN-α2a and up to 23% with IFN-α2b were obtained in Phase II trials in metastatic melanoma using various schedules and dosages.29 Accumulating clinical experience showed that responses to IFN-α can be delayed for a couple of months, and continuous treatment is more effective. One potential interpretation of these findings is that IFN-α stimulates clonal expansion of cytotoxic T cells that naturally are subjected to tumor-mediated immune suppression.30
The rationale for combining IL-2 with IFN-α resides in the up-regulation by the latter of HLA and tumor-associated antigen (TAA) expression on tumor cells, which may enhance the T-cell lysis induced by IL-2.31 However, clinical trials have not confirmed the anticipated benefits of combination therapy over HD IL-2 alone.7,32 Similarly, trials combining IL-2 with granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), IFN or IL-4 did not result in any meaningful clinical improvement as reviewed in ref.23
Other Interleukins and Cytokines
In metastatic melanoma patients, use of IL-12 was associated with limited activity in phase I trials, with one transient CR among 12 patients treated at the maximum dose level.33 Only minor responses were seen in 30% of cases in another study.34 The antitumor effects of rhIL-12 appear to be correlated with serum IFN-γ levels, and clinical benefits appear to correlate with maintaining its secretion after four weeks of treatment, as demonstrated in a trial assessing the combination of IL-12 with low-dose IL-2.35 Furthermore, recombinant cytokines such as IFN-γ,IL-1,IL-4,IL-6,TNF-α and IL-18 have been experimented with modest results in the treatment of metastatic melanoma.3638
The historical development of cellular immunotherapy targeted activation of the innate effector immune cells: macrophages, NK, and LAK. A great enthusiasm for these approaches emerged as successful results were obtained in animal models, showing an enhancement of IL-2 efficacy when combined with LAK cells derived from peripheral lymphocytes. However, clinical trials using IL-2 and LAK cells did not confirm a substantial advantage compared to high-dose IL-2 alone.18,19 In contrast, it appeared TILs had more promising activity. For example, autologous TILs plus HD bolus IL-2, with or without administration of cyclophosphamide, produced a 33% RR. Furthermore, the use of TILs generated from subcutaneous tumor deposits showed a higher RR (49%), when compared with the use of those originating from lymph nodes (17%) (P=0.006).39 Mechanistically it appears that IL-2 co-administration provides a fertile ground for TIL-expansion and/or survival in vivo.40 A note to consider are recent studies demonstrating expansion of CD4+ CD25+ T regulatory cells subsequent to administration of systemic IL-2.41 It may be therefore be worth considering Treg depletion as part of TIL+ IL-2 approaches.
A significant advancement was recognition that TIL functional parameters, such as their ability to recognize and lyse tumor targets, may correlate clinical with responses.42,43 In addition, in vivo persistence and telomere length of transferred TILs has also been correlated by some authors with the antitumor response.44,45 With the notion of TIL having activity that correlates with outcome, the next question would be how to improve on that activity? One innovative approach involved utilizing the concept of homeostatic expansion. Essentially, lymphocytes are regulated by a balance between stromal cell generated IL-7 and IL-15 and ability of the lymphocytes to uptake these cytokines. When levels of lymphocytes drop systemically, there is an excess IL-7 and IL-15 which results in activation of low-affinity receptors on lymphocytes. This causes loss of costimulatory requirement and allows previously antigen-nonreactive lymphocytes to because antigen reactive.46 Supporting this notion are numerous animal studies and human observations reviewed by Marleau et al.47 Translating this concept into clinical practice, lymphodepletion was performed by cyclophosphamide and fludarabine followed by administration of highly-selected, antigen-specific TIL. Six PRs and 4 mixed responses were achieved in 13 refractory melanoma patients.48 These results were confirmed in a larger study, where a similar regimen produced a 50% RR (8% CRs).49 The rationale for prior administration of lymphodepleting agents is to eliminate T regulatory (Treg) cells,50 as well as remove cytokine sinks,46 thus allowing for generation of costimulatory-independent tumor-targeting responses.
Intensification of the lymphodepleting regimen by using cyclophosphamide plus fludarabine along with 2 to 12 Gy of TBI before autologous TIL administration resulted in an increase in the serum levels of lymphocyte homeostatic cytokines IL-7 and IL-15. This immune activation was accompanied by clinical response rates of 52% and 72%, respectively, for the two radiation dose intensities.51 The anti-tumor responses were correlated with the number of CD8+CD27+ cells infused, as well as with their mean telomere length and their persistence in circulation at one month after administration.52 The 28% rate of CRs achieved through adoptive immunotherapy after maximum lymphodepletion sets a new standard in immunotherapy, considerably superior to the results of IL-2 alone, vaccination, or a combination of the two.
Other recent advances in adoptive cell transfer involve the use of autologous T cells engineered to express T-cell receptors with specificities for various tumor associated antigens or to secrete cytokines. Autologous T cells transduced ex vivo with anti-MART-1-TCR genes persisted for a prolonged time in vivo and led to sustained objective regression in two patients with metastatic melanoma. However, the overall RR (13%) was still inferior to those produced by the infusion of autologous TILs (50%).53 Adoptive cell transfer of TILs engineered to produce IL-2 demonstrated high IL-2 production and prolonged survival upon IL-2 withdrawal in vitro. In vivo, however, IL-2 transduced TILs elicited the same RR and persistence as unmanipulated TILs. Telomere shortening as a result of the in vitro expansion was cited as a possible cause of mediocre results.54
Achievement of durable responses with biological agents, and the possibility to complement the higher response rate of chemotherapy by prolonged duration of remissions led to development of biochemotherapy. Among potential advantages of this combination is an enhancement of immune recognition and cellular effector activity triggered by IL-2 in the setting of tumor cellular disruption and antigen release induced by chemotherapy agents. Furthermore, administration of chemotherapy first may enhance immunity and decrease the tumoral mass, therefore augmenting the effectiveness of response. Although a clear improvement in response rate (40% to 60%) resulted from the use of biochemotherapy, several randomized phase III studies produced mixed results on the duration of survival, and two recent meta-analyses did not reveal any improvement in OS.55,56 A meta-analysis of clinical trials employing various time frames between the administration of chemotherapy and biologics showed that, as the time-frame between chemo and bio components increases, the OS, survival of CRs and PRs appear to increase, but the effect is only present for the chemo-first combination. It appears possible that the interaction between components of biochemotherapy results in a double effect: an increase in the immediate response, correlated with production of nitric oxide which acts synergistically with chemotherapy in producing tumor cell killing, and an increase in survival, correlated with macrophage activation, as measured by neopterin levels.57
The goal of vaccination is to induce immune recognition against antigens expressed by tumors.58 A great variety of approaches have been utilized over the past 35 years. Despite occasional tumor responses and even suggestions of improved survival, no consistent results were obtained with the early allogeneic or autologous whole cell vaccines, cell lysates, and shed antigen preparations.59
Discoveries in characterization of tumor antigens and biology of immune reactions have shifted the focus towards construction of vaccines based on specific antigens, such as peptides, or enhancing their presentation through the use of dendritic cells. There are a limited number of completed phase III trials, and although some reported marginal benefit, no benefit, or even harm, this research has nevertheless improved our understanding of tumor immunobiology, and shed important information on the future direction of investigation.60 One concrete benefit of these studies was the appreciation that development of autoimmune manifestations, like vitiligo, may serve as a clinical marker for responsiveness to therapy and improved survival.21,61 Interestingly, as discussed in the situation of high dose IL-2 therapy, similar autoimmune-like manifestations appear in some situations to be a common feature of successful immunotherapy.62
Limited immune responses can be elicited using allogeneic and autologous tumor vaccines, and the clinical benefit of these preparations has remained modest. In early clinical trials, vaccines prepared from whole tumor cells were associated with limited activity.63 Nevertheless, continued exploration of such multivalent approaches has proceeded. In early phase II trials, vaccination with Melacine was associated with up to 10% clinical responses, while 74% of patients had either an objective remission or disease stabilization when used in conjunction with IFN-α2b.64 However, subsequent randomized controlled clinical trials comparing Melacine and IFN-α2b versus IFN-α2b alone found no significant difference in relapse-free survival (RFS) or overall survival (OS) between the two treatment arms. Median OS time exceeded 84 months in patients receiving melacine plus IFN-α2b, and was 83 months in IFN-α2b alone (P=0.56, 95% CI, 60 months to not reached).65 One possible explanation for the poor results of such polyvalent approaches may be related to various immune suppressants and angiogenic agents found in the vaccine mixture. An interesting approach to overcome this may be the use of xenogeneic vaccines. For example, it has been reported that self-tolerance to angiogeneic agents such as VEGF, FGF, and EGF may be broken by administration of xenogeneic homologues.66 Administration of xenogeneic tumor antigens has been performed clinically with promising results;67 however, to our knowledge this approach has not been evaluated in a polyvalent setting.
Peptide Vaccines
Poor efficacy of complex tumor cell-derived polyvalent vaccines has prompted interest in more defined antigens. Although highly selective approaches have the advantage that: (a) immunogenic epitopes can be selective for; and (b) epitopes can be modulated (eg, altered peptide ligands) to artificially increase immunogenicity. The first defined human cancer-testes antigen, MAGE-1, was characterized in 1991.3 Since then a long and growing list of potential targets for cancer immunotherapy has emerged. In this context, metastatic melanoma served as the first model to test immune responses to peptide vaccines.
Spontaneous T-cell responses to the native gp100 antigen have been noted in patients with metastatic melanoma who experienced tumor regression following adoptive therapy with TIL and IL-2. Rosenberg et al68 utilized a cancer vaccine consisting of immunodominant peptides derived from the gp100 melanoma-associated antigen, administered with a synthetic peptide designed to increase binding to HLA-A2 molecules. Of patients receiving the vaccine plus IL-2, 42% had an objective response. However, three different schedules of high-dose IL-2, administered with the 209-2M peptide did not produce better activity than expected with IL-2 alone (OR 14.5%, CR 8%).69 Vaccination with the synthetic modified gp100: 209 to 217 (210 M) peptide, created by altering the anchoring amino acid (methionine in place of threonine at position 2), produced an increase in specific CD8+ T cells after vaccination in 28 of 29 patients, of whom 77% remained disease-free at 23 months.70 Another approach, consisting of addition of tetanus peptide as a nonspecific helper epitope resulted in Th-cell responses in 79% of patients and an OS of 75% at 4.7 years of follow-up, which compared favorably with the expected survival.71 However, clinical responses are not always correlating with the immune activation, as exemplified by a study involving vaccination with the HLA-A*0201-restricted Mart-1/Melan-A(27 to 35) and incomplete Freund's adjuvant (IFA), where no clinical activity was observed, despite a 94% rate of CTL-specific responses.72 Conversely, no evidence of enhancement of the systemic immune response could be documented in nine patients immunized with the peptide MAGE-A12: 170 to 178 administered in IFA, although one patient had an ongoing PR.73
A paradoxical dichotomy between elicitation of immune responses and clinical activity has also been seen with the modified gp100 epitope g209-2M. While 91% of patients showed successful immunization, no clinical responses were observed. Moreover, only 16% of patients treated with IL2+g209-2M vaccination developed immune reactivity, whereas 13 of 31 (42%) demonstrated clinical responses.74 An explanation for this phenomenon can reside in trafficking of the vaccine-specific T cells to the tumor site, although it is possible that the discrepancy originated in a differential susceptibility of CTLs to in vitro sensitization.75
A recent breakthrough was seen in a multicenter, randomized, prospective trial compared HD IL-2 alone with IL-2 and gp100 209 to 217 peptide in 185 stage III/IV patients expressing HLA0201. The response rate was significantly higher in the combination treated (22.1%) as compared to the IL-2 alone treated patients (9.7%). A significant improvement was seen also for the progression free survival 2.9 months (1.7 to 4.5) compared to 1.6 months (1.5 to 1.8), respectively.76 This appears to be the first description of clinical benefit in metastatic melanoma using vaccination.
In the E1696 trial assessing multiepitope (MART-1, gp-100, and tyrosinase) vaccine alone, in combination with either GM-CSF, IFN-α2b, or in combination with both, of 115 patients who were analyzed, median survival of patients exhibiting immune response to at least one of the antigens was prolonged as compared to those lacking induction of immunity (21.3 mo vs. 13.4 mo; P=0.046). There was no difference in terms of immunity between patients receiving the vaccine alone or in combination with cytokines.77
IL-12+ Peptide Vaccines
Murine studies have shown that IL-12 promotes potent antitumor immunization when co-administered with peptides loaded onto other class I MHC+ cells, thus potentially bypassing the need to use DCs. In a Phase I clinical trial, patients with metastatic melanoma received autologous PBMCs pulsed with a MAGE-3 or Melan-A peptides and co-administered with various doses of rhIL-12. Antigen-specific CD8+ T-cell responses were demonstrated in six out of eight patients having sustained clinical responses.78 Another study found no clear dose-dependent effect of rhIL-12 on the responses to Melan-A and influenza matrix peptides administered intradermally. However, rhIL-12 was well tolerated at doses of 10 to 100 ng/kg, and 3/24 melanoma patients demonstrated clinical activity.79
GM-CSF+ Peptide Vaccines
Use of GM-CSF as a vaccine adjuvant is appealing because of its role as one of the primary growth and maturation factors for DCs. A randomized trial compared three different adjuvants in HLA*A0201+ patients with stage III or IV melanoma immunized with tyrosinase and gp100 peptides. 44% and 50% of patients immunized using QS-21 (a purified saponin) and GM-CSF, respectively, developed increased frequencies of CD8+ T cells against tyrosinase 370D peptide, compared with 0 of 9 patients immunized using IFA (P=0.045).80 Immunization against 3 HLA-A2-binding peptides of the cancer-testis antigen NY-ESO-1, a strongly immunogenic tumor antigen, followed by administration of GM-CSF resulted in 4 of 7 NY-ESO-1 antibody-negative patients developing a specific CD8+ T-cell response, and was associated with stabilization or regression of metastases in 5 of 7 cases.81 There may be some rationale for restricting dose and site of GM-CSF administration. Tumor-secreted GM-CSF has been speculated to actually play an immune suppressive role through increasing numbers of CD34 expressing myeloid suppressor cells.82,83 In addition, some tumor vaccination studies have demonstrated enhanced tumor growth and immune suppression depending on dose of GM-CSF administered.84
Recombinant (Gene-modified), Viral, and Plasmid Vaccines
Gene modified tumor vaccines are commonly designed using autologous melanoma cells that have been transfected with an immunostimulatory gene, such as the expression of an immune enhancing cytokine. Immune stimulation that translates into antitumor activity was demonstrated in a B16 melanoma model, in which irradiated tumor cells expressing murine GM-CSF (and to a lesser extend cells expressing IL-4 and 6) stimulated long-lasting, and specific anti-tumor immunity, requiring both CD4+ and CD8+ cells.85 Stimulation of immunity may have been the result of enhanced antigen presentation by local dendritic cells whose maturation was induced by tumor secreted GMCSF, although this was not formally demonstrated. In a Phase I trial investigating autologous melanoma cells engineered to secrete human GM-CSF, induction of tumor-specific responses were observed. Metastatic lesions were densely infiltrated with T lymphocytes and showed extensive destruction in 11 of 16 patients, which was associated with anti-melanoma CTL and antibody responses. One PR, one mixed response, and three minor responses were achieved, and three patients remained disease-free at 36, 36, and 20 months.86 Another approach used mixtures of autologous and allogeneic irradiated melanoma cells secreting IL-6 and sIL6-R. This evoked immune activation and promising clinical results (22% CR+PR, 32% SD).87 Several factors need to be contemplated when deciding appropriate choice of genes for tumor transfection, for example, ability to stimulate DC maturation, ability to increase immunogenicity of tumor directly, and possibility of synergizing with existing immune responses. Interestingly, in some situations, transfection of tumors with agents that are considered to be immune suppressive, such as interleukin-10, actually evoke anti-tumor immunity (reviewed in88).
An alternative approach to immunizing with gene-modified tumors is to locally transfect muscle tissue using recombinant adenovirus vector encoding tumor antigens. For example, intramuscular delivery of gp100 or MART-1 using this approach, either alone or followed by IL-2, resulted in one CR in 16 patients pertaining to the group receiving the recombinant adenovirus MART-1 alone.89 Detection of high titers of neutralizing antibodies to the adenoviral vector may explain the relatively low efficacy of this approach. In any case, the low number of patients in this study does not rule out the possibility of expanding on this approach given similar low rates of responses to other immunotherapeutic approaches.
Given that the tumor already expresses a wealth of tumor antigens, studies have been conducted to enhance tumor immunity by inducing a potent local inflammatory response in the tumor site itself. One example of this is Allovectin-7, a plasmid DNA encoding HLA-B7 and beta-2 microglobulin.90 When injected into melanoma lesions, it resulted in up to 15% rate of PRs, which occurred even at remote sites. An OS of 21.3 months was achieved with high doses of the bicistronic vaccine, and corresponded to a 12.7 months median duration of responses.91
Dendritic Cell (DCs) Vaccines
The CD8+ cytotoxic T-cell compartment, which is believed to possess major anti-tumor effector function, usually is poorly activated in cancer patients (Fig. 1). One of the postulated mechanisms is the inadequate tumor antigen presentation by DCs. This may be explained by a variety of factors including tumor secretion of factors inhibiting DC function,92,93 as well as indirect inhibition of DC function by Treg cells,94 which are increased in melanoma patients.95 Therefore, use of de novo generated dendritic cells has been part of the new strategies to enhance CTL responses (Table 2). The goal of dendritic cell vaccines is to induce a Th1 immune response and to activate CTLs, in order to facilitate tumor elimination.
TABLE 2
TABLE 2
Clinical Trials Assessing Dendritic Cell-based Vaccines for Patients With Melanoma
DCs are capable of processing and presenting peptides derived from tumor protein antigens to CD4+ and CD8+ T cells, and of regulating the activity of natural killer (NK) cells. Matured from CD14+ precursors, ex-vivo DCs are loaded with antigens in the form of whole proteins, tumor lysates, peptides, necrotic and apoptotic bodies, or messenger RNA. Specific epitopes can be pulsed in the form of synthetic HLA-binding peptides, or DNA and RNA sequences carried by viral vectors. Immature DCs are able to process antigens, but it is the mature DCs that are able to fully stimulate T cells by upregulating cytokine secretion, adhesion and costimulatory molecules (Fig. 1).113115 Animal experiments with primarily mature DCs consistently showed inducement of tumor-specific CTL responses, and occasional regression of metastases.
In a landmark study using mature DCs pulsed with Mage-3A1 peptide and a recall antigen (tetanus toxoid or tuberculin), Thurner et al116 demonstrated significant expansion of CTL in 8/11 patients with advanced stage IV disease, and objective responses in 6/11 patients. In another trial, 5 clinical responses occurred in 16 metastatic melanoma patients treated with tyrosinase 370D and gp100 210M peptides restricted to HLA class I A*0201. The peptides were modified to increase immunogenicity by altering one aminoacid from the wild type, and were pulsed into DC derived by incubation of plastic-adherent peripheral blood mononuclear cells (PBMC) with IL-4 and GMCSF. Among five patients having a CTL response to gp100 or tyrosinase, four were clinically stable or had tumor regression. Immune responses to gp100 or tyrosinase were demonstrated by gamma intereferon ELISA assay in 31% of patients.98 Vaccination with mature DCs loaded with three tumor lysates (M44, SK-MEL 28 and COLO 829), tested in a phase II trial evaluating patients with low volume or in-transit melanoma, produced two clinical responses and 4 stable disease in 33 metastatic melanoma patients. Ten patients showed evidence of enhanced anti-TAA tumor specific CD8+ T cells.109 A correlation between the enhanced immune response and survival was seen with DCs pulsed with MART1, tyrosinase, MAGE3, gp100 and influenza matrix peptides, along with keyhole limpet hemocyan (KLH). Six out of seven patients with immunity to two or fewer antigens progressed, in contrast to only 1/10 patients with immunity to more than 2 antigens.117
NK T-cell activation has also been demonstrated in phase I/II testing of vaccination of patients with metastatic melanoma with intermediate-maturity DCs engineered to express MART-1 through an adenoviral vector. CD8+ and/or CD4+ MART-1-specific T-cell responses were observed in 6/11 and 2/4, respectively, of the patients being evaluated.118 Objective responses were evident in 5 of 16 patients treated with immature DCs pulsed with tumor lysate or a cocktail of peptides in the presence of GM-CSF and IL-4. In this study, elicitation of delayed type hypersensitivity (DTH) reactions was seen in 11 patients, and recruitment of peptide-specific CTLs was demonstrated.96
In a review of 32 clinical trials on dendritic cell vaccines, Engell-Noerregaard et al119 found that clinical response (defined as CR, PR, or SD) was significantly correlated with the use of peptide antigens, use of helper antigen or adjuvant, and induction of tumor antigen-specific T cells. The majority of studies, however, show that despite repeated T-cell activation with antigen-loaded mature DCs, expansion of tumor-antigen-specific immune responses is often transient, and only rarely produces stable disease or regression of tumor metastases.97,120,121 Although significant expansions of specific CD8+ cytotoxic T lymphocytes have been demonstrated for Mage 3A1 in over 70% of patients,116 clinical results with peptide-pulsed DCs remain disappointing, indicating a failure of immunological data to translate into clinical success. Schadendorf et al122 actually reported no benefit of peptide-pulsed DC in comparison to dacarbazine (DTIC) treatment. Regulatory T cells (Tregs) may produce a potent down-regulation of anti-tumor responses that counteract the protection conferred by dendritic cell vaccines (Fig. 1). Tumors can also mediate DC suppression, and potential molecular targets are now being identified, including p44/42 MAPK, which is hyperactivated in melanoma, and its upstream activator MEK1/2. Blockade of the MEK1/2-p44/42 axis has been shown to increase IL-12 production and enhance Th1 immune responses.123
Heat-shock Proteins (HSPs)
HSPs have important immune functions: they chaperon intracellular antigenic peptides, induce maturation of DC, and activate NK, CD4+ and CD8+ T cells. Two durable CRs (559+ and 703+ d) and 3 SDs were produced in 28 metastatic melanoma patients vaccinated with autologous, tumor-derived heat shock protein gp96-peptide complexes (HSPPC-96, Oncophage). The ELISPOT assay showed an increased melanoma-specific T-cell activity in 11 of 23 patients, which correlated with clinical responses,124 but no further improvement was obtained by modulating the immune reaction with GM-CSF and IFN-α.125
More recently, however, a phase III trial enrolling 322 patients with metastatic melanoma found no difference in OS between the HSP vaccine, vitespan (an autologous tumor-derived heat shock protein gp96 peptide complex vaccine), and physician's choice of treatment, including dacarbazine, temozolomide, interleukin-2, or complete tumor resection.126
Given that mechanistically, HSP-based vaccine approaches appear to function through increasing immunogenicity of the bound peptides, it may be useful to view this therapy as a “sophisticated polyvalent lysate”. This would suggest the need for studies to calibrate the degree of immunogenicity between the various HSP-antigen fractions, as a possible next step in improving this therapeutic.
Despite a highly antigenic load resultant from structural and acquired genetic instability, melanoma and other tumors are able to avoid immune inhibitory influences. Tumor induced mechanisms of immune escape are diverse, and pose limitations to the effective use of immunotherapy. Such immune downregulatory influences probably explain the failure of preclinical models to translate in clinical efficacy when applied in vivo. Important mechanisms of immune escape include downregulation of certain components of the antigen processing machinery by tumors, including β2 microglobulin, and transport associated with antigen processing (TAP)-1 and TAP-2 peptide transporters, which are critical to MHC class I antigen presentation pathways.127129
Other postulated mechanisms for tumor escape include production of immunosuppressive factors such as the Fas ligand, TGF-β, IL-6, IL-10, PGE2, VEGF, or suppression of co-stimulatory molecules such as CD40, CD80, and CD 86, although the disturbed cellular apoptotic and proliferation cellular pathways may create myriads of immune inhibitory effects. Blocking such cellular pathways, which are disturbed as a result of genetic alterations during tumorigenesis, tumor progression, or tumoral inhibition of the effector T cells, by using small-molecule tyrosine kinase inhibitors or blocking antibodies, can result in immune augmentation and enhancement of vaccine-triggered anti-tumor immunity. In fact, a proof of principle for overcoming tumor resistance is the effectiveness of IFN in enhancing response to tumor vaccines.130 Other methods of increasing tumor immunogenicity include treatment with histone deacetylase inhibitors such as valproic acid. For example, Khan et al131 demonstrated increased expression of antigen processing machinery (TAP1, TAP2, LMP2, LMP7, Tapasin), costimulatory molecules (CD40, CD80) and MHC class I on melanoma cells after treatment with a variety of histone deacetylase inhibitors.
Another method of tumor immune evasion involves secretion of microvesicles termed “exosomes”, that have been demonstrated to contain tumor antigens on MHC I,132,133 and a variety of immune suppressive molecules such as HLA-G,134 FasL,135 as well as factors associated with inhibition of DC maturation.136 The combination of antigen with inhibitory signaling suggests the possibility that exosomes may mediate antigen-specific immune suppression. Possible mechanisms for clearing exosomes have been proposed such as hollow-fiber dialysis.137 Perhaps a more exotic means of reversing immune suppression is vaccination with xenogeneic exosomes, which would induce induce immune responses towards tumor antigens and also associated immune suppressive molecules.
Immunomodulatory Monoclonal Antibodies
As the understanding of the dynamic and complex interaction between the immune system and tumors improves, new immunotherapies targeting critical regulatory elements of the immune system can be developed in order to provide treatments with greater specificity and better safety profiles. This includes the recent development of anti-CTLA-4 monoclonal antibodies, Toll-like receptor (TLR) agonists, CD40 agonists, and anti-ganglioside monoclonal antibodies. A family of receptors on T cells serves as a natural braking mechanism for T-cell activation, functioning to reestablish homeostasis following an immune response, and to maintain peripheral self-tolerance. This includes CTLA4 and PD1.138
CTLA-4 expression on T cells outcompetes CD28 for binding to CD80 (B7–1) and CD 86 (B7–2), resulting in suppression of T-cell activation and depending on the model assessed, modulation of cytokine production.139 In addition, the high affinity binding of CTLA-4 to CD80/86 stimulates the antigen presenting cell to generate high concentrations of the immune suppressive enzyme indola-mine 2,3 deoxygenase.140 This understanding provided the rationale for development of anti-CTLA4 monoclonal antibodies that could inhibit interaction between B7 and CTLA4, thus releasing the “brakes” against T-cell activation that was hypothesized to enhance antitumor immune response. This was confirmed in animal studies showing that CTLA4 blockade enhanced antitumor T-cell function and inhibited tumor recurrence in murine prostate cancer and melanoma models.141143
Ipilimumab
Anti-CTLA4 monotherapy with ipilimumab has produced clinical response rates in 7% to 15% of patients with metastatic melanoma.144 In one study, the CTLA-4 antibody ipilimumab (formerly MDX-010), produced objective responses in 17% of patients, including 3 patients who demonstrated complete responses (ongoing at 23+, 52+, and 53+ mo).145 To enhance antitumor response, CTLA4 blockade has been investigated in various dosing regimens as well as in combination with cancer vaccines, standard therapies such as chemotherapy, and IL-2 administration (Table 3). Ipilimumab administered in conjunction with a gp-100 peptide was able to induce a 21% response rate (14% CR).146 The same antibody alone or in conjunction with dacarbazine produced durable responses in patients with melanoma, some lasting over 1 year.147 Combination therapy with ipilimumab and IL-2 showed an objective response rate of 22% in patients with metastatic melanoma. An additive, but not synergistic, effect between IL-2 and ipilimumab was observed.148
TABLE 3
TABLE 3
Clinical Trials of CTLA-4 Antibody Blockage to Stimulate Immune Responses in Melanoma
Recent studies have also noted cases of late onset objective response among patients who previously experienced stable disease or disease progression.150,154 This development prompted a suggestion to continue observation or treatment in patients with initial progressive disease or stable disease. Objective antitumor response is often associated with immune related adverse events, most commonly involving the skin and gastrointestinal tract.145,155 For example, in the study of Sanderson et al155 involving a multiple peptide vaccine in conjunction with CTLA-4 blockage, only 37.5% of patients with autoimmune effects experienced a relapse, compared to 81.8% of those without autoimmunity.
Other studies suggest that prior cytokine therapy may pose a negative prognostic factor for survival in patients later receiving treatment with anti-CTLA-4 monoclonal antibodies, although differing results have been reported between IFN and IL-2.145,156
Tremelimumab
Tremelimumab (CP-675206), a fully human immunoglobulin G-2 anti-CTLA-4 mAb, was shown in phase I and II studies to produce durable objective responses in patients with melanoma, ranging from 7% to 14% at dose levels between 0.01 to 15 mg/kg.151,152 Subsequently, a phase III randomized clinical trial was initiated to compare tremelimumab to standard chemotherapy (dacarbazine or temozolomide) in patients with advanced relapsed or refractory melanoma. Among 655 previously untreated patients, no statistically significant difference in overall survival was observed between the two treatment arms (median OS 11.8 and 10.7 for tremelimumab and chemotherapy, respectively, HR 1.04), however differences in long-term survival are still unknown.152 In the setting of advanced Stage IV melanoma, CTLA-4 blockade with tremilimumab demonstrated restoration of effector and memory CD4+ and CD8+ T cells, and induction of transient T-cell resistance to Treg-mediated suppression, which was correlated with clinical outcome.157
Increased recognition that common genetic variation in drug targets could affect clinical response to CTLA-4 blockade therapy has led to the recent incorporation of pharmacogenetic analysis to evaluate common polymorphisms in the CTLA4 gene and their influence on the response to tremelimumab. Correlation of patient genotype with clinical response has so far demonstrated inconsistent trends158,159 and further studies are necessary.
Anti-PD-1 mAbs
PD-1 is another inhibitory molecule found on the surface of T cells that is associated with tolerance induction upon binding to its ligands PD-L1 and PD-L2. PD-1 is also expressed by several tumors, including melanoma, where it inhibits antitumor responses and mediates tumor evasion.160,161 Preclinical studies demonstrate that monoclonal antibodies against PD-1 improve immune functions of tumor-specific T cells, enhance cytokine production, and increase tumor lysis.162 Phase I trials of MDX-1106/ONO-4538, a fully human anti-PD1 blocking antibody, are ongoing.
CD40 Agonist mAbs
The CD40 cell-surface costimulatory molecule is naturally expressed on dendritic cells, B lymphocytes, monocytes, even solid tumors, and has a broad range of functions. CD40 is upregulated on activated DCs. The engagement with its natural ligand CD154 (CD40L), primarily expressed on activated CD4+ T cells, triggers cytokine secretion and enhanced expression of costimulatory molecules required for efficient T-cell activation.163 It has been demonstrated in murine models that in vivo delivery of CD40-activating antibodies overcomes the immunosuppressive mechanism of tumors, increases T-cell activation, and enhances antitumor immunity, leading to regression of established tumors.164 Other preclinical studies testing CP-870,893, a recombinant human agonist monoclonal antibody against CD40, have also demonstrated enhanced in vitro anti-tumour T-cell responses, evidenced by a significant expansion of IL-2 and IFN-γ-producing cells, as assessed by ELISpot assay.165 In early phase I trials, CP-870,893 was well tolerated and was able to produce objective tumor response in 27% of melanoma patients.166
Activation of Innate Immunity (TLR Agonists)
Newer strategies to overcome tumor-induced immune suppression involve attempts to improve presentation of tumor-associated antigens through enhanced expression of co-stimulatory molecules on the surface of DCs.
Toll receptors (TLR) are signaling molecules that recognize conserved molecular patterns on common pathogens, which are able to influence the activity of DCs and induce T-cell responses. TLR-stimulating ligands are being evaluated for their potential to enhance DC activation and heighten antitumor immune responses. Attention has largely focused on TLR-9 agonists, although TLR7/8 ligands also show promise (Table 4).
TABLE 4
TABLE 4
Clinical Trials Using TLR-9 Agonist Stimulation of Immune Responses in Melanoma
The use of PF-3512676 (formerly CpG 7909), a synthetic deoxycytidyl-deoxyguanosine oligonucleotide which activates TLR-9, has been associated with a 10% PR and with one response lasting for 13+ months.168 Clinical response has been associated with the stimulation of NK-cell cytotoxicity, as well as with direct effects on increased activation of DCs, which subsequently induce potent innate immune responses via proinflammatory cytokine secretion, activation of other immune effectors (eg, NK-cells), and increased antigen presentation.170
TLR7/8 agonists demonstrated in preclinical studies an ability to markedly enhance the antitumor responses though diverse mechanisms involving maturation, activation, and/or migration of critical effector cells, including dendritic cells, B cells, T cells, NK cells, and mast cells. Dendritic cells have been shown to respond to TLR 7/8 agonists by increased secretion of IFN-α, IL-12, TNF-α, as well as upregulation of costimulatory molecules such as CD80 and CD86, increased polarization towards Th1-type responses, and enhanced tumor lysis.172174 B-cells, upon treatment with TLR7/8 agonists, are stimulated to increase production of cytokines and antibodies, as well as to upregulate CD80/86 which is essential for T-cell activation. These agonists also act directly on T cells by increased production of Th1-polarizing cytokine.175 The effects described above may render tumor cells more immunogenic and more susceptible to chemotherapy-induced tumor lysis. Inhibition of angiogenesis and promotion of apoptosis have also been associated with TLR7/8 agonists.176
Imiquimod, a synthetic TLR7 agonist, when utilized to activate DCs in a group of patients vaccinated with influenza, Melan-A, tyrosinase and NY-ESO peptides, elicited CD8+ T-cell responses in 5/8 patients, and one patient in 12 achieved a PR.177 Other cases have been reported of complete clinical clearance of locally metastatic melanoma when treated with topical 5% imiquimod178 alone, or in combination with tazarotene cream.179 These responses are likely mediated by several effects on dendritic cells, ranging from increased recruitment to the skin, enhanced migration to lymph nodes upon antigen uptake,180 and functional maturation,181 as demonstrated in preclinical studies. Alternatively, imiquimod has been demonstrated to directly increase immunogenic molecule expression of melanoma cells, as well as to induce apoptosis, thus providing an ample supply of local immunogenic proteins.182
TLR agonists are able to produce key alterations in the tumor microenvironment, but despite successful induction of Th1 antibody responses and of tumor antigen-specific CD8+ T cells, the development of clinically meaningful responses and improved survival have yet to be seen in patients with melanoma being treated with stand-alone TLR agonists. The potential use of TLR agonists as adjuvants in cancer vaccine or adoptive immunotherapy approaches are under ongoing investigation.
Depletion of Immunosuppressive Cells (Treg Depletion)
Regulatory T cells (Tregs) are immuosuppressive elements that, under normal physiological conditions, help modulate the immune response to prevent autoimmunity. In cancer, however, Tregs suppress antitumor responses of both CD4+ and CD8+ T cells, and their number in patients with Stage IV melanoma has been found to correlate inversely with survival (P=0.004).183
Two distinct populations of Tregs are recognized: naturally occurring CD4+/CD25+ Tregs (nTregs) which arise from the thymus with constitutive immunosuppressive function, and induced Tregs (iTregs) comprised of T cells that acquire an immunosuppressive function only under appropriate conditions, in the setting of an immune response.184 The most widely studied are CD4+/CD25+ Tregs. CD25 represents the alpha subunit of the IL-2 receptor, which is also highly expressed by activated T cells, thus making it a nonspecific Treg marker. The forkhead transcription factor (Foxp3) is now known to be exclusively expressed in CD4+CD25+ regulatory T cells, and is required for Treg development. Additionally, transduction of Foxp3 to conventional CD4+CD25 T cells was shown to be sufficient to confer suppressor function.185
The mechanisms by which Tregs function are not fully understood, but are thought to occur in a cell-cell contact dependent mechanisms,186 which involves IL-10, TGF-B and other cytokine secretion. A secondary messenger, cyclic adenosine monophosphate (cAMP), known to be a potent inhibitor of proliferation, also appears to be a critical component of nTreg function.187
Given their immunosuppressive effects, strategies to promote Treg depletion or inhibition have been evaluated in preclinical and human studies.
IL-21, in particular, may hold promise as an adjuvant therapy to augment response to adoptive T-cell transfer and vaccination approaches. In transfected IL-21-secreting B16 melanoma cell lines, IL-21 was found to delay tumor growth in vivo. The effect is thought to be mediated by an enhanced systemic effector and memory CD8+ T-cell responses, and a decreased accumulation of regulatory CD4+FOXP3+ T cells within the tumor microenvironment, by as much as 50%, compared to controls.188 Phase II trials of recombinant human IL-21 (rIL-21) given intravenously or subcutaneously in patients with stage IV melanoma showed acceptable safety profiles and demonstrated clinical responses, with 1 CR and 1 PR among 14 patients treated with intravenous IL-21 and 1 CR and 2 PRs among 23 patients treated with subcutaneous IL-21.189,190
Depletion of Foxp3-expressing regulatory T cells has also been demonstrated in preclinical studies using vaccination with Foxp3 mRNA-transfected dendritic cells. Strong induction of Foxp3-specific CTL responses was observed, along with as a preferential depletion of Tregs in the tumor (as opposed to the periphery), which may potentially reduce the risk for autoimmunity.191
WP1066, an inhibitor of STAT3 signaling, directly inhibits Tregs in a dose-dependent manner, an effect that was shown to promote enhanced T-cell cytotoxicity against melanoma.192 STAT3 inhibitors should be tested in combination with other immunotherapies, particularly those known to expand CD4(+) FoxP3(+) Treg populations, such as the anti-CTLA-4 monoclonal antibodies and systemic IL-2.193197 Potential synergism may exist, possibly permitting the simultaneous expansion of CD8+ T cells and inhibition of Tregs.
CD25-directed recombinant immuotoxins have also induced a significant reduction of regulatory T-cell populations, as demonstrated in preclinical studies by using RFT5-SMPT-dgA and LMB-2, respectively. Clinical studies, however, have shown only transient, partial reductions of Tregs in patients with metastatic melanoma. No objective antitumor responses were achieved with either RFT5-SMPT-dgA or LMB-2 in humans.198,199
Unconjugated mouse monoclonal antibodies against GD2 and GD3 gangliosides induced occasional responses in Phase I studies, but frequent human antimouse antibody reactions (HAMA), along with technical difficulties in production of these antibodies, have limited their clinical development. As production of human antibodies is also cumbersome, chimeric human-mouse antibodies were synthetized and have shown either minimal (anti-GD3 antibody KM871), or no clinical activity (murine anti-GD2 antibody ch14.18).200,201 Although no improvement over the use of antibody alone was seen with the concurrent administration of IL-2, a conjugated form of ch14.18 with IL-2 exerted antitumor activity in a murine model, which appeared to be mediated by MHC-I-restricted CD8+ cytotoxicity.202 The combination of ch14.18 and R24 murine antibodies administered with IL-2 produced 2 PRs (in which an anti-idiotype response to ch14.18 was elicited) and 4 SD of 23 patients with melanoma.203 Other conjugates with the toxin ricin or radioimmunoconjugate I-131 demonstrated limited clinical utility.204 Complement dependency for antibody mediated tumor cell killing is an issue since numerous tumors express high levels of complement inhibitors such as CD59.205
Various treatment modalities have been employed to reduce the risk of systemic recurrence in patients with intermediate (IIA), high (IIB-IIIA) and very high risk (stage IIIB-IIIC) patients. While patients presenting in stage IIA have a chance of recurrence of 20% to 30%, the rates of relapse are much higher for stages IIB, IIC, and III, averaging 40% to 80%. Early attempts using nonspecific immune adjuvants such as levamisole, Corynebacterium parvum, or BCG have not been proven in randomized trials to reduce the odds of recurrence.206,207 The limited clinical efficacy of adjuvant vaccination has been attributed to multiple factors, including the limited immunogenicity of the epitopes used in vaccination. Immunologically mediated tumor regression may involve tumoral modulation of the antigen processing mechanism, such as the TAP-1 and TAP-2 peptide transporters, which are critical to MHC class I antigen presentation pathways.127129
Interferons
Immunomodulatory effects of interferons (IFNs), along with the proven activity in metastatic disease, triggered considerable interest for testing this class of agents in the adjuvant setting. The E1684 trial randomized 287 patients between 52 weeks of high-dose interferon (HDI) versus observation. The first results of this trial reported in 1996 showed a significant improvement in RFS (1.72 y vs. 0.98, P=0.002) and OS in the HDI arm compared with observation (3.82 vs. 2.78 y P=0.02), leading to FDA approval of this treatment for stage IIB, IIC and III melanoma. A later update of data at 12.6 years of follow-up still shows a significant RFS advantage for HDI (HR=1.38, P=0.02), but a decrease in the OS benefit (HR=1.22, P=0.18), attributed partially to death from competing causes.208 Despite considerable toxicity, use of HDI in this setting was associated with an improvement in quality of life compared to observation only. The larger E1690 trial, designed using a cure-rate model derived from the results of E1684, employed a randomized comparison of HDI and low-dose interferon (LDI) against observation alone. Again, a significant RFS benefit was recorded for HDI compared with observation (HR=1.28, P=0.025), but no impact was seen with LDI, possibly influenced by the very high salvage rate of relapsed patients in the observation group. Furthermore, neither IFN dose led to any improvement in OS. Similar to E1684, a later follow-up analysis at 6.6 years still revealed a RFS advantage (HR=1.38, P=0.02).208 In a randomized comparison of HDI versus Gm2-klh/Qs-21 (GMK) vaccine (E1694), an initial significant advantage for both RFS and OS with HDI (HR=1.47, P=0.001 and HR=1.52, P=0.009, respectively), was maintained after 2.1 years (HR=1.33, P=0.006, and HR=1.32, P=0.04, respectively). A direct comparison of GMK alone versus GMK with either concurrent or sequential HDI (E2696) demonstrated the superiority of both HDI combinations over the vaccine alone (HR=1.75 and 1.96), reaching significance after adjusting for gender, performance status, time to resection, nodal status and age (P=0.016 and 0.03, respectively). However a survival update at 2.8 years did not confirm the advantage held initially by the two HDI groups.208
Composite data resulting from these four trials support the clinical benefit of HDI, with three trials demonstrating a significant improvement in disease-free survival (DFS), and 2 of them indicating an OS benefit.208 A pooled analysis of E1684 and E1690 data at a median follow-up of 7.2 years shows a significant superiority of HDI over observation in regard to RFS (HR=1.30, P<0.06), but not OS (HR=1.08, P=0.42), which is consistent with the survival data pooled by a large meta-analysis of eight trials comprising 3,178 patients.209 Further studies are necessary to better delineate melanoma prognostic groups and likelihood of response to adjuvant therapy before HDI can be accepted as a universal standard of care.
As discussed above in the sections regarding IL-2 and vaccine therapy, stimulation of anti-melanoma responses is associated in some cases with appearance of autoimmunity. A study by Gogas et al210 reported a correlation between the response to HDI therapy and various manifestations of autoimmunity, including antithyroid, antinuclear, anti-DNA, and anticardiolipin autoantibodies, and vitiligo. Development of autoimmunity represented an independent prognostic factor for relapse-free and overall survival (P<0.001). Although lead time bias can conduct to this correlation, similar results with CTLA-4 antibodies indicate an association between autoimmune phenomena (thyroiditis, hypophysitis, enteritis, hepatitis, and dermatitis) and prolonged survival in metastatic disease.146,155
Alternative interferon schedules have been designed in order to avoid toxicities associated with high-doses or prolonged courses of therapy. No survival benefit was achieved using an induction phase of 10 MU s.c. daily 5 days per week followed by 2 years of LDI.211 Likewise, two studies employing two and three years of LDI (3 MU s.c. three times a week), had no DFS or OS benefit compared to observation.212,213 Reduction of the duration of adjuvant HDI to 3 months (20 MU/m2 trice weekly) did not produce a median OS benefit (6.6 y for IFN-α 2a and 5.0 y for observation, P=0.40), but a possible improvement was suggested for selected high-risk node-positive patients (OS 4.1 vs. 2.7 y P=0.44). A comparison between one month induction therapy versus one full year of high-dose adjuvant interferon was reported in 364 patients with stage IIB, IIC, and III melanoma treated no later than 56 days of curative surgery with no significant differences in OS and RFS between the regimens of 1 month and 1 year of treatment.214
Based on data showing synergistic antitumor efficacy in vitro and in vivo,215 a combination of LDI-α2b and low-dose IL-2 was tested in 225 node-negative, pT3 and T4 patients, and demonstrated no superiority in regard to DFS or OS compared to LDI-α2b alone.216 Adjuvant treatment for high-risk stages IIA to IIIB with DTIC and low-dose natural interferon-a resulted in a significantly higher 7-year calculated OS rate of 51 versus 30% (P=0.007), with a greater benefit on late mortality, especially in high-risk patients.217 However, this data needs to be further confirmed.
Pegylated IFN-alpha-2b (PEG-IFN-alpha-2b) has raised interest for the chronic adjuvant treatment of melanoma given its convenient administration and positive outcomes recorded in the European trials. In a study of 1256 patients with stage III melanoma, PEG-IFN-alpha-2b (n=627) induction with 6 micrograms/kg/wk for 8 weeks followed by maintenance with 3 micrograms/kg/wk was administered against placebo for an intended total duration of 5 years. After 3.8 years of follow-up, the risk for recurrence-free survival (RFS) was reduced by 18% (hazard rate=0.82; P=0.01), along with an expected negative effect on global quality of life score.218 The exposure to Peg-IFN alpha-2b appears to be sustained during the long-term adjuvant treatment in melanoma, which is consistent with the European Organization for Research and Treatment of Cancer 18991 data indicating a significant, sustained, relapse-free survival benefit.219
High-dose Interferon (HDI) administered neoadjuvantly in doses of 10 MU/m2 three times weekly resulted in clinical responses in 55% of patients, and 50% of patients had no recurrence of their disease at over 1.5 years of follow-up. In this patient population of stage IIIB melanoma, clinical responds were associated with larger amounts of tumor infiltrating CD3+ and CD11c+ lymphocytes.220 Neoadjuvant biochemotherapy with cisplatin, vinblastine, dacarbazine, interleukin-2 and interferon-alpha 2a produced a 50% response rate and a 65% DFS at 31 months,221 and a phase III intergroup trial comparing a short intensive course of biochemotherapy (IL-2, IFN-α, cisplatin, DTIC and vinblastine) with standard HDI is currently addressing the high-risk stage III patients. Equivalent clinical results with less toxicity than HDI were obtained with a combination of intermediate-dose IFN and Melacine vaccine in stage III melanoma (RFS 31 vs. 25 mo, P=0.85).222
Microarray analysis of PBMCs gene induction in vitro may be a useful predictor of the in vivo response of T cells, NK cells or monocytes to treatment with IFN-α. Various genes involved in immune responses, nucleic acid binding and metabolism, protein catabolism, or cyclin-dependent protein kinase activity (OAS2, OASL, HERC5, ISG20, IFI44, LIR7, LGP2, MT1H, MT2A, N4BP1, PLSCR1, USP18, TREX1, ZCCHC2) are activated in both settings, therefore being potential markers of patient response to interferon.223
Vaccines
Several melanoma-specific vaccines have been prepared from whole cells,224226 or from antigens shed from allogeneic cell lines.227 A prolongation of survival in comparison to historical controls or to patients not developing an immune response to the vaccine was reported in the initial studies. However, randomized phase III studies did not confirm a significant benefit.
In a 689 patients trial comparing an allogeneic melanoma cell lysate vaccine, Melacine, to observation in patients with stage II disease, a similar 5-year disease-free survival was reported (65% for the vaccine and 63% for observation arm).228 An OS analysis could only be performed in a follow-up study, which showed a survival advantage for the HLA-A2+ and/or C3+ group, but no significant benefit for the overall cohort of patients.229
Canvaxin is an allogeneic whole cell vaccine that was tested in double blind trials of post resection patients with high probability of relapse. Initial nonrandomized studies demonstrated promising results.230 2602 AJCC stage III melanoma patients who underwent lymphadenectomy were enrolled in a study where 935 received Canvaxin vaccine between 1984 and 1998, while 1667 historical controls did not. A significantly higher median OS and 5-year OS was observed in patients receiving the vaccine (56.4 vs. 31.9 mo and 49% vs. 37%, respectively; P=0.0001). The difference was maintained when patients treated with Canvaxin were matched with non-PV patients by six covariates forming 739 pairs.231 Another study involving 150 stage IV melanoma patients treated adjuvantly with Canvaxin after surgical resection demonstrated induction of delayed-type hypersensitivity (DTH), which correlated with the OS (39% for vaccinated and 19% for non-vaccinated patients) (P=0.0001). In a multivariate analysis model, vaccine therapy was the most significant prognostic variable (P=0.0001).232 However, subsequent prospective randomized trials showed less favorable results, with a trend towards decreased survival in treated patients. Among 1656 patients with resected stage III and IV melanoma, Canvaxin failed to demonstrate improvements in overall survival (OS), as compared to BCG.233
Similarly, a polyvalent, shed-antigen Bystryn vaccine did not produce a significant statistical improvement in median OS, even after adjusting for risk factors. Thirty-eight patients with stage III melanoma with a particularly poor prognosis were immunized intradermally in a 2:1 vaccine/placebo ratio every 3 weeks×4, monthly×3, every 3 months×2, and then every 6 months for 5 years or until disease progression. At 2.5 years median length of observation, the median time to disease progression was 2.5 times longer in the active arm (P=0.03). Median overall survival was 40% longer in the active treatment group (3.8 vs. 2.7 y, P=nonsignificant).234
Interest in targeting more specific surface antigens has resulted in vaccines derived from GM2 gangliosides, a well-defined melanoma associated antigen. Anti-GM2 antibodies have been detected in approximately 5% of melanoma patients, and their presence was associated with an increase in the relapse-free survival (RFS).235 An IgM antibody response was obtained in 85% of patients with resected stage III melanoma who were immunized with purified GM2 adherent to BCG. An improved DFS was observed for patients with anti-GM-2 titers >1:40, but such a benefit could not be confirmed in a subsequent randomized phase III trial comparing GM2+BCG vaccine with BCG alone.236 However, when the six patients who produced GM2 antibodies before randomization were excluded, an increased DFS of 23% (P=0.02) and a trend toward longer OS were observed for the GM2+BCG. Another phase III randomized trial comparing the efficacy of high-dose interferon alfa-2b therapy (HDI) versus vaccination with GM2 (GMK) demonstrated an overall benefit for HDI in terms of RFS and OS in melanoma patients. Antibody responses to GM2, however, were associated with a trend toward improved RFS and OS.237
However, no survival benefit was noted in two large phase III trials which used adjuvant GM2-KLH with QS21 adjuvant. A negative effect on survival might have been observed in the E1694 trial, where 880 patients with resected stage IIB and III melanoma have been randomized between GM2-KLH vaccine and HD IFNa-2b. Lower RFS (relapse-free survival) and OS (overall survival) were seen in the GM2-KLH arm.237 A negative effect on the rate of development of distant metastases and a lower OS (HR 1.57, P=0.03) were observed in an European study European Organization for Research and Treatment of Cancer 18961 involving 1314 patients with resected stage II (T3-4N0M0) disease who were randomized between the GM2-KLH vaccine and placebo, although the disease-free survival was similar in the two groups.238 Both studies were closed prematurely because of the apparent detrimental effect on survival of the GM2-KLH vaccine.
The most immunogenic peptides among 12 cancertestis and melanocyte differentiation proteins tested are tyrosinase, gp100, MAGE-A1, and MAGE-A10, and it was determined that administration of multiple peptides is safe and immunogenic.239 An ongoing Intergroup trial (E4697) is currently testing vaccination with HLA-A2-restricted peptides (tyrosinase, gp210M, and MART-1), with GM-CSF used either alone (in HLA-A2- patients), or added to the peptide vaccine for potential synergy.
Anti-ganglioside Monoclonal Antibodies
A humoral response can be generated against the monoclonal antibody active sites. Anti-GD3 antibodies were induced in 3 of 14 patients immunized with BEC2, an anti-idiotypic monoclonal antibody that mimics GD3 and BCG, but the immunogenicity of anti-idiotypic monoclonal antibodies has generally remained low. A 71% survival and a 64% disease-free survival at 2.4 years of follow-up were considered to be encouraging results.240
TLR Agonists
In a study involving 24 patients with stage I to III melanoma, local administration of PF-3512676 (formerly CpG 7909), a synthetic deoxycytidyl-deoxyguanosine oligonucleotide which activates TLR-9, has induced melanoma-specific CD8+ T-cell responses against at least one melanoma-associated antigen in the draining lymph node in 50% of the patients receiving the compound, vs. none of the control patients injected with saline (P=0.01). Clinical response has been associated with enhanced local and systemic melanoma-specific CD8+ T-cell reactivity and NK cell mobilization.241 Intradermally injected PF-3512676 appears promising as an adjuvant therapy for early-stage melanoma, being associated with increased dendritic cell activation, enhanced type I cytokine secretion, and a significant reduction in CD4+CD25+ Tregs in sentinal lymph nodes.242
Granulocyte-macrophage Colony Stimulating Factor (GM-CSF)
The use of GM-CSF in the adjuvant treatment of melanoma is based on its differentiating activity on DC and formation of cytotoxic macrophages. A better survival rate of 38.0 versus 12.2 mo was obtained in a single-arm study of s.c. GM-CSF (P<0.001) compared to historical controls, with only one of 48 node-positive patients discontinuing the drug due to a grade 2 injection site reaction.243 The increase of IL-2 receptor expression on T-lymphocytes in response to GM-CSF was postulated to act synergistically with formation of LAK and TIL cells by IL-2. Early results in a trial assessing the efficacy of this combination indicated a DFS of 93.7% at a median follow-up of 14 months, and a good tolerability of the combination.244 GM-CSF has been reported to also possess immune suppressive effects depending on concentration, therefore, a word of caution must be added when discussing these trials.84
Progress in identifying tumor epitopes of heightened immunogenicity and advances in deciphering the homeostasis of immune responses has lead to a new age of melanoma immunotherapy. Recent important steps represent the recognition of tumor immune evasion mechanisms, which resulted in the clinical use of anti-CTLA4 and anti-PD1 antibodies; understanding of the importance of costimulatory signals, which was translated into the use of CD40 agonist mAbs; and appreciation of the importance of innate immune activation, causing investigators to seek stimulation of dendritic cells by various TLR agonists. Defining of the role played by immune adjuvants and the influence of booster doses has represented important additions to the development of anti-tumor immunology. Finally, the recognition of the role played by Treg cells in the formation of immune responses and their interference with immune effectors resulted in new strategies to deplete or interfere with their function.
Through accepting that immune-based approaches lead to limited responses, which often do not exceed a 15% response rate threshold, the hypothesis of a decisive role played by tumor-induced immunosuppression, has been gaining acceptance. Such understanding explains the poor clinical results observed even in well-designed, controlled clinical studies. However, much disappointment resulted over time as the results of a plethora of small phase I and II trials designed to assess the safety and biologic properties of immunotherapeutic agents appeared to have created breakthroughs in clinical responses. A delay in implementing the latest immunological knowledge in Phase III trials, as well as the absence of adequate control study populations further flawed the scientific and clinical value of many of the recent trials. In addition to these obstacles comes the fact that the much-sought correlation between tumor responses with the presence of immunological responses is also not reliably demonstrated across studies. However, this yet faint association raises hope that the clinical efficacy of immunotherapy may be increased once the mechanisms of tumor suppression are better understood and addressed. This also highlights the urgent need to validate standardized biomarker/immune monitoring methods so that trials can be accurately compared, as well as to develop more accurate diagnostic biomarkers. Currently the International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) to address these two issues through a systematic analysis of available technologies. Two working groups have been created245 with the goal to perform high-throughput screening of clinical samples in order to identify predictors of immune responsiveness, clinical responsiveness, and survival in an era where the response evaluation criteria in solid tumors criteria may not be entirely reflective of the clinical benefit, identifying markers that predict the risks of toxicity to treatment, and identifying mechanistic biomarkers which will help characterizing the mechanism of action of immunotherapeutic approaches. A strategy to observe the common modifications that occur during response to therapy was proposed, which consists of analyzing samples relevant to the genetic background of the patients, the modified phenotypes of immune cells in relation to the natural evolution of the neoplasia, and the tumor response at local and distant sites. Identified promising immune monitoring techniques for the future are PET scans of activated T cells, or analysis of the proteins produced during immune activation and tumor response, which can be performed either non-invasively or through a minimal peripheral blood collection.245
The possibility to engineer better immune interactions and to boost positive feedback loops predicts a new coming of age of immunotherapy. Demonstration of immune responses to tumor-associated antigens, along with the possibility to follow the T-lymphocyte activation during immune stimulation in fact opens a new age in testing and enhancing immune stimulating approaches with proficiency. The contribution of fundamental research, along with the discovery of more potent immune stimulation strategies, will probably be able to separate the anti-tumor responses from the generation of autoimmunity. Insidiously coming of age, melanoma immunotherapy has a future which is ultimately dependent on the understanding of the contradictory and complex influences that govern the immune responses, and in particular the immunosuppressive barriers.
ACKNOWLEDGMENTS
The authors like to thank Famela Ramos for assistance in literature collection and critical review of the manuscript.
Footnotes
All authors have confirmed there is no financial conflict of interest in regards to this work.
1. Touloukian CE, Leitner WW, Topalian SL, et al. Identification of a MHC class II-restricted human gp100 epitope using DR4-IE transgenic mice. J Immunol. 2000;164:3535–3542. [PMC free article] [PubMed]
2. Topalian SL, Rivoltini L, Mancini M, et al. Human CD4+ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene. Proc Natl Acad Sci USA. 1994;91:9461–9465. [PubMed]
3. Van der Bruggen P, Traversari C, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science. 1991;254:1643–1647. [PubMed]
4. Houghton AN, Gold JS, Blachere NE. Immunity against cancer: lessons learned from melanoma. Curr Opin Immunol. 2001;13:134–140. [PubMed]
5. Parkinson DR, Abrams JS, Wiernik PH, et al. Interleukin-2 therapy in patients with metastatic malignant melanoma: a phase II study. J Clin Oncol. 1990;8:1650–1656. [PubMed]
6. Legha SS, Gianan MA, Plager C, et al. Evaluation of interleukin-2 administered by continuous infusion in patients with metastatic melanoma. Cancer. 1996;77:89–96. [PubMed]
7. Sparano JA, Fisher RI, Sunderland M, et al. Randomized phase III trial of treatment with high-dose interleukin-2 either alone or in combination with interferon alfa-2a in patients with advanced melanoma. J Clin Oncol. 1993;11:1969–1677. [PubMed]
8. Smith FO, Downey SG, Klapper JA, et al. Treatment of metastatic melanoma using interleukin-2 alone or in conjunction with vaccines. Clin Cancer Res. 2008;14:5610–5618. [PMC free article] [PubMed]
9. Agarwala SS, Tarhini AA, Kirkwood JM, et al. Phase II trial of sequential temozolomide (TMZ) and high-dose bolus (HDB) IL-2 in patients with metastatic melanoma. Journal of Clinical Oncology. 2006;24:18S–8037.
10. Sosman JA, Carrillo C, Urba WJ, et al. Three phase II cytokine working group trials of gp100 (210M) peptide plus high-dose interleukin-2 in patients with HLA-A2-positive advanced melanoma. J Clin Oncol. 2008;26:2292–2298. [PMC free article] [PubMed]
11. Kaufmann R, Spieth K, Leiter U, et al. Temozolomide in combination with interferon-alfa versus temozolomide alone in patients with advanced metastatic melanoma: a randomized, phase III, multicenter study from the Dermatologic Cooperative Oncology Group. J Clin Oncol. 2005;23:9001–9007. [PubMed]
12. Bar J, Yerushalmi R, Shapira-Frummer R, et al. Concurrent chemobiotherapy with cisplatin, dacarbazine, decrescendo interleukin-2 and interferon alpha2b in patients with metastatic melanoma. Oncol Rep. 2008;20:1533–1538. [PubMed]
13. Hauschild A, Dummer R, Ugurel S, et al. Combined treatment with pegylated interferon-alpha-2a and dacarbazine in patients with advanced metastatic melanoma: a phase 2 study. Cancer. 2008;113:1404–1411. [PubMed]
14. Spieth K, Kaufmann R, Dummer R, et al. Temozolomide plus pegylated interferon alfa-2b as first-line treatment for stage IV melanoma: a multicenter phase II trial of the Dermatologic Cooperative Oncology Group (DeCOG) Ann Oncol. 2008;19:801–806. [PubMed]
15. Alatrash G, Hutson TE, Molto L, et al. Clinical and immunologic effiects of subcutaneously administered interleukin-12 and interferon alfa-2b: phase I trial of patients with metastatic renal cell carcinoma or malignant melanoma. J Clin Oncol. 2004;22:2891–2900. [PubMed]
16. Senzer NN, Kaufman H, Amatruda T, et al. Phase II clinical trial with a second generation, GM-CSF encoding, oncolytic herpesvirus in unresectable metastatic melanoma. J Clin Oncol. 2009;27:5763–5771. [PubMed]
17. Rosenberg SA, Yang JC, Topalian SL. Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2. JAMA. 1994;271:907–913. [PubMed]
18. Rosenberg SA, Lotze MT, Muul LM. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med. 1987;316:889–897. [PubMed]
19. Rosenberg SA, Lotze MT, Yang JC, et al. Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer. J Natl Cancer Inst. 1993;85:622–632. [PubMed]
20. Atkins MB, Kunkel L, Sznol M, et al. High-dose recombinant interleukin-2 therapy in patients with metastatic melanoma: long-term survival update. Cancer J Sci Am. 2000;6(suppl 1):S11–S14. [PubMed]
21. Phan GQ, Attia P, Steinberg SM, et al. Factors associated with response to high-dose interleukin-2 in patients with metastatic melanoma. J Clin Oncol. 2001;19:3477–3482. [PubMed]
22. Sabatino M, Kim-Schulze S, Panelli MC, et al. Serum vascular endothelial growth factor and fibronectin predict clinical response to high-dose interleukin-2 therapy. J Clin Oncol. 2009;27:2645–2652. [PMC free article] [PubMed]
23. Atkins MB, Shet A, Sosman JA. IL-2 clinical applications. In: De Vita VT Jr, Hellman S, Rosenberg SA, et al., editors. Biologic Therapy of Cancer Principles and Practice. 3rd ed Vol. 50. JB Lippincott; Philadelphia: 2000.
24. Maellaro E, Pacenti L, Del Bello B, et al. Diffierent effiects of interferon-alpha on melanoma cell lines: a study on telomerase reverse transcriptase, telomerase activity and apoptosis. Br J Dermatol. 2003;148:1115–1124. [PubMed]
25. Fleischmann CM, Stanton GJ, Fleischmann WR., Jr Enhanced in vivo sensitivity of in vitro interferon-treated B16 melanoma cells to CD8 cells and activated macrophages. J Interferon Cytokine Res. 1996;16:805–812. [PubMed]
26. Streck CJ, Ng CY, Zhang Y, et al. Interferon-mediated anti-angiogenic therapy for neuroblastoma. Cancer Lett. 2005;228:163–170. [PubMed]
27. Sabel MS, Sondak VK. Is there a role for adjuvant high-dose interferon-alpha-2b in the management of melanoma? Drugs. 2003;63:1053–1058. [PubMed]
28. Sondak VK, Redman BG. Chapter 16: Pharmacology of Cancer Biotherapeutics: Section 1: Interferons. In: De Vita VT Jr, Hellman S, Rosenberg SA, editors. Cancer, Principals and Practice of Oncology. 7th ed JB Lippincot; Philadelphia: 2000.
29. Morton DL, Essner R, Kirkwood JM, et al. In: The Skin: Malignant Melanoma in Cancer Medicine. 6th ed Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. BC Decker; Hamilton, ON: 2003. pp. 1973–1997.
30. Astsaturov I, Petrella T, Bagriacik EU, et al. Amplification of virus-induced antimelanoma T-cell reactivity by high-dose interferon-alpha2b: implications for cancer vaccines. Clin Cancer Res. 2003;9:4347–4355. [PubMed]
31. Cangemi G, Morandi B, D'Agostino A, et al. IFN-alpha mediates the up-regulation of HLA class I on melanoma cells without switching proteasome to immunoproteasome. Int Immunol. 2003;15:1415–1421. [PubMed]
32. Marincola FM, White DE, Wise AP, et al. Combination therapy with interferon alfa-2a and interleukin-2 for the treatment of metastatic cancer. J Clin Oncol. 1995;13:1110–1122. [PubMed]
33. Atkins MB, Robertson MJ, Gordon M, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res. 1997;3:409–417. [PubMed]
34. Bajetta E, Del Vecchio M, Mortarini R, et al. Pilot study of subcutaneous recombinant human interleukin 12 in meta-static melanoma. Clin Cancer Res. 1998;4:75–85. [PubMed]
35. Gollob JA, Veenstra KG, Parker RA, et al. Phase I trial of concurrent twice-weekly recombinant human interleukin-12 plus low-dose IL-2 in patients with melanoma or renal cell carcinoma. J Clin Oncol. 2003;21:2564–2573. [PubMed]
36. Schiller JH, Pugh M, Kirkwood JM, et al. Eastern cooperative group trial of interferon gamma in metastatic melanoma: an innovative study design. Clin Cancer Res. 1996;2:29–36. [PubMed]
37. Atkins MB. Immunotherapy and experimental approaches for metastatic melanoma. Hematol Oncol Clin North Am. 1998;12:877–902. viii. [PubMed]
38. Robertson MJ, Mier J, Logan T, et al. Tolerability and anti-tumor activity of recombinant human IL-18 (rhIL-18) administered as five daily intravenous infusions in patients with solid tumors. Proc Am Soc Clin Oncology. 2004;22:14S. Abstr. 2553.
39. Ichim CV. Revisiting immunosurveillance and immunostimulation: Implications for cancer immunotherapy. J Transl Med. 2005;3:8. [PMC free article] [PubMed]
40. Yee C, Thompson JA, Byrd D, et al. Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effiect of transferred T cells. Proc Natl Acad Sci U S A. 2002;99:16168–16173. [PubMed]
41. Lemoine FM, Cherai M, Giverne C, et al. Massive expansion of regulatory T-cells following interleukin 2 treatment during a phase I–II dendritic cell-based immunotherapy of metastatic renal cancer. Int J Oncol. 2009;35:569–581. [PubMed]
42. Rosenberg SA, Yannelli JR, Yang JC, et al. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2. J Natl Cancer Inst. 1994;86:1159–1166. [PubMed]
43. Aebersold P, Hyatt C, Johnson S, et al. Lysis of autologous melanoma cells by tumor-infiltrating lymphocytes: association with clinical response. J Natl Cancer Inst. 1991;83:932–937. [PubMed]
44. Robbins PF, Dudley ME, Wunderlich J, et al. Cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy. J Immunol. 2004;173:7125–7130. [PMC free article] [PubMed]
45. Zhou J, Shen X, Huang J, et al. Telomere length of transferred lymphocytes correlates with in vivo persistence and tumor regression in melanoma patients receiving cell transfer therapy. J Immunol. 2005;175:7046–7052. [PMC free article] [PubMed]
46. Klebanoff CA, Khong HT, Antony PA, et al. Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol. 2005;26:111–117. [PMC free article] [PubMed]
47. Marleau AM, Sarvetnick N. T cell homeostasis in tolerance and immunity. J Leukoc Biol. 2005;78:575–584. [PubMed]
48. Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science. 2002;298:850–854. [PMC free article] [PubMed]
49. Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol. 2005;23:2346–2357. [PMC free article] [PubMed]
50. Van der Most RG, Currie AJ, Mahendran S, et al. Tumor eradication after cyclophosphamide depends on concurrent depletion of regulatory T cells: a role for cycling TNFR2-expressing effector-suppressor T cells in limiting effective chemotherapy. Cancer Immunol Immunother. 2009;58:1219–1228. [PubMed]
51. Dudley ME, Yang JC, Sherry R, et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol. 2008;26:5233–5239. [PMC free article] [PubMed]
52. Rosenberg SA, Dudley ME. Adoptive cell therapy for the treatment of patients with metastatic melanoma. Curr Opin Immunol. 2009;21:233–240. [PMC free article] [PubMed]
53. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006;314:126–129. [PMC free article] [PubMed]
54. Heemskerk B, Liu K, Dudley ME, et al. Adoptive cell therapy for patients with melanoma, using tumor-infiltrating lymphocytes genetically engineered to secrete interleukin-2. Hum Gene Ther. 2008;19:496–510. [PMC free article] [PubMed]
55. Keilholz U, Conradt C, Legha SS, et al. Results of interleukin-2-based treatment in advanced melanoma: a case record-based analysis of 631 patients. J Clin Oncol. 1998;16:2921–2929. [PubMed]
56. Allen I, Kupelnick B, Kumashiro M. Efficacy of interleukin-2 in the treatment of metastatic melanoma-systematic review and meta-analysis. Sel Cancer Ther. 1998;1:168–173.
57. Alexandrescu DT, Dutcher JP, Wiernik PH. Metastatic melanoma: is biochemotherapy the future? Med Oncol. 2005;22:101–111. [PubMed]
58. Yannelli JR, Wroblewski JM. On the road to a tumor cell vaccine: 20 years of cellular immunotherapy. Vaccine. 2004;23:97–113. [PubMed]
59. Terando AM, Faries MB, Morton DL. Vaccine therapy for melanoma: current status and future directions. Vaccine. 2007;25(suppl 2):B4–B16. [PubMed]
60. Riley LB, Agarwala SS. Melanoma vaccines. Expert Rev Vaccines. 2008;7:937–949. [PubMed]
61. Rosenberg SA, White DE. Vitiligo in patients with melanoma: normal tissue antigens can be targets for cancer immunotherapy. J Immunother Emphasis Tumor Immunol. 1996;19:81–84. [PubMed]
62. Wang E, Monaco A, Monsurro V, et al. Antitumor vaccines, immunotherapy and the immunological constant of rejection. IDrugs. 2009;12:297–301. [PMC free article] [PubMed]
63. Hersey P. Melanoma vaccines. Current status and future prospects. Drugs. 1994;47:373–382. [PubMed]
64. Vaishampayan U, Abrams J, Darrah D, et al. Active immunotherapy of metastatic melanoma with allogeneic melanoma lysates and interferon alpha. Clin Cancer Res. 2002;8:3696–3701. [PubMed]
65. Mitchell MS, Abrams J, Thompson JA, et al. Randomized trial of an allogeneic melanoma lysate vaccine with low-dose interferon Alfa-2b compared with high-dose interferon Alfa-2b for Resected stage III cutaneous melanoma. J Clin Oncol. 2007;25:2078–2085. [PubMed]
66. Pan J, Jin P, Yan J, et al. Anti-angiogenic active immunotherapy: a new approach to cancer treatment. Cancer Immunol Immunother. 2008;57:1105–1114. [PubMed]
67. Yuan J, Ku GY, Gallardo HF, et al. Safety and immunogenicity of a human and mouse gp100 DNA vaccine in a phase I trial of patients with melanoma. Cancer Immun. 2009;9:5. [PMC free article] [PubMed]
68. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med. 1998;4:321–327. [PMC free article] [PubMed]
69. Ernstoff M, Carrillo C, Urba W, et al. A cytokine working group (CWG) 3-arm phase II trial of gp100 (209–2M) peptide +high dose (HD) interleukin-2 (IL-2) in HLA-A2+ (A2+) advanced melanoma patients (pts) Proc Am Soc Clin Oncology. 2005;23:16S. Abstr. 7504.
70. Smith JW, II, Walker EB, Fox BA, et al. Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8+ T-cell responses. J Clin Oncol. 2003;21:1562–1573. [PubMed]
71. Slingluff CL, Jr, Yamshchikov G, Neese P, et al. Phase I trial of a melanoma vaccine with gp100 (280–288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes. Clin Cancer Res. 2001;7:3012–3024. [PubMed]
72. Cormier JN, Salgaller ML, Prevette T, et al. Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART-1/Melan A. Cancer J Sci Am. 1997;3:37–44. [PMC free article] [PubMed]
73. Bettinotti MP, Panelli MC, Ruppe E, et al. Clinical and immunological evaluation of patients with metastatic melanoma undergoing immunization with the HLA-Cw*0702-associated epitope MAGE-A12:170–178. Int J Cancer. 2003;105:210–216. [PubMed]
74. Roberts JD, Niedzwiecki D, Carson WE, et al. Phase 2 study of the g209-2M melanoma peptide vaccine and low-dose interleukin-2 in advanced melanoma: Cancer and Leukemia Group B 509901. J Immunother. 2006;29:95–101. [PubMed]
75. Lee KH, Wang E, Nielsen MB, et al. Increased vaccine-specific T cell frequency after peptide-based vaccination correlates with increased susceptibility to in vitro stimulation but does not lead to tumor regression. J Immunol. 1999;163:6292–6300. [PubMed]
76. Schwartzentruber D. A phase III multi-institutional randomized study of immunization with the gp100:209-217(210M) peptide followed by high-dose IL-2 compared with high-dose IL-2 alone in patients with metastatic melanoma. J Clin Oncol. 2009;27(suppl):18s. abstr CRA9011.
77. Kirkwood JM, Lee S, Moschos SJ, et al. Immunogenicity and antitumor effects of vaccination with peptide vaccine± granulocyte-monocyte colony-stimulating factor and/or IFN-alpha2b in advanced metastatic melanoma: Eastern Cooperative Oncology Group Phase II Trial E1696. Clin Cancer Res. 2009;15:1443–1451. [PMC free article] [PubMed]
78. Gajewski TF, Fallarino F, Ashikari A, et al. Immunization of HLA-A2+ melanoma patients with MAGE-3 or MelanA peptide-pulsed autologous peripheral blood mononuclear cells plus recombinant human interleukin 12. Clin Cancer Res. 2001;7(3 suppl):895s–901s. [PubMed]
79. Cebon J, Jager E, Shackleton MJ, et al. Two phase I studies of low dose recombinant human IL-12 with Melan-A and influenza peptides in subjects with advanced malignant melanoma. Cancer Immun. 2003;3:7. [PubMed]
80. Schaed SG, Klimek VM, Panageas KS, et al. T-cell responses against tyrosinase 368–376(370D) peptide in HLA*A0201+ melanoma patients: randomized trial comparing incomplete Freund's adjuvant, granulocyte macrophage colony-stimulating factor, and QS-21 as immunological adjuvants. Clin Cancer Res. 2002;8:967–972. [PubMed]
81. Jager E, Gnjatic S, Nagata Y, et al. Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers. Proc Natl Acad Sci U S A. 2000;97:12198–12203. [PubMed]
82. Young MR, Wright MA, Lozano Y, et al. Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34+ natural suppressor cells. Int J Cancer. 1997;74:69–74. [PubMed]
83. Young MR, Wright MA, Matthews JP, et al. Suppression of T cell proliferation by tumor-induced granulocyte-macro-phage progenitor cells producing transforming growth factor-beta and nitric oxide. J Immunol. 1996;156:1916–1922. [PubMed]
84. Parmiani G, Castelli C, Pilla L, et al. Opposite immune functions of GM-CSF administered as vaccine adjuvant in cancer patients. Ann Oncol. 2007;18:226–232. [PubMed]
85. Dranoff G, Jaffee E, Lazenby A, et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A. 1993;90:3539–3543. [PubMed]
86. Soiffer R, Lynch T, Mihm M, et al. Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma. Proc Natl Acad Sci U S A. 1998;95:13141–13146. [PubMed]
87. Nawrocki S, Murawa P, Malicki J, et al. Genetically modified tumour vaccines (GMTV) in melanoma clinical trials. Immunol Lett. 2000;74:81–86. [PubMed]
88. Mocellin S, Marincola FM, Young HA. Interleukin-10 and the immune response against cancer: a counterpoint. J Leukoc Biol. 2005;78:1043–1051. [PubMed]
89. Rosenberg SA, Zhai Y, Yang JC, et al. Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gp100 melanoma antigens. J Natl Cancer Inst. 1998;90:1894–1900. [PMC free article] [PubMed]
90. Bergen M, Chen R, Gonzalez R. Efficacy and safety of HLA-B7/beta-2 microglobulin plasmid DNA/lipid complex (Allovectin-7) in patients with metastatic melanoma. Expert Opin Biol Ther. 2003;3:377–384. [PubMed]
91. Richards JM, Bedikian A, Gonzalez R, et al. High-dose Allovectin-7® in patients with advanced metastatic melanoma: final phase 2 data and design of phase 3 registration trial. Proc Am Soc Clin Oncology. 2005;23:16S. Abstr. 7543.
92. Ilkovitch D, Lopez DM. Immune modulation by melanoma-derived factors. Exp Dermatol. 2008;17:977–985. [PubMed]
93. Kim R, Emi M, Tanabe K. Functional roles of immature dendritic cells in impaired immunity of solid tumour and their targeted strategies for provoking tumour immunity. Clin Exp Immunol. 2006;146:189–196. [PubMed]
94. Min WP, Zhou D, Ichim TE, et al. Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance. J Immunol. 2003;170:1304–1312. [PubMed]
95. Nicholaou T, Ebert LM, Davis ID, et al. Regulatory T-cell-mediated attenuation of T-cell responses to the NY-ESO-1 ISCOMATRIX vaccine in patients with advanced malignant melanoma. Clin Cancer Res. 2009;15:2166–2173. [PubMed]
96. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med. 1998;4:328–332. [PubMed]
97. Banchereau J, Palucka AK, Dhodapkar M, et al. Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine. Cancer Res. 2001;61:6451–6458. [PubMed]
98. Lau R, Wang F, Jeffery G, et al. Phase I trial of intravenous peptide-pulsed dendritic cells in patients with metastatic melanoma. J Immunother. 2001;24:66–78. [PubMed]
99. Schuler-Thurner B, Schultz ES, Berger TG, et al. Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopre-served, peptide-loaded monocyte-derived dendritic cells. J Exp Med. 2002;195:1279–1288. [PMC free article] [PubMed]
100. Smithers M, O'Connell K, MacFadyen S, et al. Clinical response after intradermal immature dendritic cell vaccination in metastatic melanoma is associated with immune response to particulate antigen. Cancer Immunol Immunother. 2003;52:41–52. [PubMed]
101. Slingluff CL, Jr, Petroni GR, Yamshchikov GV, et al. Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J Clin Oncol. 2003;21:4016–4026. [PubMed]
102. O'Rourke MG, Johnson M, Lanagan C, et al. Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine. Cancer Immunol Immunother. 2003;52:387–395. [PubMed]
103. Ribas A, Glaspy JA, Lee Y, et al. Role of dendritic cell phenotype, determinant spreading, and negative costimula-tory blockade in dendritic cell-based melanoma immunotherapy. J Immunother. 2004;27:354–367. [PubMed]
104. Gorin I, Prince M, Grob J, et al. A phase I/II study of a multivalent dendritic cell vaccine in patients with metastatic melanoma. Journal of Clinical Oncology. 2005;23:16S. ASCO Annual Meeting Proceedings. Part I of II, 2542.
105. Akiyama Y, Tanosaki R, Inoue N, et al. Clinical response in Japanese metastatic melanoma patients treated with peptide cocktail-pulsed dendritic cells. J Transl Med. 2005;3:4. [PMC free article] [PubMed]
106. Palucka AK, Ueno H, Connolly J, et al. Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity. J Immunother. 2006;29:545–557. [PubMed]
107. Ridolfi R, Petrini M, Fiammenghi L, et al. Improved overall survival in dendritic cell vaccination-induced immunoreactive subgroup of advanced melanoma patients. J Transl Med. 2006;4:36. [PMC free article] [PubMed]
108. Salcedo M, Bercovici N, Taylor R, et al. Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate. Cancer Immunol Immunother. 2006;55:819–829. [PubMed]
109. Ross M, Camacho LH, Hersh EM, et al. Clinical and Immunological responses in patients with malignant melanoma treated with a dendritic cell-based vaccine. Preliminary report from a multi-institutional phase II clinical trial. Journal of Clinical Oncology. 2007;25(18S (June 20 Supplement)):3004. 2007 ASCO Annual Meeting Proceedings. Part I.
110. O'Rourke MG, Johnson MK, Lanagan CM, et al. Dendritic cell immunotherapy for stage IV melanoma. Melanoma Res. 2007;17:316–322. [PubMed]
111. Petenko NN, Mikhaylova I, Chkadua GZ, et al. Dendritic cell based vaccine therapy of melanoma. Journal of Clinical Oncology. 2007;25(18S (June 20 Supplement)):3077. 2007 ASCO Annual Meeting Proceedings (Post-Meeting Edition)
112. Wei YC, Stephenson JJ, Li K, et al. A phase II clinical study of dendritoma vaccination combined with low dose inter-leukin-2 in advanced melanoma patients. Journal of Clinical Oncology. 2007;25(18S):3051. ASCO Annual Meeting Proceedings Part I.
113. Jonuleit H, Giesecke-Tuettenberg A, Tuting T, et al. A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection. Int J Cancer. 2001;93:243–251. [PubMed]
114. de Vries IJ, Lesterhuis WJ, Scharenborg NM, et al. Maturation of dendritic cells is a prerequisite for inducing immune responses in advanced melanoma patients. Clin Cancer Res. 2003;9:5091–5100. [PubMed]
115. Dhodapkar MV, Krasovsky J, Steinman RM, et al. Mature dendritic cells boost functionally superior CD8(+) T-cell in humans without foreign helper epitopes. J Clin Invest. 2000;105:R9–R14. [PMC free article] [PubMed]
116. Thurner B, Haendle I, Roder C, et al. Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med. 1999;190:1669–1678. [PMC free article] [PubMed]
117. Banchereau J, Fay J, Pascual V, et al. Dendritic cells: controllers of the immune system and a new promise for immunotherapy. Novartis Found Symp. 2003;252:226–235. discussion 35–8, 57–67. [PubMed]
118. Butterfield LH, Comin-Anduix B, Vujanovic L, et al. Adenovirus MART-1-engineered autologous dendritic cell vaccine for metastatic melanoma. J Immunother. 2008;31:294–309. [PMC free article] [PubMed]
119. Engell-Noerregaard L, Hansen TH, Andersen MH, et al. Review of clinical studies on dendritic cell-based vaccination of patients with malignant melanoma: assessment of correlation between clinical response and vaccine parameters. Cancer Immunol Immunother. 2009;58:1–14. [PubMed]
120. Rosenberg SA, Sherry RM, Morton KE, et al. Tumor progression can occur despite the induction of very high levels of self/tumor antigen-specific CD8+ T cells in patients with melanoma. J Immunol. 2005;175:6169–6176. [PubMed]
121. Tuettenberg A, Becker C, Huter E, et al. Induction of strong and persistent MelanA/MART-1-specific immune responses by adjuvant dendritic cell-based vaccination of stage II melanoma patients. Int J Cancer. 2006;118:2617–2627. [PubMed]
122. Schadendorf D, Ugurel S, Schuler-Thurner B, et al. Dacarbazine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) in first-line treatment of patients with metastatic melanoma: a randomized phase III trial of the DC study group of the DeCOG. Ann Oncol. 2006;17:563–570. [PubMed]
123. Jackson AM, Mulcahy LA, Zhu XW, et al. Tumour-mediated disruption of dendritic cell function: inhibiting the MEK1/2-p44/42 axis restores IL-12 production and Th1-generation. Int J Cancer. 2008;123:623–632. [PubMed]
124. Belli F, Testori A, Rivoltini L, et al. Vaccination of metastatic melanoma patients with autologous tumor-derived heat shock protein gp96-peptide complexes: clinical and immunologic findings. J Clin Oncol. 2002;20:4169–4180. [PubMed]
125. Parmiani G. Phase II study of HSPPC-96 in combination with GM-CSF and IFN-a in stage IV malignant melanoma. Proc Am Soc Clin Oncology. 2004
126. Testori A, Richards J, Whitman E, et al. Phase III comparison of vitespen, an autologous tumor-derived heat shock protein gp96 peptide complex vaccine, with physician's choice of treatment for stage IV melanoma: the C-100-21 Study Group. J Clin Oncol. 2008;26:955–962. [PubMed]
127. Chang CC, Ogino T, Mullins DW, et al. Defective human leukocyte antigen class I-associated antigen presentation caused by a novel beta2-microglobulin loss-of-function in melanoma cells. J Biol Chem. 2006;281:18763–18773. [PubMed]
128. Chang CC, Campoli M, Restifo NP, et al. Immune selection of hot-spot beta 2-microglobulin gene mutations, HLA-A2 allospecificity loss, and antigen-processing machinery component down-regulation in melanoma cells derived from recurrent metastases following immunotherapy. J Immunol. 2005;174:1462–1471. [PMC free article] [PubMed]
129. Dissemond J, Gotte P, Mors J, et al. Association of TAP1 downregulation in human primary melanoma lesions with lack of spontaneous regression. Melanoma Res. 2003;13:253–258. [PubMed]
130. Spaner DE. Amplifying cancer vaccine responses by modifying pathogenic gene programs in tumor cells. J Leukoc Biol. 2004;76:338–351. [PubMed]
131. Khan AN, Gregorie CJ, Tomasi TB. Histone deacetylase inhibitors induce TAP, LMP, Tapasin genes and MHC class I antigen presentation by melanoma cells. Cancer Immunol Immunother. 2008;57:647–654. [PMC free article] [PubMed]
132. Andre F, Schartz NE, Movassagh M, et al. Malignant effusions and immunogenic tumour-derived exosomes. Lancet. 2002;360:295–305. [PubMed]
133. Mitchell PJ, Welton J, Staffurth J, et al. Can urinary exosomes act as treatment response markers in prostate cancer? J Transl Med. 2009;7:4. [PMC free article] [PubMed]
134. Riteau B, Faure F, Menier C, et al. Exosomes bearing HLAG are released by melanoma cells. Hum Immunol. 2003;64:1064–1072. [PubMed]
135. Valenti R, Huber V, Iero M, et al. Tumor-released micro-vesicles as vehicles of immunosuppression. Cancer Res. 2007;67:2912–2915. [PubMed]
136. Yu S, Liu C, Su K, et al. Tumor exosomes inhibit differentiation of bone marrow dendritic cells. J Immunol. 2007;178:6867–6875. [PubMed]
137. Ichim TE, Zhong Z, Kaushal S, et al. Exosomes as a tumor immune escape mechanism: possible therapeutic implications. J Transl Med. 2008;6:37. [PMC free article] [PubMed]
138. Brown JA, Dorfman DM, Ma FR, et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol. 2003;170:1257–1266. [PubMed]
139. Alegre ML, Shiels H, Thompson CB, et al. Expression and function of CTLA-4 in Th1 and Th2 cells. J Immunol. 1998;161:3347–3356. [PubMed]
140. Munn DH, Sharma MD, Mellor AL. Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J Immunol. 2004;172:4100–4110. [PubMed]
141. Hurwitz AA, Foster BA, Kwon ED, et al. Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade. Cancer Res. 2000;60:2444–2448. [PubMed]
142. Kwon ED, Foster BA, Hurwitz AA, et al. Elimination of residual metastatic prostate cancer after surgery and adjunctive cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade immunotherapy. Proc Natl Acad Sci U S A. 1999;96:15074–15079. [PubMed]
143. Van Elsas A, Hurwitz AA, Allison JP. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med. 1999;190:355–366. [PMC free article] [PubMed]
144. Fang L, Lonsdorf AS, Hwang ST. Immunotherapy for advanced melanoma. J Invest Dermatol. 2008;128:2596–2605. [PubMed]
145. Downey SG, Klapper JA, Smith FO, et al. Prognostic factors related to clinical response in patients with metastatic melanoma treated by CTL-associated antigen-4 blockade. Clin Cancer Res. 2007;13(22 Pt 1):6681–6688. [PMC free article] [PubMed]
146. Phan GQ, Yang JC, Sherry RM, et al. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci U S A. 2003;100:8372–8377. [PubMed]
147. Fischkoff SA, Hersh E, Weber J, et al. Durable responses and long-term progression-free survival observed in a phase II study of MDX-010 alone or in combination with dacarbazine (DTIC) in metastatic melanoma. Proc Am Soc Clin Oncology. 2005;23:16S. Abstr. 7525.
148. Maker AV, Phan GQ, Attia P, et al. Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: a phase I/II study. Ann Surg Oncol. 2005;12:1005–1016. [PMC free article] [PubMed]
149. Attia P, Phan GQ, Maker AV, et al. Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4. J Clin Oncol. 2005;23:6043–6053. [PMC free article] [PubMed]
150. Weber JS, Hersh E, Yellin M, et al. The efficacy and safety of ipilimumab (MDX-010) in patients with unresectable stage III or stage IV malignant melanoma. Journal of Clinical Oncology. 2007;25(18S (June 20 Supplement)):8523. 2007 ASCO Annual Meeting Proceedings Part I.
151. Ribas A, Camacho LH, Lopez-Berestein G, et al. Antitumor activity in melanoma and anti-self responses in a phase I trial with the anti-cytotoxic T lymphocyte-associated antigen 4 monoclonal antibody CP-675, 206. J Clin Oncol. 2005;23:8968–8977. [PubMed]
152. Ribas A, Antonia S, Sosman J, et al. Results of a phase II clinical trial of 2 doses and schedules of CP-675, 206, an anti-CTLA4 monoclonal antibody, in patients (pts) with advanced melanoma. J Clin Oncol. 2008;26:1011s.
153. Kirkwood JM, Lorigan P, Hersey P, et al. A phase II trial of tremelimumab (CP-675,206) in patients with advanced refractory or relapsed melanoma. Clin Cancer Res. 2010;16:1042–1048. [PubMed]
154. Hamid O, Urba WJ, Yellin M, et al. Kinetics of response to ipilimumab (MDX-010) in patients with stage III/IV melanoma. Journal of Clinical Oncology. 2007;25(18S (June 20 Supplement)):8525. 2007 ASCO Annual Meeting Proceedings Part I.
155. Sanderson K, Scotland R, Lee P, et al. Autoimmunity in a phase I trial of a fully human anti-cytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma. J Clin Oncol. 2005;23:741–750. [PubMed]
156. Comin-Anduix B, Lee Y, Jalil J, et al. Detailed analysis of immunologic effects of the cytotoxic T lymphocyte-associated antigen 4-blocking monoclonal antibody tremelimumab in peripheral blood of patients with melanoma. J Transl Med. 2008;6:22–36. [PMC free article] [PubMed]
157. Menard C, Ghiringhelli F, Roux S, et al. Ctla-4 blockade confers lymphocyte resistance to regulatory T-cells in advanced melanoma: surrogate marker of efficacy of tremelimumab? Clin Cancer Res. 2008;14:5242–5249. [PubMed]
158. Richmond J, Gao F, Wood L, et al. Pharmacogenetic analysis of CTLA4 gene polymorphisms and response to tremelimumab in patients with advanced melanoma. Journal of Clinical Oncology. 2008 Presented at: 44th annual meeting of the American Society of Clinical Oncology. Abstract 14003.
159. Breunis WB, Tarazona-Santos E, Chen R, et al. Influence of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) common polymorphisms on outcome in treatment of melanoma patients with CTLA-4 blockade. J Immunother. 2008;31:586–590. [PMC free article] [PubMed]
160. Ohigashi Y, Sho M, Yamada Y, et al. Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2 expression in human esophageal cancer. Clin Cancer Res. 2005;11:2947–2953. [PubMed]
161. Iwai Y, Ishida M, Tanaka Y, et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A. 2002;99:12293–12297. [PubMed]
162. Blank C, Kuball J, Voelkl S, et al. Blockade of PD-L1 (B7-H1) augments human tumor-specific T cell responses in vitro. Int J Cancer. 2006;119:317–327. [PubMed]
163. O'Sullivan B, Thomas R. CD40 and dendritic cell function. Crit Rev Immunol. 2003;23:83–107. [PubMed]
164. Sotomayor EM, Borrello I, Tubb E, et al. Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40. Nat Med. 1999;5:780–787. [PubMed]
165. Hunter TB, Alsarraj M, Gladue RP, et al. An agonist antibody specific for CD40 induces dendritic cell maturation and promotes autologous anti-tumour T-cell responses in an in vitro mixed autologous tumour cell/lymph node cell model. Scand J Immunol. 2007;65:479–486. [PubMed]
166. Vonderheide RH, Flaherty KT, Khalil M, et al. Clinical activity and immune modulation in cancer patients treated with CP-870, 893, a novel CD40 agonist monoclonal antibody. J Clin Oncol. 2007;25:876–883. [PubMed]
167. Kruit WH, van Ojik H, Portielje J, et al. Phase I/II study with CpG 7909 as adjuvant to vaccination with MAGE-3 protein in patients with MAGE-3 positive tumors. Journal of Clinical Oncology. Proc Am Soc Clin Oncol. 2002;21 abstr 1854.
168. Wagner SN, Pashenkov M, Goess G, et al. TLR-9-targeted CpG immunostimulatory treatment of metastatic melanoma: a phase II trial with CpG 7909 (promune) Journal of Clinical Oncology. Proc Am Soc Clin Oncology. 2004;22:14S. Abstr. 7513.
169. Wagner S, Weber J, Redman B, et al. CPG 7909, a TLR9 Agonist Immunomodulator in Metastatic Melanoma: A Randomized Phase II Trial Comparing Two Doses and in Combination with DTIC. Journal of Clinical Oncology. 2005;23:16S. ASCO Annual Meeting Proceedings. Part I of II. 7526.
170. Pashenkov M, Goess G, Wagner C, et al. Phase II trial of a toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma. J Clin Oncol. 2006;24:5716–5724. [PubMed]
171. Hofmann MA, Kors C, Audring H, et al. Phase 1 evaluation of intralesionally injected TLR9-agonist PF-3512676 in patients with basal cell carcinoma or metastatic melanoma. J Immunother. 2008;31:520–527. [PubMed]
172. Lehner M, Morhart P, Stilper A, et al. Efficient chemokine-dependent migration and primary and secondary IL-12 secretion by human dendritic cells stimulated through Toll-like receptors. J Immunother. 2007;30:312–322. [PubMed]
173. Birmachu W, Gleason RM, Bulbulian BJ, et al. Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists. BMC Immunol. 2007;8:26. [PMC free article] [PubMed]
174. Stary G, Bangert C, Tauber M, et al. Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells. J Exp Med. 2007;204:1441–1451. [PMC free article] [PubMed]
175. Ramakrishna V, Vasilakos JP, Tario JD, et al. Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells. J Transl Med. 2007;5:5. [PMC free article] [PubMed]
176. Li VW, Li WW, Talcott KE, et al. Imiquimod as an antiangiogenic agent. J Drugs Dermatol. 2005;4:708–717. [PubMed]
177. Shackleton M, Davis ID, Hopkins W, et al. The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptide vaccination with adjuvant Flt3 ligand. Cancer Immun. 2004;4:9. [PubMed]
178. Steinmann A, Funk JO, Schuler G, et al. Topical imiquimod treatment of a cutaneous melanoma metastasis. J Am Acad Dermatol. 2000;43:555–556. [PubMed]
179. Shistik G, Prakash AV, Fenske NA, et al. Treatment of locally metastatic melanoma: a novel approach. J Drugs Dermatol. 2007;6:830–832. [PubMed]
180. Suzuki H, Wang B, Shivji GM, et al. Imiquimod, a topical immune response modifier, induces migration of Langerhans cells. J Invest Dermatol. 2000;114:135–141. [PubMed]
181. Burns RP, Ferbel B, Tomai M, et al. The imidazoquinolines, imiquimod and R-848, induce functional, but not phenotypic, maturation of human epidermal Langerhans' cells. Clin Immunol. 2000;94:13–23. [PubMed]
182. Schon MP, Wienrich BG, Drewniok C, et al. Death receptor-independent apoptosis in malignant melanoma induced by the small-molecule immune response modifier imiquimod. J Invest Dermatol. 2004;122:1266–1276. [PubMed]
183. Baumgartner JM, Gonzalez R, Lewis KD, et al. Increased survival from stage IV melanoma associated with fewer regulatory T cells. J Surg Res. 2008 [PubMed]
184. Fehervari Z, Sakaguchi S. CD4+ Tregs and immune control. J Clin Invest. 2004;114:1209–1217. [PMC free article] [PubMed]
185. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003;4:330–336. [PubMed]
186. Miyara M, Sakaguchi S. Natural regulatory T cells: mechanisms of suppression. Trends Mol Med. 2007;13:108–116. [PubMed]
187. Bopp T, Becker C, Klein M, et al. Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression. J Exp Med. 2007;204:1303–1310. [PMC free article] [PubMed]
188. Kim-Schulze S, Kim HS, Fan Q, et al. Local IL-21 promotes the therapeutic activity of effector T cells by decreasing regulatory T cells within the tumor microenvironment. Mol Ther. 2009;17:380–388. [PubMed]
189. Davis ID, Brady B, Kefford R, et al. Activity of recombinant human interleukin-21 (rIL-21) in patients (pts) with stage IV malignant melanoma (MM) without prior treatment: clinical data from a phase IIa study. J Clin Oncol. 2008;26
190. Schmidt H, Selby P, Mouritzen U, et al. Subcutaneous (SC) dosing of recombinant human interleukin-21 (rIL-21) is safe and has clinical activity: results from a dose-escalation study in stage 4 melanoma (MM) or renal cell cancer (RCC) J Clin Oncol. 2008 May 20;26(suppl) abstr 3041.
191. Nair S, Boczkowski D, Fassnacht M, et al. Vaccination against the forkhead family transcription factor Foxp3 enhances tumor immunity. Cancer Res. 2007;67:371–380. [PubMed]
192. Kong LY, Wei J, Sharma AK, et al. A novel phosphorylated STAT3 inhibitor enhances T cell cytotoxicity against melanoma through inhibition of regulatory T cells. Cancer Immunol Immunother. 2008 [PMC free article] [PubMed]
193. Kavanagh B, O'Brien S, Lee D, et al. CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion. Blood. 2008;112:1175–1183. [PubMed]
194. Van der Vliet HJ, Koon HB, Yue SC, et al. Effects of the administration of high-dose interleukin-2 on immunoregulatory cell subsets in patients with advanced melanoma and renal cell cancer. Clin Cancer Res. 2007;13:2100–2108. [PubMed]
195. Cesana GC, DeRaffele G, Cohen S, et al. Characterization of CD4+CD25+ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol. 2006;24:1169–1177. [PubMed]
196. Zhou G, Drake CG, Levitsky HI. Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines. Blood. 2006;107:628–636. [PubMed]
197. Ahmadzadeh M, Rosenberg SA. IL-2 administration increases CD4+ CD25(hi) Foxp3+ regulatory T cells in cancer patients. Blood. 2006;107:2409–2414. [PubMed]
198. Powell DJ, Attia P, Ghetie V, et al. Partial reduction of human FOXP3+ CD4 T cells in vivo after CD25-directed recombinant immunotoxin administration. J Immunother. 2008;31:189–198. [PMC free article] [PubMed]
199. Powell DJ, Felipe-Silva A, Merino MJ, et al. Administration of a CD25-directed immunotoxin, LMB-2, to patients with metastatic melanoma induces a selective partial reduction in regulatory T cells in vivo. J Immunol. 2007;179:4919–4928. [PMC free article] [PubMed]
200. Scott AM, Lee FT, Hopkins W, et al. Specific targeting, biodistribution, and lack of immunogenicity of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma: results of a phase I trial. J Clin Oncol. 2001;19:3976–3987. [PubMed]
201. Scott AM, Liu Z, Murone C, et al. Immunological effects of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma. Cancer Immun. 2005;5:3. [PubMed]
202. Becker JC, Pancook JD, Gillies SD, et al. T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy. J Exp Med. 1996;183:2361–2366. [PMC free article] [PubMed]
203. Choi BS, Sondel PM, Hank JA, et al. Phase I trial of combined treatment with ch14.18 and R24 monoclonal antibodies and interleukin-2 for patients with melanoma or sarcoma. Cancer Immunol Immunother. 2006;55:761–774. [PubMed]
204. Cheng JD, Adams GP, Robinson MK, et al. Chapter 16: Pharmacology of Cancer Biotherapeutics: Section 4: Monoclonal Antibodies. In: De Vita VT Jr, Hellman S, Rosenberg SA, et al., editors. Biologic Therapy of Cancer Principles and Practice. 7th ed. JB Lippincot; Philadelphia: 2003. p. 576.
205. Brasoveanu LI, Altomonte M, Fonsatti E, et al. Levels of cell membrane CD59 regulate the extent of complement-mediated lysis of human melanoma cells. Lab Invest. 1996;74:33–42. [PubMed]
206. Parkinson DR. Levamisole as adjuvant therapy for melanoma: quo vadis? J Clin Oncol. 1991;9:716–717. [PubMed]
207. Lipton A, Harvey HA, Balch CM, et al. Corynebacterium parvum versus bacille Calmette-Guerin adjuvant immunotherapy of stage II malignant melanoma. J Clin Oncol. 1991;9:1151–1156. [PubMed]
208. Kirkwood JM, Manola J, Ibrahim J, et al. A pooled analysis of eastern cooperative oncology group and intergroup trials of adjuvant high-dose interferon for melanoma. Clin Cancer Res. 2004;10:1670–1677. [PubMed]
209. Lens MB, Dawes M. Interferon alfa therapy for malignant melanoma: a systematic review of randomized controlled trials. J Clin Oncol. 2002;20:1818–1825. [PubMed]
210. Gogas H, Ioannovich J, Dafni U, et al. Prognostic significance of autoimmunity during treatment of melanoma with interferon. N Engl J Med. 2006;354:709–718. [PubMed]
211. Eggermont AM. The role interferon-alpha in malignant melanoma remains to be defined. Eur J Cancer. 2001;37:2147–2153. [PubMed]
212. Cascinelli N, Belli F, MacKie RM, et al. Effect of long-term adjuvant therapy with interferon alpha-2a in patients with regional node metastases from cutaneous melanoma: a randomised trial. Lancet. 2001;358:866–869. [PubMed]
213. Hancock BW, Wheatley K, Harris S, et al. Adjuvant interferon in high-risk melanoma: the AIM HIGH Study—United Kingdom Coordinating Committee on Cancer Research randomized study of adjuvant low-dose extended-duration interferon Alfa-2a in high-risk resected malignant melanoma. J Clin Oncol. 2004;22:53–61. [PubMed]
214. Pectasides D, Dafni U, Bafaloukos D, et al. Randomized phase III study of 1 month versus 1 year of adjuvant high-dose interferon alfa-2b in patients with resected high-risk melanoma. J Clin Oncol. 2009;27:939–944. [PubMed]
215. Von Rohr A, Ghosh AK, Thatcher N, et al. Immunomodulation during prolonged treatment with combined interleukin-2 and interferon-alpha in patients with advanced malignancy. Br J Cancer. 1993;67:163–171. [PMC free article] [PubMed]
216. Hauschild A, Weichenthal M, Balda BR, et al. Prospective randomized trial of interferon alfa-2b and interleukin-2 as adjuvant treatment for resected intermediate- and high-risk primary melanoma without clinically detectable node metastasis. J Clin Oncol. 2003;21:2883–2888. [PubMed]
217. Stadler R, Luger T. Long term survival benefit after adjuvant treatment of high risk cutaneous melanoma with dacarbazine and low dose natural interferon alpha: a controlled, randomized, multicentre trial. Journal of Clinical Oncology. Proc Am Soc Clin Oncology. 2005 Abstr. 7516. [PubMed]
218. Bottomley A, Coens C, Suciu S, et al. Adjuvant therapy with pegylated interferon alfa-2b versus observation in resected stage III melanoma: a phase III randomized controlled trial of health-related quality of life and symptoms by the European Organisation for Research and Treatment of Cancer Melanoma Group. J Clin Oncol. 2009;27:2916–2923. [PubMed]
219. Eggermont AM, Bouwhuis MG, Kruit WH, et al. Serum concentrations of pegylated interferon alpha-2b in patients with resected stage III melanoma receiving adjuvant pegylated interferon alpha-2b in a randomized phase III trial (EORTC 18991) Cancer Chemother Pharmacol. 2010;65:671–677. [PubMed]
220. Moschos SJ, Edington HD, Land SR, et al. Neoadjuvant treatment of regional stage IIIB melanoma with high-dose interferon alfa-2b induces objective tumor regression in association with modulation of tumor infiltrating host cellular immune responses. J Clin Oncol. 2006;24:3164–3171. [PubMed]
221. Buzaid AC, Colome M, Bedikian A, et al. Phase II study of neoadjuvant concurrent biochemotherapy in melanoma patients with local-regional metastases. Melanoma Res. 1998;8:549–556. [PubMed]
222. Mitchell MS, Abrams J, Kashani-Sabet M, et al. Interim analysis of a phase III stratified randomized trial of Melacine +low-dose Intron-A versus high-dose Intron-A for resected stage III melanoma. Journal of Clinical Oncology. Proc Am Soc Clin Oncology. 2003;22 Abstr. 2851.
223. Zimmerer JM, Lesinski GB, Ruppert AS, et al. Gene expression profiling reveals similarities between the in vitro and in vivo responses of immune effector cells to IFN-alpha. Clin Cancer Res. 2008;14:5900–5906. [PMC free article] [PubMed]
224. Mitchell MS. Perspective on allogeneic melanoma lysates in active specific immunotherapy. Semin Oncol. 1998;25:623–635. [PubMed]
225. Morton DL, Hoon DS, Nizze JA, et al. Polyvalent melanoma vaccine improves survival of patients with metastatic melanoma. Ann N Y Acad Sci. 1993;690:120–134. [PubMed]
226. Lotem M, Peretz T, Drize O, et al. Autologous cell vaccine as a post operative adjuvant treatment for high-risk melanoma patients (AJCC stages III and IV). The new American Joint Committee on Cancer. Br J Cancer. 2002;86:1534–1539. [PMC free article] [PubMed]
227. Bystryn JC. Clinical activity of a polyvalent melanoma antigen vaccine. Recent Results Cancer Res. 1995;139:337–348. [PubMed]
228. Sondak VK, Liu PY, Tuthill RJ, et al. Adjuvant immunotherapy of resected, intermediate-thickness, node-negative melanoma with an allogeneic tumor vaccine: overall results of a randomized trial of the Southwest Oncology Group. J Clin Oncol. 2002;20:2058–2066. [PubMed]
229. Sondak VK, Sosman J, Unger JM, et al. Significant impact of HLA class I allele expression on outcome in melanoma patients treated with an allogeneic melanoma cell lysate vaccine. Final analysis of SWOG-9035. Journal of Clinical Oncology. Proc Am Soc Clin Oncology. 2004;22:14S. Abstr. 7501.
230. Barth A, Morton DL. The role of adjuvant therapy in melanoma management. Cancer. 1995;75(2 suppl):726–734. [PubMed]
231. Morton DL, Hsueh EC, Essner R, et al. Prolonged survival of patients receiving active immunotherapy with Canvaxin therapeutic polyvalent vaccine after complete resection of melanoma metastatic to regional lymph nodes. Ann Surg. 2002;236:438–448. discussion 448–449. [PubMed]
232. Hsueh EC, Essner R, Foshag LJ, et al. Prolonged survival after complete resection of disseminated melanoma and active immunotherapy with a therapeutic cancer vaccine. J Clin Oncol. 2002;20:4549–4554. [PubMed]
233. Morton DL, Mozzillo N, Thompson JF, et al. An international, randomized, phase III trial of bacillus Calmette-Guerin (BCG) plus allogeneic melanoma vaccine (MCV) or placebo after complete resection of melanoma metastatic to regional or distant sites. Journal of Clinical Oncology. Proc ASCO. 2007;25:18S. Abstract 8508.
234. Bystryn JC, Zeleniuch-Jacquotte A, Oratz R, et al. Double-blind trial of a polyvalent, shed-antigen, melanoma vaccine. Clin Cancer Res. 2001;7:1882–1887. [PubMed]
235. Livingston PO, Natoli EJ, Calves MJ, et al. Vaccines containing purified GM2 ganglioside elicit GM2 antibodies in melanoma patients. Proc Natl Acad Sci U S A. 1987;84:2911–2915. [PubMed]
236. Livingston PO, Wong GY, Adluri S, et al. Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglio-side. J Clin Oncol. 1994;12:1036–1044. [PubMed]
237. Kirkwood JM, Ibrahim JG, Sosman JA, et al. High-dose interferon alfa-2b significantly prolongs relapse-free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with resected stage IIB-III melanoma: results of intergroup trial E1694/S9512/C509801. J Clin Oncol. 2001;19:2370–2380. [PubMed]
238. Eggermont A, Suciu S, Ruka W, et al. EORTC 18961: Postoperative adjuvant ganglioside GM2-KLH21 vaccination treatment versus observation in stage II (T3-T4N0M0) melanoma: 2nd interim analysis led to an early disclosure of the results (abstract) J Clin Oncol. 2008;26:484s.
239. Slingluff CL, Petroni G, Bullock KA, et al. Immunological results of a phase II randomized trial of multipeptide vaccines for melanoma. Proc Am Soc Clin Oncology. 2004;22:14S. Abstr. 7503.
240. McCaffery M, Yao TJ, Williams L, et al. Immunization of melanoma patients with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: enhanced immunogenicity when combined with adjuvant. Clin Cancer Res. 1996;2:679–686. [PubMed]
241. Molenkamp BG, Sluijter BJ, Van Leeuwen PA, et al. Local administration of PF-3512676 CpG-B instigates tumor-specific CD8+ T-cell reactivity in melanoma patients. Clin Cancer Res. 2008;14:4532–4542. [PubMed]
242. Molenkamp BG, Van Leeuwen PA, Meijer S, et al. Intradermal CpG-B activates both plasmacytoid and myeloid dendritic cells in the sentinel lymph node of melanoma patients. Clin Cancer Res. 2007;13:2961–2969. [PubMed]
243. Spitler LE, Grossbard ML, Ernstoff MS, et al. Adjuvant therapy of stage III and IV malignant melanoma using granulocyte-macrophage colony-stimulating factor. J Clin Oncol. 2000;18:1614–1621. [PubMed]
244. Elias EG, Zapas JL, Beam SL, et al. Granulocyte-macro-phage colony stimulating factor (GM-CSF) & interleukin-2 (IL-2) combination as adjuvant therapy in cutaneous melanoma. Early results of a phase II clinical trial. Proc Am Soc Clin Oncology. 2004;22:14S. Abstr. 7559.
245. Butterfield LH, Disis ML, Fox BA, et al. A systematic approach to biomarker discovery; preamble to “the iSBTc-FDA taskforce on immunotherapy biomarkers” J Transl Med. 2008;6:81. [PMC free article] [PubMed]