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Medicine » Oncology » "Treatment of Metastatic Melanoma", book edited by Rachael Morton, ISBN 978-953-307-574-7, Published: October 5, 2011 under CC BY 3.0 license. © The Author(s).

Chapter 6

Chemocentric Chemoimmunotherapy: A New Concept in Melanoma Immunotherapy

By Paul Howell, Soroosh Radfar, Yixiang Wang and Hung Khong
DOI: 10.5772/20018

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Chemocentric Chemoimmunotherapy: A New Concept in Melanoma Immunotherapy

Paul Howell1, Soroosh Radfar3, Yixiang Wang1 and Hung Khong1

1. Introduction

According to the American Cancer Society, 68,130 new cases of melanoma and 8,700 deaths were predicted for 2010 (Jemal et al., 2010). Although melanoma comprises only 5% of all skin cancers, it is by far the deadliest, responsible for 75% of skin cancer-related deaths, and its incidence is steadily increasing worldwide. Primary cutaneous melanoma has an excellent outcome with early surgical intervention, but regional and distant metastatic disease have a much more dismal prognosis. Current five-year survival rates equal 91% for all disease sites, including 98% for local disease, 62% for regional disease (including regional lymph node involvement), and only 15% for those with distant metastasis (median survival is only 7.5 months). Unfortunately, for those patients who will develop locally advanced and metastatic melanoma, current treatment options are limited in scope and effectiveness; thus, the overall poor mortality rates for these patients emphasize the need for better treatment modalities, methods of relapse prevention and earlier recognition of pre-neoplastic and neoplastic cells (Atallah & Flaherty, 2006; Balch et al., 2001a, Balch et al., 2001b; Blesa et al., 2011).

Currently, there are no curative therapies for metastatic melanoma. Until March 25, 2011, the U.S. Food and Drug Administration (FDA) had only approved two drugs for the treatment of metastatic melanoma, the DNA alkylating agent dacarbazine and high-dose Interleukin-2 (IL-2). Dacarbazine alkylates and crosslinks DNA in all phases of the cell cycle, impairing DNA function, inducing cell-cycle arrest and apoptosis (Bajetta et al., 2002), and is a common chemotherapeutic option for multiple cancers (Seam et al., 2009; Yang & Chapman, 2009). IL-2 activates endogenous anti-tumor-reactive T cells and natural killer (NK) cells. Notably, circulating NK cells from stage IV melanoma patients were recently shown to exhibit a unique phenotypic and functional character toward melanoma cells, and this unique character was altered in patients after treatment with chemotherapy (Fregni et al., 2011). Dacarbazine induces objective tumor regression in 15-20% of metastatic melanoma patients with a median survival of only 8-9 months and a 2-3% complete response rate, and single agent bolus IL-2, while providing a 20-30% objective response rate, creates significant long-term response in only 5% of patients treated, is fraught with toxicity and costly. Combination low-dose IL-2 and chemotherapy and single-agent high-dose IL-2 produce a similarly low complete response (Atkins et al., 2000; Tsao et al., 2004). Other cytotoxic compounds have resulted in poor response rates and significant adverse effects, including temozolomide, cisplatin, carboplatin, vinca alkaloids, taxanes, and nitrosoureas (Bafaloukos et al., 2002; Bajetta et al., 2002; Blesa et al., 2011; Khayat et al., 2002; Middleton et al., 2000). Notable agents of promise currently undergoing testing are anti-CD40, anti-PD-1L, 1-MT (1-methyl-D-tryptophan), and the BRAF-inhibitor PLX4032 (Bollag et al., 2010; Comin-Anduix; Flaherty et al., 2010; Fonsatti et al., 2010). On March 25, 2011, the U. S. Food and Drug Administration approved ipilimumab (Yervoy™, Bristol-Myers Squibb Company, New York, NY, USA), a monoclonal antibody against the inhibitory lymphocyte receptor, CTLA4, as an injection for the treatment of unresectable or metastatic melanoma (Hodi et al., 2010).

Advanced melanomas are infamously resistant to chemotherapy, principally due to the presence of inherent primary anti-apoptotic and acquired cellular chemoresistance mechanisms (Serrone & Hersey, 1999). Of note, a recent study by Rosner et al. (Rosner et al., 2011) described a means to circumvent such apoptosis resistance. Without activating the apoptosis signaling cascade or utilizing other apoptotic nucleases, recombinant deoxyribonuclease I (DNaseI), engineered with a nuclear localization signal and mutated actin-binding site to avoid entering its inactive bound form, was effective in inducing apoptosis in an apoptosis-resistant melanoma cell model with 70-100% killing efficiency, emphasizing its potential application in many of the known apoptosis-resistant cancers. Surgical resection of primary and known metastatic lesions is still the most effective means of improving overall survival of melanoma patients, though palliative care is standard for patients with metastases after first-line dacarbazine or other physician-preferred therapy, as survival is very rare (Rass et al., 2008; Tagawa et al., 2006; Yang et al., 2006). Surgery in partnership with radiation is standard for locoregional control, while additional chemical-, cell- or antibody- based therapies, or some combination thereof, are commonly attempted for systemic eradication of tumor cells in the blood or at unspecified sites in the body to combat current disease and prevent relapse and metastatic implantation. Melanoma has attracted a lot of interest from immunologists due to a heightened frequency over other solid tumors to display fast, spontaneous, complete tumor regression associated with a specific cellular immune response (Kadison & Morton, 2003). Chemoimmunotherapy, tumor vaccines and other chemotherapeutic combinations have shown improvement in objective response rates for melanoma patients, but they have not yet been able to show significant survival benefits (Bajetta et al., 2006; Eton et al., 2002; Kaufmann et al., 2005). Indeed, a recent meta-analysis of 18 studies comprising 2,625 participants revealed improved objective response rates in metastatic melanoma patients treated with chemoimmunotherapy compared with chemotherapy, but no improvement was noted in overall survival benefit (Sasse et al., 2007). Additionally, hematological and non-hematological toxicities were greatest in those undergoing chemoimmunotherapy.

While the historic chemoimmunotherapeutic approach has been to sensitize tumor cells to immunotherapy with administration of chemotherapeutic compounds, we have described a high degree of chemosensitization with pre-treatment of non-tumor-Ag-specific CD4+ T cells (Radfar et al., 2009), our termed ‘chemocentric chemoimmunotherapy’ strategy. This technique may provide a basis for development of novel methods of selectively reducing the chemoresistance of virtually all tumor cell types, given the lack of requirement for tumor-Ag-specific T cells. We will present an overview of conventional chemoimmunotherapy development for melanoma and examine the functional significance of chemocentric chemoimmunotherapy for melanoma and other cancers.

2. Background

It has long been part of the clinical canon that chemotherapy drugs negatively affect immunological systems, inhibiting anti-tumor immune activity by a variety of related mechanisms. However, the intricacies of the chemo-immuno interaction provide areas of opportunity for utilizing chemo drugs to enhance the immune response. These cytotoxic drugs tend to target dividing lymphocytes, necessary for development of an immune response, but they also deplete CD4+/CD25+ regulatory T cells (T reg), potentially enhancing the response. Again, conversely, lymphodepletion triggers homeostatic T cell reconstitution which proceeds to create T cells recognizing tumor as self. Opportunities arise during the depletion of T reg and the heightened formation of T cells, allowing an avenue to both infuse the patient with tumor-reactive T cells which are now not impeded by T reg and the potential to create within the patient a large quantity of T cells reactive to some tumor antigen(s).

Conventional cancer chemotherapy is capable of inducing the death of immunologically sensitive tumor cells by utilizing the host innate and adaptive immune responses, e.g. through the induction of immunoregulatory cytokines (Bracci et al., 2007), and some of these drugs can alter cellular pathways of immune suppression and tolerance in a dose- and time-dependent manner, e.g. inducing homeostatic proliferation (Bracci et al., 2007; Proietti et al., 1998), or modulate the extracellular release of certain proteins upon cell death, e.g. HMGB1 release and subsequent tumor survival/metastasis-promoting inflammation is inhibited by oxaliplatin (Dong et al., 2007). Additionally, some chemo drugs can modulate the expression of tumor antigens and antigen processing/presentation machinery on tumor cells. For example, 5-Fluorouracil (5-FU) has been shown to induce carcinoembryonic antigen (CEA) expression in colon and breast cancer cells (Correale et al., 2003) while 5-aza-2’-deoxycytidine can induce the expression of cancer testis antigens and the cell surface MHC class I complex in melanoma and other cancers (Adair & Hogan, 2009; Coral et al., 2002; Fonsatti et al., 2007; Natsume et al., 2008). Notably, direct intralesional injection of the MHC class I complex, which allows tumor immune evasion when defective in numerous cancers (Lampen & van Hall, 2011; Maleno et al., 2011), via high-dose Allovectin-7® (Vical Inc., San Diego, CA, USA), a cationic lipid-formulated bicistronic plasmid encoding MHC-I components β-2 microglobulin and HLA-B7, in 127 recurrent or previously unresponsive to chemotherapy stage III and IV melanoma patients tested for efficacy in a recent phase II dose escalation study produced an objective response of 11.8% with median duration of response of 13.8 months (Bedikian et al., 2010). Tissue from two responding patients was noted for the absence of melanoma upon histological analysis, and the therapy was well tolerated.

Drugs of complementary activity and dissimilar toxicities are routinely combined to enhance individual anti-tumor effects. In the 1960s, effective combination antibiotic therapy for tuberculosis influenced the combination treatment strategy of acute lymphocytic leukemia and lymphoma. A number of significant combination chemotherapeutic regimens were developed in the 1980s, including CVD (cisplatin, vinblastine, and dacarbazine) and the Dartmouth regimen (cisplatin, carmustine, dacarbazine, and tamoxifen) (Del Prete et al., 1984; Legha et al., 1989). Notably, according to two independent clinical trials of the 1990s, CVD and Dartmouth were not shown to significantly increase overall survival relative to dacarbazine alone (Buzaid et al., 1993; Chapman et al., 1999). Although dacarbazine and cisplatin-based combination chemotherapy/biochemotherapy regimens combining IFN and/or IL-2 are now recommended by the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology for Melanoma for stage III and IV melanomas, promising increases in response rates notwithstanding (Riker & Howell, 2010a, Riker & Howell 2010b), significant improvement in long-term responses has yet to be seen.

Early efforts to combine chemotherapy with immunomodulators, particularly cancer vaccines, were discounted given the understood detrimental myelosuppressive properties of many chemotherapeutic compounds (Emens, 2008). This suppression was purported to be short-lived and reversible, as the replenishing of leukocytes following chemotherapy would show, by Harris et al. in 1976 (Harris et al., 1976). As reviewed by Sinkovics and Horvath (Sinkovics & Horvath, 2006), around this time, the M.D. Anderson Hospital Melanoma-Sarcoma Service submitted multiple grant applications to the NCI for combination chemoimmunotherapeutic intervention strategies for patients with metastatic melanomas and sarcomas. Despite NCI rejection, clinical trials began, utilizing the findings of Lindenmann and Klein (Lindenmann & Klein, 1967) from nearly a decade earlier which showed an acquired immunity to tumor formation in mice injected with the cell culture lysates of oncolytic myxo- or paramyxovirus-infected tumor cells. Sinkovics et al. further adapted this strategy for the post-chemotherapy administration of cultured tumor cell lysates infected with oncolytic, attenuated PR8 influenza A virus. PR8 viral oncosylates, the immunotherapeutic suspension, were comprised of UV-irradiated, endotoxin-free tumor cell cultures with low levels of live PR8 virus. ‘Tumor-specific active immunization’ with viral oncosylates two-to-three weeks post-chemotherapy resulted in heightened rates of remission with moderate, occasionally significant increases in survival.

Early phase clinical trials have been able to show response rates of up to 50% for chemoimmunotherapies (Atkins et al., 2003; Eton et al., 2002; Keilholz et al., 1997; Rosenberg et al., 1999); however, the duration of responses has been limited in most cases to less than a year. Importantly, systemic toxicities and adverse side effects tend to be heightened with chemoimmunotherapeutic interventions in relation to chemotherapy or immunotherapy alone. Furthermore, many of the most common chemotherapies were not inherently designed to take into account the entire tumor-host microenvironment and the effects of such therapies in conjunction with locally and systemically active immunological mechanisms. Designing treatment course strategy according to the character of the tumor-host microenvironment milieu is vital for its long-term efficacy (Formenti, 2010; Pages et al., 2009; Zitvogel et al., 2008). Today, thirty years after the first modern chemoimmunotherapy clinical trials were initiated, the impasse that is melanoma chemoresistance has continued to stymie significant, clinically-applicable advancement in this therapeutic field. Given the recurring roadblocks of current treatment strategies, traditional methods of chemotherapy and immunotherapy use are being radically adjusted to achieve a better overall clinical outcome.

3. Conventional chemoimmunotherapy for the treatment of melanoma

Immunotherapy can play an important role in the treatment of cancer, and combinations of chemotherapy and immunotherapy have been investigated in many clinical trials. Although some phase III trials have shown mixed results regarding duration of survival, meta-analyses have found no improvement in overall survival (Allen et al., 1998; Ives et al., 2007; Keilholz et al., 1998; Sasse et al., 2007). Chemoimmunotherapy, or biochemotherapy, has been extensively studied in melanoma. Conventional chemoimmunotherapeutic strategies include two major categories. The first category uses cytokines such as IL-2 and/or IFN- in combination with chemotherapy. This experimental strategy was not supported by strong preclinical evidence and has not been shown to be superior to chemotherapy alone (Atkins et al., 2003; Eton et al., 2002; Keilholz et al., 1997, 2005; Rosenberg et al., 1999). The second category is a strategy that we have termed ‘immunocentric chemoimmunotherapy’. In this approach, the primary or central focus is on immunotherapy, with chemotherapy playing a peripheral role in immunomodulation. Using this approach, melanoma patients have been successfully treated with non-myeloablative, lymphodepleting chemotherapy and adoptive cell transfer with tumor-Ag specific lymphocytes and high dose IL-2 (Dudley et al., 2002, 2005). Although this strategy is promising, it is not widely applicable to non-melanoma cancers due to the difficulty in generating large numbers of tumor-Ag specific tumor-infiltrating lymphocytes. To date, there have been no studies using chemoimmunotherapy that have shown a significant improvement in overall survival (Atkins et al., 2003, 2008; Bajetta et al., 2006; Eton et al., 2002; Kielholz et al., 1997, 1998, 2005; Middleton et al., 2007; Punt et al., 2006; Ridolofi et al., 2002; Rosenberg et al., 1999), and the development of immunotherapy for the treatment of a broad range of cancer types is still lacking. Given the proven capacity of many already developed and thoroughly tested, well characterized chemotherapeutic agents to have significant, specific and intricate influence over global and localized immune response, adjustments to the multitudes of chemoimmunotherapeutic strategic fronts are currently being made in anticipation of a clinically relevant breakthrough.

3.1. Cytokines and chemotherapy

Activated T cells amplify cytokines that are essential for an effective immune response (Szabo et al., 2003). These include IL-2, TNF-α, IL-21 (by activated CD4+ T cells), GM-CSF, IFN-γ, and other as yet unidentified cytokines. In addition to their immunomodulatory activities, some cytokines can also have direct effects on tumor cells and/or tumor vasculature. TNF-α is known to induce hemorrhagic necrosis in tumors (Carswell et al., 1975; Old, 1985) and has also been shown to induce apoptotic and necrotic tumor cell death in vitro (Helson et al., 1975; Laster et al., 1988; Rubin et al., 1988; Sugarman et al., 1985). The hemorrhagic necrotic effect of TNF-α has been shown to be more potent when used in combination with chemotherapy in a rat sarcoma model (Manusama et al., 1996). However, in these studies, TNF-α had no direct activity against tumor cell lines in vitro and demonstrated no synergy with chemotherapy, acting instead on the tumor vasculature (van Horssen et al., 2006; Watanabe et al., 1988). The IFNs can exert direct effects on the proliferation, differentiation, and apoptosis of tumor cells (Chawla-Sarkar et al., 2001, 2003; Detjen et al., 2001; Gooch et al, 2000; Pfeffer et al., 1998). However, the response to the IFNs varies considerably, depending on the tumor histology, and resistance to the IFNs has been reported in several tumor types (Burke et al., 1999; Harvat & Jetten, 1996; Kaplan et al., 1998; Phan-Bich et al., 1997).

Several early-phase trials of cytokines combined with cisplatin-based therapies have shown an overall response rate of ~50%, though such responses are of very short duration and often complicated by a high degree of systemic toxicity and adverse side effects. Over the past 15 years, numerous cisplatin-based cytokine combination regimens have been assessed (Atkins et al., 2002, 2003; Atzpodien et al., 2002; Buzaid et al., 1993; Flaherty et al., 2001; Legha et al., 1989, 1998; McDermott et al., 2000; Pejcic et al., 2010; Rosenberg et al., 1999, 2011; Thompson et al., 1997). Notably, in a phase II trial from 1997, Thompson et al. describe the development and utility of an outpatient chemoimmunotherapy treatment regimen of monthly cycles of intravenous cisplatin, carmustine, dacarbazine, and tamoxifen plus self-administered subcutaneous recombinant IL-2 and IFN-α for 32 metastatic melanoma patients, 30 assessed for clinical response (Thompson et al., 1997). After three months, they note a complete response rate of 13% and a 30% partial response, and toxicity was primarily restricted to minor fever and nausea with only a few instances of hospitalization for toxicity management.

Rosenberg et al. (Rosenberg et al., 1999) reported in 1999 on their phase II clinical trial testing tamoxifen, cisplatin, and dacarbazine in 52 metastatic melanoma patients, while the 50 patients in the other arm received the same therapy followed by IL-2 and IFN-2b. The chemoimmunotherapy arm exhibited 44% total response rate while the chemotherapy arm was 27%; however, of the seven complete responders, only three received the cytokines, indicating no significant impact of cytokines on complete response, and median duration of response was less than six months. Even more disappointing, most of the toxicities were reported for the chemoimmunotherapy group. Median duration of survival was 10.7 months for those receiving cytokines compared to 15.8 months for those without. A regimen of concurrent treatment with CVD chemotherapy, IL-2 and low-dose IFN-α has shown to be better tolerated and more practical than sequential treatment of CVD followed by IL-2/IFN-α (Atkins et al., 2002; Eton et al., 2002; Legha et al., 1998), though randomized phase III trials of this CVD/IL-2/IFN-α combination show no improvement in response or survival over CVD and modified CVD chemotherapy regimens alone (Atkins et al., 2003; Keilholz et al., 2005). In regard to pre-treatment of a chemoimmunotherapeutic regimen with chemotherapy, Punt et al. (Punt et al., 2006) reported on a randomized, phase II clinical trial of metastatic melanoma patients receiving either dacarbazine, cisplatin, IFN- and IL-2 or the same combination after an initial treatment of two cycles of dacarbazine, finding no difference between the two groups in terms of median overall survival.

O’Day et al. (O’Day et al., 2009) treated 133 chemotherapy-naïve metastatic melanoma patients with CVD, decrescendo IL-2, and IFN-α2b with granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine support. Maintenance biotherapy with low-dose IL-2 and GM-CSF followed by intermittent pulses of decrescendo IL-2 over 12 months was provided for those without disease progression. The overall response rate was 44% with 8% complete response and 36% partial response. Stable disease was noted for 29% of patients; median progression-free survival was 9 months, and median survival was 13.5 months with 12-month and 24-month survival rates at 57% and 23%, respectively, providing evidence of above average long-term survival rates for these patients. Further randomized trials need to be carried out.

A recent multicenter phase II chemoimmunotherapy trial of the Dermatologic Cooperative Oncology Group (DeCOG) testing the multi-kinase inhibitor sorafenib in combination with pegylated-IFN-α2b (PEG-IFN-α2b) in 55 patients with metastatic melanoma resulted in two patients with partial response and 14 with stable disease (29.1% disease control rate in intent-to-treat population) (Egberts et al., 2011). Median progression-free survival was 2.5 months, and toxicities were primarily hematological, including one treatment-related bleeding complication leading to death. A smaller study of 22 metastatic melanoma patients receiving a regimen of CVD with PEG-IFN-α2b, subcutaneous IL-2 and oral 13-cis-retinoic acid, with maintenance biotherapy for those with clinical response after six courses, provided 12 objective responses with seven maintenance-receiving patients having stable disease after six months (Recchia et al., 2008). Median progression-free and overall survivals were 23.3 and 45.7 months, respectively, with mostly hematological toxicities and 21% of patients exhibiting grade 2 autoimmune reactions.

3.2. Immunocentric chemoimmunotherapy

Another conventional strategy is one that we have termed ‘immunocentric chemoimmunotherapy’. This strategy is an immunotherapy-focused approach which uses chemotherapy as an immunomodulator to enhance the effect of cancer vaccines or adoptive cell transfer therapy. Based on this approach, recent studies using chemotherapy to prepare the host before the infusion of ex vivo activated, melanoma Ag-specific tumor-infiltrating lymphocytes and high dose IL-2 have resulted in impressive response rates.

3.2.1. Cancer vaccines

Cancer vaccines are promising for their abilities to elicit both humoral (antibody) and cellular (T cell) immune responses (Finn, 2008; Schoenfeld et al., 2010) specifically against tumor cells, as well as for their minimal side effects and the potential for development of immunologic memory allowing for a long-term, durable response to treatment without the need for continuous therapy (Yannelli & Wroblewski, 2004). The presence of cell surface antigens and receptors, capable of activating a myriad of intracellular biochemical pathways, makes possible the development of cancer vaccines with highly stringent criteria for target tumor cell selection and may assist in the focused delivery and activity of chemotherapeutic agents. The recent FDA-approval of sipuleucel-T (Provenge®; Dendreon Inc., Seattle, WA, USA), the first personalized therapeutic cancer vaccine, for the treatment of advanced prostate cancer, has opened the doors for novel vaccine development for other cancers, contributing to the heightened interest in targeted immune-based study of cancer systems.

Nistico et al. (Nistico et al., 2009) recently reported on their phase I/II pilot study testing 36 HLA-A2+ disease-free, stage II to IV melanoma patients with standard-dose dacarbazine administered one day before vaccination with HLA-A2 restricted melanoma antigen A (melan-A/MART-1) and gp100 melanoma peptide vaccination compared with vaccination alone. Dacarbazine significantly increased the numbers of peptide-specific CD8+ effector memory T cells which recognize and lyse HLA-A2+/melan-A+ tumor cells, indicating the enhancement of effective CD8+ T cell recognition of vaccine peptides after treatment with a chemotherapy drug in patients with melanoma. Another more recent pilot clinical trial (Palermo et al., 2010) by the Nistico group examined the role of dacarbazine again as pre-treatment to this vaccine therapy, noting a progressive widening of the T cell receptor repertoire diversity with concomitant high avidity and tumor reactivity in melan-A–specific T cell clones of patients treated with this chemoimmunotherapy and a trend toward longer survival. Alternatively, patients receiving vaccine alone exhibited a tendency to narrowing the T cell receptor repertoire diversity and a decrease of tumor lytic activity in one patient.

3.2.2. Adoptive cell transfer

One of the most exciting avenues for melanoma therapy is adoptive cell transfer of tumor-reactive T cells harvested from the tumor itself, expanded and activated ex vivo using various methods (e.g., high concentrations of IL-2) and then transferred back into the patient. The success of adoptive cell transfer depends on the specificity of T cells for the tumor and their ability to survive and proliferate in the environment. Notably, intratumoral injection of toll-like receptor agonists were found to enhance the potency of activated adoptive cell transfer against a murine model with established subcutaneous melanoma tumors, acting to enhance IFN-γ production and subsequent killing of the now more immunogenic tumor cells by adoptively transferred T cells (Amos et al., 2011). Metastatic melanoma patients have benefited in certain settings with adoptive cell transfer of tumor-infiltrating lymphocytes in combination with chemotherapeutic lymphodepletion, with an impressive objective response rate of 50-70% (Dudley & Rosenberg, 2007; Gattinoni et al., 2006; Hershkovitz et al., 2010; Khattar et al., 2009; Rosenberg et al., 2008, 2011; Rosenberg & Dudley, 2009). Given the promising results of adoptive cell transfer strategies in melanoma and other cancers, many notable contributions are being made toward a more discrete understanding of this field.

During expansion of tumor-infiltrating lymphocytes for infusion, CD8+ T cells are driven to differentiate into effector cells, losing key costimulatory molecules such as CD28 and CD27. Costimulation with CD137/4-1BB was recently shown to significantly increase lymphocyte survival during melanoma adoptive cell transfer and improve the anti-tumor response (Hernandez-Chacon et al., 2011). Inozume et al. (Inozume et al., 2010) reported on the recovery of function of PD-1 cell surface receptor protein on CD8+ T cells in melanoma digests after ex vivo conditioning with IL-2, leading to the production of much more tumor-specific IFN-γ compared with PD-1⁻ T cells and the proposal of PD-1 functional operation or presence being a capable biomarker for selecting tumor-specific lymphocytes from melanomas. As a common practice in adoptive cell transfer prior to infusion of these lymphocytes, patient lymphodepletion of T reg and other lymphocytes in competition for cytokines allows for a much more potent response to activated tumor-specific T cells (Dudley et al., 2002, Wrzesiniski et al., 2010). Adoptive cell transfer with autologous tumor-infiltrating lymphocytes, conditioned with cyclophosphamide and fludarabine, and IL-2 tested in 50 metastatic melanoma patients selected to receive either 2-Gy (non-myeloablative) or 12-Gy (myeloablative) of total body irradiation resulted in response rates of 49%, 52% and 72% for patients not receiving radiation and those receiving either 2- or 12-Gy, respectively (Dudley et al., 2008). The authors note the possibility for increased tumor-infiltrating lymphocyte activity, proliferation, and/or persistence in the body and at the tumor site as a result of rising cytokine availability following irradiation, having seen a significant increase of IL-7 and IL-15 in the serum following the myeloablative regimen. This trial is part of three whose combined and complete results were recently published by Rosenberg et al. (Rosenberg et al. 2011), providing impressive objective response and overall survival rates. In a panel of 93 heavily pretreated metastatic melanoma patients (86% with visceral metastases and 95% recurring after prior therapy), they found adoptive cell transfer of autologous lymphocytes plus high-dose IL-2, administered within one day following a lymphodepletion regimen of cyclophosphamide and fludarabine or either 2- or 12-Gy of total body irradiation, to produce 52 objective responses (56%) with 20 (22%) complete responses, 19 of which lasted a span of three to seven years.

Other clinical studies have utilized genetically engineered peripheral T cells in place of tumor-infiltrating lymphocytes. Due to limitations in generating tumor-specific T cells for adoptive cell transfer, Morgan et al. (Morgan et al., 2006) reported on the specific conference of tumor recognition by autologous lymphocytes from peripheral blood of 15 metastatic melanoma patients using a retrovirus that encodes a T cell receptor recognizing a tumor antigen. Adoptive cell transfer of these transduced cells after lymphodepletion resulted in durable engraftment of more than 10% of peripheral blood lymphocyte levels for over two months after infusion. In two patients demonstrating objective regression of metastatic melanoma lesions, circulating engineered cells were maintained at high levels at one year after infusion.

Such viral T cell receptor engineering is currently being studied for advanced melanoma and general (Chinnasamy et al., 2010) cancer therapy by Dr. Steven A. Rosenberg’s group and colleagues in The National Cancer Institute’s Surgery Branch (Rosenberg & Dudley, 2009). Indeed, they have recently described the selective delivery of IL-12, noted for its limited immunostimulatory clinical application due to toxicity, into the tumor microenvironment upon tumor-Ag recognition by T cell receptors on genetically engineered lymphocytes, enhancing in vivo melanoma tumor regression without toxicity (Zhang et al., 2011). Another recent report from this group describes adoptive cell transfer of autologous T cells with a T cell receptor against the NY-ESO-1 cancer/testis antigen, expressed in ~25% of patients with melanoma and other epithelial cancers, resulting in an objective response in five of 11 patients with NY-ESO-1-expressing tumors, with two complete responses of greater than one year (Robbins et al., 2011).

3.3. Cyclophosphamide for melanoma immunocentric chemoimmunotherapy

Cyclophosphamide has been an invaluable chemotherapeutic agent in studying immunocentric methodology and application. Numerous reports have shown the synergistic capabilities of cyclophosphamide in combination with immunotherapeutics, describing its ability to induce cytokine expression, type I IFNs and homeostatic proliferation of B and T cells and to deplete the T reg population from the tumor site, increasing the possibility of CD8+ T cell-mediated targeting and destruction of tumor cells (Berd et al., 1986, 1990, 2004; Bracci et al., 2007; Dummer et al., 2002; Ercolini et al., 2005; Ghiringhelli et al., 2004; Livingston et al., 1987, 1994; Maine & Mule, 2002; North, 1982; Nowak et al., 2006; Proietti et al., 1998; Schiavoni et al., 2000; Turk & Parker, 1982; Zitvogel et al., 2008). An important study in 2005 showed non-myeloablative lymphodepleting chemotherapy with cyclophosphamide and fludarabine conditioning of tumor-infiltrating lymphocytes followed by infusion and subsequent high-dose IL-2 treatment to produce a 51% overall response rate in 35 metastatic melanoma patients previously having received IL-2 therapy, all but one being refractory to IL-2 (Dudley et al., 2005).

A surge of recent studies have expounded on the role of cyclophosphamide in T reg modulation and immune therapies that can take advantage of the T reg depleted tumor environment. Combination cyclophosphamide and an agonist antibody against the co-stimulatory CD4+ Foxp3+ T reg cell receptor OX40 has recently been shown to induce apoptosis of the tumor T reg population and induce the influx of CD8+ T cells resulting in a potent anti-tumor response in an in vivo B16 melanoma model (Hirschhorn-Cymerman et al., 2009). Furthermore, Kohlmeyer et al. (Kohlmeyer et al., 2009) reported chemotherapeutic preconditioning with cyclophosphamide prior to adoptive cell transfer and viral vaccination followed by adjuvant peritumoral injections of immunostimulatory nucleic acids to be a highly effective chemoimmunotherapeutic regimen, resulting in complete regression of primary and lung metastatic lesions in the normally adoptive cell transfer-resistant genetically engineered Hgf-Cdk4R24C metastatic melanoma mouse model.

In following the combination of chemotherapy and adoptive cell transfer with immune-enhancing cytokines, such as IL-2 (Dudley et al., 2002, 2005; Dudley & Rosenberg, 2007; Mihalyo et al., 2004), and the recent characterization of IL-21 with adoptive cell transfer by Hinrichs et al. (Hinrichs et al., 2008), another study describes the boosting of immunocentric therapeutic efficacy with IL-21 (Petersen et al., 2010), an immune-enhancing cytokine that, unlike IL-2, does not support proliferation of activated T reg nor activation-induced cell death (Spolski & Leonard, 2008; Waldmann, 2006). As collective data would indicate, IL-21 likely enhances the T reg-depleting capabilities of cyclophosphamide by abrogating T reg development, via the inhibition of IL-2 secretion, and function (Elsaesser et al., 2009; Fantini et al., 2007; Hinrichs et al., 2008; Peluso et al., 2007; Piao et al., 2008; Yi et al., 2009). Mice with established tumors, subsequently treated with cyclophosphamide and adoptive cell transfer and/or daily injections of IL-21, exhibited better early tumor growth inhibition up to five days after the last (day 7) IL-21 injection and increased circulating tumor-specific T cells with the IL-21 administration. The authors note the potential for continued tumor growth stunting with continued IL-21 injection as well as the importance of timing considerations for this particular design (Skak et al., 2009). A similar study by Salem et al. (Salem et al., 2007) showed a more pronounced effect using peptide vaccination and naïve T cells, possibly a more effective option than pre-primed cells for adoptive cell transfer (Hinrichs et al., 2008).

4. Chemocentric chemoimmunotherapy

We have developed a third strategy for combining chemo- and immunotherapy, termed ‘chemocentric chemoimmunotherapy’ (Radfar et al., 2009). In this model, chemotherapy plays the central effector role, while immunotherapy is used to sensitize the tumor and its microenvironment to the cytotoxic effect of chemotherapy. This strategy employs the use of nonspecifically activated CD4+ T lymphocytes (aCD4) as a chemosensitizer of tumor cells followed by treatment with chemotherapeutic drugs. The rationale for this strategy is based on the known ability of activated T cells to secrete multiple cytokines that can regulate proliferation and/or apoptosis of tumor cells and the ability of activated T cells to exert direct activity on tumor cells through apoptotic pathways such as the Fas/Fas ligand pathway; for instance, tumor cell apoptosis is initiated when the Fas ligand, present on activated T cells, binds the Fas receptor, present on tumor cells. This model does not depend on Ag-specific activation of T cells and is, therefore, potentially applicable to a wide variety of tumor types and patients regardless of their HLA status. Indeed, preclinical studies examining the role of chemocentric chemoimmunotherapy in melanoma, breast, colon and prostate cancer cell lines have shown a dramatic enhancement of induced tumor-cell apoptosis. With the added advantage of ease of use and cost effectiveness in the clinic, chemocentric chemoimmunotherapy is a promising future methodology that is widely applicable, in terms of patient character schema, cost and basic technical utility, in the clinical setting.

4.1. Chemosensitization with nonspecifically activated CD4+ T cells

According to conventional wisdom, it is counterintuitive to administer chemotherapy immediately after cell therapy because the transferred immune cells will be eliminated by chemotherapy. Although this may be true in the setting of conventional immunocentric chemoimmunotherapy, where immunotherapy plays the major effector role and chemotherapy is used to prepare the host, this approach is rational in the context of chemocentric chemoimmunotherapy, where chemotherapy exerts the principal effector function and immunotherapy is used transiently for the purpose of presensitization.

We have recently demonstrated that presensitization of tumor cells with nonspecifically activated CD4+ T cells (aCD4) greatly enhanced the cytotoxic effect of chemotherapy in both in vitro and in vivo models (Radfar et al., 2009). This activity was observed for all seven tumor cell lines as well as all four chemotherapeutic agents tested. Soluble factors secreted from the activated CD4+ T cells were found to be responsible for the observed effect, with IFN-γ playing a major role in the chemosensitization of tumor cells. IFN-γ by itself, however, was consistently inferior to activated CD4+ T cells in the chemosensitization of tumor cells.

For three human metastatic melanoma, two human breast, one prostate and one colon cancer cell line, treated with 5-FU, temozolomide (TMZ), carboplatin (Carbo), or paclitaxel, chemosensitization with aCD4 had a dramatic impact on tumor cell viability and in vivo tumor formation. TMZ, the oral imidazotetrazine derivative of dacarbazine that is also capable of crossing the blood-brain barrier, ultimately produces the same dacarbazine compound after intake and has been shown in clinical trial to be similarly effective in treating metastatic melanoma as dacarbazine (Middleton et al., 2000). Treatment of the A375 melanoma cell line with TMZ after presensitization with aCD4 resulted in near complete cell death, with residual cell viability as low as 5%, compared to 67% viability for cells treated with TMZ that were not presensitized with aCD4. Similar study of two more melanoma cell lines provided comparable results. Further tests using Carbo, a drug with minimal activity against melanoma, resulted in a decrease of melanoma cell viability of ~83% at the optimal aCD4 concentration, compared with no change in viability after Carbo treatment alone. Non-melanoma cell lines treated with paclitaxel, Carbo or 5-FU all gave similar results.

Presensitization was found to be mediated in large part via cytokines, principally IFN-γ, and not through cell-cell contact. aCD4 supernatants were screened for 13 common Th1 and Th2 pro- and anti-inflammatory cytokines. IL-1b, IL-5, and IL-12p70 levels were negative or minimally expressed. The remaining 10 were tested for in combination against aCD4 alone for their chemosensitizing ability, assessed based on viability after TMZ treatment. There was no increase in chemosensitizing ability of combination IL-2/-4 over cells without cytokine addition, and combinations IL-6/-8 and IL-10/-12p40/-13 produced enhanced activity of 7% and 8%, respectively. Combination IFN-γ/TNF-α/GM-CSF significantly enhanced the cytotoxic activity of TMZ by 17%. While most individual cytokines provided little to no enhancement of TMZ cytotoxicity, IFN-γ in combination with other cytokines consistently provided the greatest impact on chemosensitization, and IFN-γ combinations showed no improvement over IFN-γ itself; however, the effects of IFN-γ by itself or in combination lagged the single combination of all 10 tested cytokines. Administration of aCD4 alone prior to TMZ treatment as a positive control yielded the best results, showing significant enhancement of TMZ cytotoxic cell killing over the 10-cytokine combination.

Lysates from A375 cells presensitized with aCD4 followed by treatment with TMZ were analyzed via a transwell system for cytoplasmic histone-associated DNA fragments as a measure of apoptosis. Melanoma cells treated with TMZ alone or aCD4 alone resulted in slightly higher levels of DNA fragments, ~49 arbitrary units (AU) and 54 AU, respectively, as compared 23 AU for nontreated cells. However, when A375 cells were presensitized with aCD4 and treated with TMZ, the level of DNA fragments detected, 1041 AU, was more than 21-fold over that of the TMZ alone group, 19-fold over the aCD4 alone group, and 45-fold over the nontreatment control. In addition, a significantly higher level of caspase-8 activity, ~2300 RFU, was detected in cells in the experimental group compared with the control groups, i.e., no treatment (524.4 + 3.5 RFU), TMZ treatment only (508.6 + 28.3 RFU), or aCD4 treatment only (392.7 + 4.0 RFU). A reduction in the relative expression of Bcl-2 protein was also detected in A375 cells treated with aCD4 alone or the combination of aCD4 followed by TMZ, but not in cells treated with TMZ alone. These data demonstrated that presensitization of tumor cells with aCD4 led to a decrease in Bcl-2, and that treatment with aCD4 and TMZ together resulted in enhanced caspase-8 activity, resulting in significant reduction in cell viability through an increase in apoptosis. The combination of aCD4 and chemotherapy yielded dramatic results at 24 hours in viability experiments. To evaluate true pro-apoptotic potential of aCD4 presensitization, apoptosis assays were performed at earlier time points, 12 hours instead of 24 hours after treatment with chemotherapy. For caspase-8 activity, chemotherapy treatment was stopped after 5 to 6 hours. Even at these early time points when aCD4 alone or chemotherapy alone had no discernable effects, the combination of aCD4 and chemotherapy resulted in a dramatic increase in apoptosis and caspase-8 activity.

Two human tumor xenograft models provided evidence of significant tumor growth delay for melanoma and breast cancer after presensitization with aCD4 and subsequent treatment with TMZ or paclitaxel, respectively. The melanoma xenograft also displayed significant growth inhibition. In the first model, injections of aCD4 were made intratumorally, followed 48 hours later with intraperitoneal administration of TMZ, to athymic nude mice bearing human melanoma A375 xenograft. There was a significantly pronounced delay in tumor growth compared with mice receiving no treatment or either treatment alone. Similarly, mice bearing aggressive MDA-MB-231 human breast tumors treated with aCD4 followed by paclitaxel had significant delay in tumor growth compared with each of the control groups. The difference between aCD4 plus paclitaxel versus paclitaxel alone was statistically significant. Interestingly, aCD4 alone had a significant growth inhibition effect on the melanoma xenograft but not on the breast cancer xenograft. This was consistent with the known immuno sensitivity of melanoma compared with other solid tumors. To assess toxicity resulting from the experimental treatment, animal weights were measured and recorded during the course of the study. No statistically significant difference in weight was found between the experimental group and control groups up to day 30 after initiation of treatment.

Some recent studies may help shed light onto the possible mechanisms that underlie the dramatic chemosensitizing effect of aCD4. Inflammation and inflammatory cytokines, as represented by a mixture of IL-1β, TNF-α, and IFN-γ, were shown to generate nitric oxide through the induction of nitric oxide synthase, resulting in global inhibition of DNA repair activity in cholangiocarcinoma cells (Jaiswal et al., 2000). Perrotta et al. (Perrotta et al., 2007) further described chemosensitization through dendritic-cell-mediated intratumoral administration of nitric oxide in a B16 mouse melanoma model. Other studies demonstrated that IFN-β or IL-24 could overcome TMZ resistance in neuroblastoma and melanoma, respectively, through down-regulation of the DNA repair enzyme O6-methylguanine-DNA methyltransferase expression and activity (Rosati et al., 2008; Zheng et al., 2008). Additionally, several recent clinical studies have reported improved response rates and survival with salvage chemotherapy in patients who previously received cancer vaccination and developed an immune response (Antonia et al., 2006; Arlen et al., 2006; Gribben et al., 2005; Schlom et al., 2007; Wheeler et al., 2004). Our model may provide a plausible mechanism to explain these observations as well as to facilitate further understanding, design, and development of improved methods for chemoimmunotherapy in cancer.

5. Conclusion

Despite intense research efforts, scientists are still struggling with the simplest principles of tumor cell proliferation, cell cycle kinetics and the genetic basis of malignant transformation and tumor progression. In particular, treatment options for melanoma are few, and those with metastatic disease have been afforded only meager therapeutic options in the past, maintaining a disappointing 15% five-year survival rate. In somewhat of a contradiction, melanoma is now at the forefront of targeted and immunological therapeutic study, evident by the surging number of relevant publications and the recent addition of ipilimumab to a short list of FDA-approved therapies for metastatic melanoma patients. One of the most popular and theoretically promising of these directions currently being studied is combination chemoimmunotherapy.

While many chemoimmunotherapeutic regimens have resulted in lackluster responses, our novel adaptation of this methodology for melanoma and other cancers, utilizing non-tumor-specific activation of CD4+ T cells as a chemosensitizer, has provided exciting results. We have shown dramatic increases in the efficacies of four functionally unique chemotherapeutic drugs in seven cell lines comprising four different cancer types with our ‘chemocentric chemoimmunotherapeutic’ strategy. After presensitization with aCD4, cytotoxicity was greatly enhanced for all chemo drugs, both in vitro and in vivo, against all cell types tested. In addition, we recently described the presensitization of tumor cells with aCD4 prior to gamma-irradiation to significantly enhance cancer cell growth inhibition (Wang et al., 2010). Soluble factors released by aCD4, particularly IFN-γ, were primarily responsible for the observed activity, and TNF-α, though inactive by itself, significantly augmented the radiosensitizing activity of IFN-γ.

Not only are the time and monetary costs of producing non-tumor-specific activated CD4+ T cells far and away less than the current standard of activating against a specific set of patient tumor cell antigens, but, as our data shows, aCD4 may also be applied in patients with a variety of cancer types in synergistic conjunction with the most currently effective and applicable chemotherapy for the individual patient. Regardless of the cell line used or its sensitivity or resistance to a particular chemotherapeutic drug, in all our tested cases, presensitization with aCD4 greatly enhanced the cytotoxicity of the drugs, resulting in near complete cell death. Therefore, patients with a typically chemoresistant cancer, such as metastatic melanoma, may be able to benefit from enhanced chemotherapeutic efficacy with aCD4 presensitization. Additionally, utilization of a much broader range of chemotherapeutic agents may be possible for patients who might normally have only a small number of available options, specific for their tumor type and physical condition. Patients may thus be able to receive treatment sooner, accessing the readily available wealth of well-studied chemotherapeutic drugs without the need to await new drug development and testing. To this end, drug selection may ultimately be better tailored to patient tolerance, creating a therapeutic strategy with greater anti-tumor activity, therefore, less time for development of chemoresistance, and better tolerance which will allow more patients to remain on their particular regimen for its appropriate duration. Indeed, the utilization of this novel technique may be able to provide a more effective and efficient means to combat melanoma and other cancers.


1 - S. J. Adair, K. T. Hogan, 2009 Treatment of Ovarian Cancer Cell Lines with 5-Aza-2’-Deoxycytidine Upregulates the Expression of Cancer-Testis Antigens and Class I Major Histocompatibility Complex-Encoded Molecules, Cancer Immunol Immunother, 58 4 589601 , 1432-0851
2 - I. Allen, B. Kupelnick, M. Kumashiro, et al. 1998 Efficacy of Interleukin-2 in the Treatment of Metastatic Melanoma-Systematic Review and Meta-Analysis. Sel Cancer Ther, 1 168173
3 - S. M. Amos, H. J. Pegram, J. A. Westwood, et al. 2011 Adoptive Immunotherapy Combined with Intratumoral TLR Agonist Delivery Eradicates Established Melanoma in Mice, Cancer Immunol Immunother, 60 5 671683 , 1432-0851
4 - S. J. Antonia, N. Mirza, I. Fricke, et al. 2006 Combination of P53 Cancer Vaccine with Chemotherapy in Patients with Extensive Stage Small Cell Lung Cancer, Clin Cancer Res, 12 3 Pt 1, 878887 , 1078-0432
5 - P. M. Arlen, J. L. Gulley, C. Parker, et al. 2006 A Randomized Phase Ii Study of Concurrent Docetaxel Plus Vaccine Versus Vaccine Alone in Metastatic Androgen-Independent Prostate Cancer, Clin Cancer Res, 12 4 12601269 , 1078-0432
6 - E. Atallah, L. Flaherty, 2005 Treatment of Metastatic Malignant Melanoma, Curr Treat Options Oncol, 6 3 185193 , 1527-2729
7 - M. B. Atkins, L. Kunkel, M. Sznol, et al. 2000 High-Dose Recombinant Interleukin-2 Therapy in Patients with Metastatic Melanoma: Long-Term Survival Update, Cancer J Sci Am, 6 Suppl 1, S11S14 , 1081-4442
8 - M. B. Atkins, J. A. Gollob, J. A. Sosman, et al. 2002 A Phase II Pilot Trial of Concurrent Biochemotherapy with Cisplatin, Vinblastine, Temozolomide, Interleukin 2, and IFN-Alpha 2b in Patients with Metastatic Melanoma, Clin Cancer Res, 8 10 30753081 , 1078-0432
9 - M. B. Atkins, J. Hsu, S. Lee, et al. 2008 Phase III Trial Comparing Concurrent Biochemotherapy with Cisplatin, Vinblastine, Dacarbazine, Interleukin-2, and Interferon Alfa-2b with Cisplatin, Vinblastine, and Dacarbazine Alone in Patients with Metastatic Malignant Melanoma (E3695): A Trial Coordinated by the Eastern Cooperative Oncology Group, J Clin Oncol, 26 35 57485754 , 1527-7755
10 - J. Atzpodien, K. Neuber, D. Kamanabrou, et al. 2002 Combination Chemotherapy with or without s.c. IL-2 and IFN-Alpha: Results of a Prospectively Randomized Trial of the Cooperative Advanced Malignant Melanoma Chemoimmunotherapy Group (Acimm), Br J Cancer, 86 2 179184 , 0007-0920
11 - D. Bafaloukos, G. Aravantinos, G. Fountzilas, et al. 2002 Docetaxel in Combination with Dacarbazine in Patients with Advanced Melanoma, Oncology, 63 4 333337 , 0030-2414
12 - E. Bajetta, M. Del Vecchio, C. Bernard-Marty, et al. 2002 Metastatic Melanoma: Chemotherapy, Semin Oncol, 29 5 427445 , 0093-7754
13 - E. Bajetta, M. Del Vecchio, P. Nova, et al. 2006 Multicenter Phase III Randomized Trial of Polychemotherapy (CVD Regimen) Versus the Same Chemotherapy (Ct) Plus Subcutaneous Interleukin-2 and Interferon-Alpha2b in Metastatic Melanoma, Ann Oncol, 17 4 571577 , 0923-7534
14 - C. M. Balch, A. C. Buzaid, S. J. Soong, et al. 2001 Final Version of the American Joint Committee on Cancer Staging System for Cutaneous Melanoma, J Clin Oncol, 19 16 36353648 , 0732-183X
15 - C. M. Balch, S. J. Soong, J. E. Gershenwald, et al. 2001 Prognostic Factors Analysis of 17,600 Melanoma Patients: Validation of the American Joint Committee on Cancer Melanoma Staging System, J Clin Oncol, 19 16 36223634 , 0732-183X
16 - A. Y. Bedikian, J. Richards, D. Kharkevitch, et al. 2010 A Phase 2 Study of High-Dose Allovectin-7 in Patients with Advanced Metastatic Melanoma, Melanoma Res, 20 3 218226 , 1473-5636
17 - D. Berd, H. C. Maguire, Jr , M. J. Mastrangelo, 1986 Induction of Cell-Mediated Immunity to Autologous Melanoma Cells and Regression of Metastases after Treatment with a Melanoma Cell Vaccine Preceded by Cyclophosphamide, Cancer Res, 46 5 25722577 , 0008-5472
18 - D. Berd, H. C. Maguire, Jr , P. Mccue, et al. 1990 Treatment of Metastatic Melanoma with an Autologous Tumor-Cell Vaccine: Clinical and Immunologic Results in 64 Patients, J Clin Oncol, 8 11 18581867 , 0732-183X
19 - D. Berd, T. Sato, H. C. Maguire, Jr , et al. 2004 Immunopharmacologic Analysis of an Autologous, Hapten-Modified Human Melanoma Vaccine, J Clin Oncol, 22 3 403415 , 0732-183X
20 - J. M. Blesa, E. G. Pulido, V. A. Candel, et al. 2011 Melanoma: From Darkness to Promise, Am J Clin Oncol, 34 2 179187 , 1537-453X
21 - G. Bollag, P. Hirth, J. Tsai, et al. 2010 Clinical Efficacy of a Raf Inhibitor Needs Broad Target Blockade in BRAF-Mutant Melanoma, Nature, 467 7315 596599 , 1476-4687
22 - L. Bracci, F. Moschella, P. Sestili, et al. 2007 Cyclophosphamide Enhances the Antitumor Efficacy of Adoptively Transferred Immune Cells through the Induction of Cytokine Expression, B-Cell and T-Cell Homeostatic Proliferation, and Specific Tumor Infiltration, Clin Cancer Res, 13 2 Pt 1, 644653 , 1078-0432
23 - F. Burke, P. D. Smith, M. R. Crompton, et al. 1999 Cytotoxic Response of Ovarian Cancer Cell Lines to IFN-Gamma Is Associated with Sustained Induction of IRF-1 and P21 mRNA, Br J Cancer, 80 8 12361244 , 0007-0920
24 - A. C. Buzaid, S. S. Legha, R. Winn, et al. 1993 Cisplatin (C), Vinblastine (V), Dacarbazine (D) (CVD) Versus Dacarbazine Alone in Metastatic Melanoma: Preliminary Results Phase III Cancer Community Oncology Program (CCOP). Proc Annu Meet Am Soc Clin Oncol, 12 1328
25 - E. A. Carswell, L. J. Old, R. L. Kassel, et al. 1975 An Endotoxin-Induced Serum Factor That Causes Necrosis of Tumors, Proc Natl Acad Sci U S A, 72 9 36663670 , 0027-8424
26 - P. B. Chapman, L. H. Einhorn, M. L. Meyers, et al. 1999 Phase III Multicenter Randomized Trial of the Dartmouth Regimen Versus Dacarbazine in Patients with Metastatic Melanoma, J Clin Oncol, 17 9 27452751 , 0732-183X
27 - M. Chawla-Sarkar, D. W. Leaman, E. C. Borden, 2001 Preferential Induction of Apoptosis by Interferon (IFN)-Beta Compared with IFN-Alpha2: Correlation with Trail/Apo2l Induction in Melanoma Cell Lines, Clin Cancer Res, 7 6 18211831 , 1078-0432
28 - M. Chawla-Sarkar, D. J. Lindner, Y. F. Liu, et al. 2003 Apoptosis and Interferons: Role of Interferon-Stimulated Genes as Mediators of Apoptosis, Apoptosis, 8 3 237249 , 1360-8185
29 - D. Chinnasamy, Z. Yu, M. R. Theoret, et al. 2010 Gene Therapy Using Genetically Modified Lymphocytes Targeting VEGFR-2 Inhibits the Growth of Vascularized Syngenic Tumors in Mice, J Clin Invest, 120 11 39533968 , 1558-8238
30 - B. Comin-Anduix, T. Chodon, H. Sazegar, et al. 2010 The Oncogenic BRAF Kinase Inhibitor Plx4032/Rg7204 Does Not Affect the Viability or Function of Human Lymphocytes across a Wide Range of Concentrations, Clin Cancer Res, 16 24 60406048 , 1078-0432
31 - S. Coral, L. Sigalotti, M. Altomonte, et al. 2002 5-Aza-2’-Deoxycytidine-Induced Expression of Functional Cancer Testis Antigens in Human Renal Cell Carcinoma: Immunotherapeutic Implications, Clin Cancer Res, 8 8 26902695 , 1078-0432
32 - P. Correale, A. Aquino, A. Giuliani, et al. 2003 Treatment of Colon and Breast Carcinoma Cells with 5-Fluorouracil Enhances Expression of Carcinoembryonic Antigen and Susceptibility to Hla-a(*)02.01 Restricted, CEA-Peptide-Specific Cytotoxic T Cells in Vitro, Int J Cancer, 104 4 437445 , 0020-7136
33 - S. A. Del Prete, L. H. Maurer, J. O’donnell, et al. 1984 Combination Chemotherapy with Cisplatin, Carmustine, Dacarbazine, and Tamoxifen in Metastatic Melanoma, Cancer Treat Rep, 68 11 14031405 , 0361-5960
34 - K. M. Detjen, K. Farwig, M. Welzel, et al. 2001 Interferon Gamma Inhibits Growth of Human Pancreatic Carcinoma Cells Via Caspase-1 Dependent Induction of Apoptosis, Gut, 49 2 251262 , 0017-5749
35 - Xda. E. Dong, N. Ito, M. T. Lotze, et al. 2007 High Mobility Group Box I (HMGB1) Release from Tumor Cells after Treatment: Implications for Development of Targeted Chemoimmunotherapy, J Immunother, 30 6 596606 , 1524-9557
36 - M. E. Dudley, J. R. Wunderlich, P. F. Robbins, et al. 2002 Cancer Regression and Autoimmunity in Patients after Clonal Repopulation with Antitumor Lymphocytes, Science, 298 5594 850854 , 1095-9203
37 - M. E. Dudley, J. R. Wunderlich, J. C. Yang, et al. 2005 Adoptive Cell Transfer Therapy Following Non-Myeloablative but Lymphodepleting Chemotherapy for the Treatment of Patients with Refractory Metastatic Melanoma, J Clin Oncol, 23 10 23462357 , 0732-183X
38 - M. E. Dudley, S. A. Rosenberg, 2007 Adoptive Cell Transfer Therapy, Semin Oncol, 34 6 524531 , 0093-7754
39 - M. E. Dudley, J. C. Yang, R. Sherry, et al. 2008 Adoptive Cell Therapy for Patients with Metastatic Melanoma: Evaluation of Intensive Myeloablative Chemoradiation Preparative Regimens, J Clin Oncol, 26 32 52335239 , 1527-7755
40 - W. Dummer, A. G. Niethammer, R. Baccala, et al. 2002 T Cell Homeostatic Proliferation Elicits Effective Antitumor Autoimmunity, J Clin Invest, 110 2 185192 , 0021-9738
41 - F. Egberts, R. Gutzmer, S. Ugurel, et al. 2011 Sorafenib and Pegylated Interferon-{Alpha}2b in Advanced Metastatic Melanoma: A Multicenter Phase II Decog Trial, Ann Oncol, 15698041
42 - H. Elsaesser, K. Sauer, D. G. Brooks, 2009 Il-21 Is Required to Control Chronic Viral Infection, Science, 324 5934 15691572 , 1095-9203
43 - L. A. Emens, 2008 Chemotherapy and Tumor Immunity: An Unexpected Collaboration, Front Biosci, 13 249257 , 1093-4715
44 - A. M. Ercolini, B. H. Ladle, E. A. Manning, et al. 2005 Recruitment of Latent Pools of High-Avidity Cd8(+) T Cells to the Antitumor Immune Response, J Exp Med, 201 10 15911602 , 0022-1007
45 - O. Eton, S. S. Legha, A. Y. Bedikian, et al. 2002 Sequential Biochemotherapy Versus Chemotherapy for Metastatic Melanoma: Results from a Phase III Randomized Trial, J Clin Oncol, 20 8 20452052 , 0732-183X
46 - M. C. Fantini, A. Rizzo, D. Fina, et al. 2007 Il-21 Regulates Experimental Colitis by Modulating the Balance between Treg and Th17 Cells, Eur J Immunol, 37 11 31553163 , 0014-2980
47 - O. J. Finn, 2008 Cancer Immunology, N Engl J Med, 358 25 27042715 , 1533-4406
48 - L. E. Flaherty, M. Atkins, J. Sosman, et al. 2001 Outpatient Biochemotherapy with Interleukin-2 and Interferon Alfa-2b in Patients with Metastatic Malignant Melanoma: Results of Two Phase II Cytokine Working Group Trials, J Clin Oncol, 19 13 31943202 , 0732-183X
49 - K. T. Flaherty, I. Puzanov, K. B. Kim, et al. 2010 Inhibition of Mutated, Activated BRAF in Metastatic Melanoma, N Engl J Med, 363 9 809819 , 1533-4406
50 - E. Fonsatti, H. J. Nicolay, L. Sigalotti, et al. 2007 Functional up-Regulation of Human Leukocyte Antigen Class I Antigens Expression by 5-Aza-2’-Deoxycytidine in Cutaneous Melanoma: Immunotherapeutic Implications, Clin Cancer Res, 13 11 33333338 , 1078-0432
51 - E. Fonsatti, M. Maio, M. Altomonte, et al. 2010 Biology and Clinical Applications of CD40 in Cancer Treatment, Semin Oncol, 37 5 517523 , 1532-8708
52 - S. C. Formenti, 2010 Immunological Aspects of Local Radiotherapy: Clinical Relevance, Discov Med, 9 45 119124 , 1944-7930
53 - G. Fregni, A. Perier, G. Pittari, et al. 2011 Unique Functional Status of Natural Killer Cells in Metastatic Stage IV Melanoma Patients and Its Modulation by Chemotherapy. Clin Cancer Res, 17 9 26282637
54 - L. Gattinoni, D. J. Powell, Jr , S. A. Rosenberg, et al. 2006 Adoptive Immunotherapy for Cancer: Building on Success, Nat Rev Immunol, 6 5 383393 , 1474-1733
55 - F. Ghiringhelli, N. Larmonier, E. Schmitt, et al. 2004 Cd4+Cd25+ Regulatory T Cells Suppress Tumor Immunity but Are Sensitive to Cyclophosphamide Which Allows Immunotherapy of Established Tumors to Be Curative, Eur J Immunol, 34 2 336344 , 0014-2980
56 - J. L. Gooch, R. E. Herrera, D. Yee, 2000 The Role of P21 in Interferon Gamma-Mediated Growth Inhibition of Human Breast Cancer Cells, Cell Growth Differ, 11 6 335342 , 1044-9523
57 - J. G. Gribben, D. P. Ryan, R. Boyajian, et al. 2005 Unexpected Association Between Induction of Immunity to the Universal Tumor Antigen CYP1B1 and Response to Next Therapy, Clin Cancer Res, 11 12 44304436 , 1078-0432
58 - J. Harris, D. Sengar, T. Stewart, et al. 1976 The Effect of Immunosuppressive Chemotherapy on Immune Function in Patients with Malignant Disease, Cancer, 37 2 Suppl, 10581069 , 0008-543X
59 - B. L. Harvat, A. M. Jetten, 1996 Gamma-Interferon Induces an Irreversible Growth Arrest in Mid-G1 in Mammary Epithelial Cells Which Correlates with a Block in Hyperphosphorylation of Retinoblastoma, Cell Growth Differ, 7 3 289300 , 1044-9523
60 - L. Helson, S. Green, E. Carswell, et al. 1975 Effect of Tumour Necrosis Factor on Cultured Human Melanoma Cells, Nature, 258 5537 731732 , 0028-0836
61 - J. A. Hernandez-Chacon, Y. Li, R. C. Wu, et al. 2011 Costimulation Through the CD137/4-1bb Pathway Protects Human Melanoma Tumor-Infiltrating Lymphocytes from Activation-Induced Cell Death and Enhances Antitumor Effector Function, J Immunother, 34 3 236250 , 1537-4513
62 - L. Hershkovitz, J. Schachter, A. J. Treves, et al. 2010 Focus on Adoptive T Cell Transfer Trials in Melanoma, Clin Dev Immunol, 2010 260267
63 - C. S. Hinrichs, R. Spolski, C. M. Paulos, et al. 2008 Il-2 and Il-21 Confer Opposing Differentiation Programs to Cd8+ T Cells for Adoptive Immunotherapy, Blood, 111 11 53265333 , 1528-0020
64 - D. Hirschhorn-Cymerman, G. A. Rizzuto, T. Merghoub, et al. 2011 OX40 engagement and chemotherapy combination provides potent antitumor immunity with concomitant regulatory T cell apoptosis. J Exp Med, 206 5 11031116
65 - F. S. Hodi, S. J. O’day, D. F. Mcdermott, et al. 2010 Improved Survival with Ipilimumab in Patients with Metastatic Melanoma, N Engl J Med, 363 8 711723 , 1533-4406
66 - T. Inozume, K. Hanada, Q. J. Wang, et al. 2010 Selection of Cd8+Pd-1+ Lymphocytes in Fresh Human Melanomas Enriches for Tumor-Reactive T Cells, J Immunother, 33 9 956964 , 1537-4513
67 - N. J. Ives, R. L. Stowe, P. Lorigan, et al. 2007 Chemotherapy Compared with Biochemotherapy for the Treatment of Metastatic Melanoma: A Meta-Analysis of 18 Trials Involving 2,621 Patients, J Clin Oncol, 25 34 54265434 , 1527-7755
68 - M. Jaiswal, N. F. Larusso, L. J. Burgart, et al. 2000 Inflammatory Cytokines Induce DNA Damage and Inhibit DNA Repair in Cholangiocarcinoma Cells by a Nitric Oxide-Dependent Mechanism, Cancer Res, 60 1 184190 , 0008-5472
69 - A. Jemal, R. Siegel, J. Xu, et al. 2010 Cancer Statistics, 2010, CA Cancer J Clin, 60 5 277300 , 1542-4863
70 - A. S. Kadison, D. L. Morton, 2003 Immunotherapy of Malignant Melanoma, Surg Clin North Am, 83 2 343370 , 0039-6109
71 - D. H. Kaplan, V. Shankaran, A. S. Dighe, et al. 1998 Demonstration of an Interferon Gamma-Dependent Tumor Surveillance System in Immunocompetent Mice, Proc Natl Acad Sci U S A, 95 13 75567561 , 0027-8424
72 - R. Kaufmann, K. Spieth, U. Leiter, et al. 2005 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, 23 35 90019007 , 0732-183X
73 - U. Keilholz, S. H. Goey, C. J. Punt, et al. 1997 Interferon Alfa-2a and Interleukin-2 with or without Cisplatin in Metastatic Melanoma: A Randomized Trial of the European Organization for Research and Treatment of Cancer Melanoma Cooperative Group, J Clin Oncol, 15 7 25792588 , 0732-183X
74 - U. Keilholz, C. Conradt, S. S. Legha, et al. 1998 Results of Interleukin-2-Based Treatment in Advanced Melanoma: A Case Record-Based Analysis of 631 Patients, J Clin Oncol, 16 9 29212929 , 0732-183X
75 - U. Keilholz, C. J. Punt, M. Gore, et al. 2005 Dacarbazine, Cisplatin, and Interferon-Alfa-2b with or without Interleukin-2 in Metastatic Melanoma: A Randomized Phase III Trial (18951) of the European Organisation for Research and Treatment of Cancer Melanoma Group, J Clin Oncol, 23 27 67476755 , 0732-183X
76 - M. Khattar, W. Chen, S. M. Stepkowski, 2009 Expanding and Converting Regulatory T Cells: A Horizon for Immunotherapy, Arch Immunol Ther Exp (Warsz), 57 3 199204 , 1661-4917
77 - D. Khayat, C. Bernard-Marty, J. B. Meric, et al. 2002 Biochemotherapy for Advanced Melanoma: Maybe It Is Real, J Clin Oncol, 20 10 24112414 , 0732-183X
78 - J. Kohlmeyer, M. Cron, J. Landsberg, et al. 2009 Complete Regression of Advanced Primary and Metastatic Mouse Melanomas Following Combination Chemoimmunotherapy, Cancer Res, 69 15 62656274 , 1538-7445
79 - M. H. Lampen, T. Van Hall, 2011 Strategies to Counteract MHC-I Defects in Tumors, Curr Opin Immunol, 23 2 293298 , 1879-0372
80 - S. M. Laster, J. G. Wood, L. R. Gooding, 1988 Tumor Necrosis Factor Can Induce Both Apoptic and Necrotic Forms of Cell Lysis, J Immunol, 141 8 26292634 , 0022-1767
81 - S. S. Legha, S. Ring, N. Papadopoulos, et al. 1989 A Prospective Evaluation of a Triple-Drug Regimen Containing Cisplatin, Vinblastine, and Dacarbazine (CVD) for Metastatic Melanoma, Cancer, 64 10 20242029 , 0008-543X
82 - S. S. Legha, S. Ring, O. Eton, et al. 1998 Development of a Biochemotherapy Regimen with Concurrent Administration of Cisplatin, Vinblastine, Dacarbazine, Interferon Alfa, and Interleukin-2 for Patients with Metastatic Melanoma, J Clin Oncol, 16 5 17521759 , 0732-183X
83 - J. Lindenmann, P. A. Klein, 1967 Viral Oncolysis: Increased Immunogenicity of Host Cell Antigen Associated with Influenza Virus, J Exp Med, 126 1 93108 , 0022-1007
84 - P. O. Livingston, S. Cunningham-Rundles, G. Marfleet, et al. 1987 Inhibition of Suppressor-Cell Activity by Cyclophosphamide in Patients with Malignant Melanoma, J Biol Response Mod, 6 4 392403 , 0732-6580
85 - P. O. Livingston, G. Y. Wong, S. Adluri, et al. 1994 Improved Survival in Stage III Melanoma Patients with Gm2 Antibodies: A Randomized Trial of Adjuvant Vaccination with Gm2 Ganglioside, J Clin Oncol, 12 5 10361044 , 0732-183X
86 - G. N. Maine, J. J. Mule, 2002 Making Room for T Cells, J Clin Invest, 110 2 157159 , 0021-9738
87 - I. Maleno, N. Aptsiauri, T. Cabrera, et al. 2011 Frequent Loss of Heterozygosity in the Beta2-Microglobulin Region of Chromosome 15 in Primary Human Tumors, Immunogenetics, 63 2 6571 , 1432-1211
88 - E. R. Manusama, P. T. Nooijen, J. Stavast, et al. 1996 Synergistic Antitumour Effect of Recombinant Human Tumour Necrosis Factor Alpha with Melphalan in Isolated Limb Perfusion in the Rat, Br J Surg, 83 4 551555 , 0007-1323
89 - D. F. Mc Dermott, J. W. Mier, D. P. Lawrence, et al. 2000 A Phase II Pilot Trial of Concurrent Biochemotherapy with Cisplatin, Vinblastine, Dacarbazine, Interleukin 2, and Interferon Alpha-2b in Patients with Metastatic Melanoma, Clin Cancer Res, 6 6 22012208 , 1078-0432
90 - M. R. Middleton, J. J. Grob, N. Aaronson, et al. 2000 Randomized Phase III Study of Temozolomide Versus Dacarbazine in the Treatment of Patients with Advanced Metastatic Malignant Melanoma, J Clin Oncol, 18 1 158166 , 0732-183X
91 - M. Middleton, A. Hauschild, D. Thomson, et al. 2007 Results of a Multicenter Randomized Study to Evaluate the Safety and Efficacy of Combined Immunotherapy with Interleukin-2, Interferon-{Alpha}2b and Histamine Dihydrochloride Versus Dacarbazine in Patients with Stage IV Melanoma, Ann Oncol, 18 10 16911697 , 1569-8041
92 - M. A. Mihalyo, A. D. Doody, J. P. Mcaleer, et al. 2004 In Vivo Cyclophosphamide and IL-2 Treatment Impedes Self-Antigen-Induced Effector CD4 Cell Tolerization: Implications for Adoptive Immunotherapy, J Immunol, 172 9 53385345 , 0022-1767
93 - R. A. Morgan, M. E. Dudley, J. R. Wunderlich, et al. 2006 Cancer Regression in Patients after Transfer of Genetically Engineered Lymphocytes, Science, 314 5796 126129 , 1095-9203
94 - A. Natsume, T. Wakabayashi, K. Tsujimura, et al. 2008 The DNA Demethylating Agent 5-Aza-2’-Deoxycytidine Activates NY-ESO-1 Antigenicity in Orthotopic Human Glioma, Int J Cancer, 122 11 25422553 , 1097-0215
95 - P. Nistico, I. Capone, B. Palermo, et al. 2009 Chemotherapy Enhances Vaccine-Induced Antitumor Immunity in Melanoma Patients, Int J Cancer, 124 1 130139 , 1097-0215
96 - R. J. North, 1982 Cyclophosphamide-Facilitated Adoptive Immunotherapy of an Established Tumor Depends on Elimination of Tumor-Induced Suppressor T Cells, J Exp Med, 155 4 10631074 , 0022-1007
97 - A. K. Nowak, R. A. Lake, B. W. Robinson, 2006 Combined Chemoimmunotherapy of Solid Tumours: Improving Vaccines?, Adv Drug Deliv Rev, 58 8 975990 , 0169-409X
98 - S. J. O’day, M. B. Atkins, P. Boasberg, et al. 2009 Phase II Multicenter Trial of Maintenance Biotherapy after Induction Concurrent Biochemotherapy for Patients with Metastatic Melanoma, J Clin Oncol, 27 36 62076212 , 1527-7755
99 - L. J. Old, 1985 Tumor Necrosis Factor (TNFf), Science, 230 4726 630632 , 0036-8075
100 - F. Pages, J. Galon, M. C. Dieu-Nosjean, et al. 2009 Immune Infiltration in Human Tumors: A Prognostic Factor That Should Not Be Ignored, Oncogene, 29 8 10931102 , 1476-5594
101 - B. Palermo, D. Del Bello, A. Sottini, et al. 2010 Dacarbazine Treatment before Peptide Vaccination Enlarges T-Cell Repertoire Diversity of Melan-A-Specific, Tumor-Reactive CTL in Melanoma Patients, Cancer Res, 70 18 70847092 , 1538-7445
102 - I. Pejcic, S. Vrbic, S. Filipovic, et al. 2010 Concurrent Chemoimmunotherapy: Is It Still the Best Option for the Treatment of Metastatic Melanoma in Patients with Good Performance Status?, J Buon, 15 3 470474 , 1107-0625
103 - I. Peluso, M. C. Fantini, D. Fina, et al. 2007 IL-21 Counteracts the Regulatory T Cell-Mediated Suppression of Human CD4+ T Lymphocytes, J Immunol, 178 2 732739 , 0022-1767
104 - C. Perrotta, L. Bizzozero, S. Falcone, et al. 2007 Nitric Oxide Boosts Chemoimmunotherapy Via Inhibition of Acid Sphingomyelinase in a Mouse Model of Melanoma, Cancer Res, 67 16 75597564 , 0008-5472
105 - C. C. Petersen, J. E. Diernaes, A. Skovbo, et al. 2010 Interleukin-21 Restrains Tumor Growth and Induces a Substantial Increase in the Number of Circulating Tumor-Specific T Cells in a Murine Model of Malignant Melanoma, Cytokine, 49 1 8088 , 1096-0023
106 - L. M. Pfeffer, C. A. Dinarello, R. B. Herberman, et al. 1998 Biological Properties of Recombinant Alpha-Interferons: 40th Anniversary of the Discovery of Interferons, Cancer Res, 58 12 24892499 , 0008-5472
107 - L. Phan-Bich, A. Buard, J. F. Petit, et al. 1997 Differential Responsiveness of Human and Rat Mesothelioma Cell Lines to Recombinant Interferon-Gamma, Am J Respir Cell Mol Biol, 16 2 178186 , 1044-1549
108 - W. H. Piao, Y. H. Jee, R. L. Liu, et al. 2008 IL-21 Modulates CD4+ CD25+ Regulatory T-Cell Homeostasis in Experimental Autoimmune Encephalomyelitis, Scand J Immunol, 67 1 3746 , 1365-3083
109 - E. Proietti, G. Greco, B. Garrone, et al. 1998 Importance of Cyclophosphamide-Induced Bystander Effect on T Cells for a Successful Tumor Eradication in Response to Adoptive Immunotherapy in Mice, J Clin Invest, 101 2 429441 , 0021-9738
110 - C. J. Punt, S. Suciu, M. A. Gore, et al. 2006 Chemoimmunotherapy with Dacarbazine, Cisplatin, Interferon-Alpha2b and Interleukin-2 Versus Two Cycles of Dacarbazine Followed by Chemoimmunotherapy in Patients with Metastatic Melanoma: A Randomised Phase II Study of the European Organization for Research and Treatment of Cancer Melanoma Group, Eur J Cancer, 42 17 29912995 , 0959-8049
111 - S. Radfar, Y. Wang, H. T. Khong, 2009 Activated CD4+ T Cells Dramatically Enhance Chemotherapeutic Tumor Responses in Vitro and in Vivo, J Immunol, 183 10 68006807 , 1550-6606
112 - K. Rass, W. Tilgen, 2008 Treatment of Melanoma and Nonmelanoma Skin Cancer, Adv Exp Med Biol, 624 296318 , 0065-2598
113 - F. Recchia, G. Candeloro, S. Necozione, et al. 2008 Multicenter Phase II Study of Chemoimmunotherapy in the Treatment of Metastatic Melanoma, Anticancer Drugs, 19 2 201207 , 0959-4973
114 - R. Ridolfi, V. Chiarion-Sileni, M. Guida, et al. 2002 Cisplatin, Dacarbazine with or without Subcutaneous Interleukin-2, and Interferon Alpha-2b in Advanced Melanoma Outpatients: Results from an Italian Multicenter Phase III Randomized Clinical Trial, J Clin Oncol, 20 6 16001607 , 0732-183X
115 - A. I. Riker, P. M. . Howell, June, 2010 Treatment Options for Patients with Stage III & IV Melanoma: Part I: A Look at Existing Neoadjuvant and Adjuvant Treatments, In: Contemporary Oncology, 16.06.2010, Available from
116 - A. I. Riker, P. M. . Howell, June, 2010 Treatment Options for Patients with Stage III & IV Melanoma: Part II: Experimental Treatments for Stage III/IV or Refractory/Recurrent Melanoma, In: Contemporary Oncology, 03.06.2010, Available from
117 - P. F. Robbins, R. A. Morgan, S. A. Feldman, et al. 2011 Tumor Regression in Patients with Metastatic Synovial Cell Sarcoma and Melanoma Using Genetically Engineered Lymphocytes Reactive with NY-ESO-1, J Clin Oncol, 29 7 917924 , 1527-7755
118 - S. F. Rosati, R. F. Williams, L. C. Nunnally, et al. 2008 Ifn-Beta Sensitizes Neuroblastoma to the Antitumor Activity of Temozolomide by Modulating O6-Methylguanine DNA Methyltransferase Expression, Mol Cancer Ther, 7 12 38523858 , 1535-7163
119 - S. A. Rosenberg, J. C. Yang, D. J. Schwartzentruber, et al. 1999 Prospective Randomized Trial of the Treatment of Patients with Metastatic Melanoma Using Chemotherapy with Cisplatin, Dacarbazine, and Tamoxifen Alone or in Combination with Interleukin-2 and Interferon Alfa-2b, J Clin Oncol, 17 3 968975 , 0732-183X
120 - S. A. Rosenberg, N. P. Restifo, J. C. Yang, et al. 2008 Adoptive Cell Transfer: A Clinical Path to Effective Cancer Immunotherapy, Nat Rev Cancer, 8 4 299308 , 1474-1768
121 - S. A. Rosenberg, M. E. Dudley, 2009 Adoptive Cell Therapy for the Treatment of Patients with Metastatic Melanoma, Curr Opin Immunol, 21 2 233240 , 1879-0372
122 - S. A. Rosenberg, J. C. Yang, R. M. Sherry, et al. 2011 Durable Complete Responses in Heavily Pretreted Patients with Metastatic Melanoma Using T Cell Transfer Immunotherapy, Clin Cancer Res, 10780432
123 - K. Rosner, M. F. Kasprzak, A. C. Horenstein, et al. 2011 Engineering a Waste Management Enzyme to Overcome Cancer Resistance to Apoptosis: Adding Dnase1 to the Anti-Cancer Toolbox, Cancer Gene Ther, 18 5 346357 , 1476-5500
124 - B. Y. Rubin, L. J. Smith, G. R. Hellermann, et al. 1988 Correlation between the Anticellular and DNA Fragmenting Activities of Tumor Necrosis Factor, Cancer Res, 48 21 60066010 , 0008-5472
125 - M. L. Salem, A. N. Kadima, S. A. El -Naggar, et al. 2007 Defining the Ability of Cyclophosphamide Preconditioning to Enhance the Antigen-Specific CD8+ T-Cell Response to Peptide Vaccination: Creation of a Beneficial Host Microenvironment Involving Type I IFNs and Myeloid Cells, J Immunother, 30 1 4053 , 1524-9557
126 - A. D. Sasse, E. C. Sasse, L. G. Clark, et al. 2007 Chemoimmunotherapy Versus Chemotherapy for Metastatic Malignant Melanoma, Cochrane Database Syst Rev, 1 CD005413 1469-493X
127 - G. Schiavoni, F. Mattei, T. Di Pucchio, et al. 2000 Cyclophosphamide Induces Type I Interferon and Augments the Number of CD44(hi) T Lymphocytes in Mice: Implications for Strategies of Chemoimmunotherapy of Cancer, Blood, 95 6 20242030 , 0006-4971
128 - J. Schlom, P. M. Arlen, J. L. Gulley, 2007 Cancer Vaccines: Moving Beyond Current Paradigms, Clin Cancer Res, 13 13 37763782 , 1078-0432
129 - J. Schoenfeld, M. Jinushi, Y. Nakazaki, et al. 2010 Active Immunotherapy Induces Antibody Responses That Target Tumor Angiogenesis, Cancer Res, 70 24 1015010160 , 1538-7445
130 - P. Seam, J. E. Janik, D. L. Longo, et al. 2009 Role of Chemotherapy in Hodgkin’s Lymphoma, Cancer J, 15 2 150154 , 1528-9117
131 - L. Serrone, P. Hersey, 1999 The Chemoresistance of Human Malignant Melanoma: An Update, Melanoma Res, 9 1 5158 , 0960-8931
132 - J. G. Sinkovics, J. C. Horvath, 2006 Evidence Accumulating in Support of Cancer Vaccines Combined with Chemotherapy: A Pragmatic Review of Past and Present Efforts, Int J Oncol, 29 4 765777 , 1019-6439
133 - K. Skak, H. Sondergaard, K. S. Frederiksen, et al. 2009 In Vivo Antitumor Efficacy of Interleukin-21 in Combination with Chemotherapeutics, Cytokine, 48 3 231238 , 1096-0023
134 - R. Spolski, W. J. Leonard, 2008 Interleukin-21: Basic Biology and Implications for Cancer and Autoimmunity, Annu Rev Immunol, 26 5779 , 0732-0582
135 - B. J. Sugarman, B. B. Aggarwal, P. E. Hass, et al. 1985 Recombinant Human Tumor Necrosis Factor-Alpha: Effects on Proliferation of Normal and Transformed Cells in Vitro, Science, 230 4728 943945 , 0036-8075
136 - S. J. Szabo, B. M. Sullivan, S. L. Peng, et al. 2003 Molecular Mechanisms Regulating Th1 Immune Responses, Annu Rev Immunol, 21 713758 , 0732-0582
137 - S. T. Tagawa, E. Cheung, W. Banta, et al. 2006 Survival Analysis after Resection of Metastatic Disease Followed by Peptide Vaccines in Patients with Stage IV Melanoma, Cancer, 106 6 13531357 , 0008-543X
138 - J. A. Thompson, P. J. Gold, A. Fefer, 1997 Outpatient Chemoimmunotherapy for the Treatment of Metastatic Melanoma, Semin Oncol, 24 1 Suppl 4, S44S48 , 0093-7754
139 - H. Tsao, M. B. Atkins, A. J. Sober, 2004 Management of Cutaneous Melanoma, N Engl J Med, 351 10 9981012 , 1533-4406
140 - J. L. Turk, D. Parker, 1982 Effect of Cyclophosphamide on Immunological Control Mechanisms, Immunol Rev, 65 99113 , 0105-2896
141 - R. Van Horssen, Hagen. T. L. Ten, A. M. Eggermont, 2006 TNF-Alpha in Cancer Treatment: Molecular Insights, Antitumor Effects, and Clinical Utility, Oncologist, 11 4 397408 , 1083-7159
142 - T. A. Waldmann, 2006 The Biology of Interleukin-2 and Interleukin-15: Implications for Cancer Therapy and Vaccine Design, Nat Rev Immunol, 6 8 595601 , 1474-1733
143 - Y. Wang, S. Radfar, H. T. Khong, 2010 Activated CD4+ T Cells Enhance Radiation Effect through the Cooperation of Interferon-Gamma and TNF-Alpha, BMC Cancer, 10 60 1471-2407
144 - N. Watanabe, Y. Niitsu, H. Umeno, et al. 1988 Toxic Effect of Tumor Necrosis Factor on Tumor Vasculature in Mice, Cancer Res, 48 8 21792183 , 0008-5472
145 - C. J. Wheeler, A. Das, G. Liu, et al. 2004 Clinical Responsiveness of Glioblastoma Multiforme to Chemotherapy after Vaccination, Clin Cancer Res, 10 16 53165326 , 1078-0432
146 - C. Wrzesinski, C. M. Paulos, A. Kaiser, et al. 2010 Increased Intensity Lymphodepletion Enhances Tumor Treatment Efficacy of Adoptively Transferred Tumor-Specific T Cells, J Immunother, 33 1 17 , 1537-4513
147 - A. S. Yang, P. B. Chapman, 2009 The History and Future of Chemotherapy for Melanoma, Hematol Oncol Clin North Am, 23 3 583597 , 1558-1977
148 - H. G. Yang, B. L. Hu, L. Xiao, et al. 2011 Dendritic Cell-Directed Lentivector Vaccine Induces Antigen-Specific Immune Responses against Murine Melanoma, Cancer Gene Ther, 18 5 370380 , 1476-5500
149 - J. R. Yannelli, J. M. Wroblewski, 2004 On the Road to a Tumor Cell Vaccine: 20 Years of Cellular Immunotherapy, Vaccine, 23 1 97113 , 0264-410X
150 - J. S. Yi, M. Du, A. J. Zajac, 2009 A Vital Role for Interleukin-21 in the Control of a Chronic Viral Infection, Science, 324 5934 15721576 , 1095-9203
151 - L. Zhang, S. P. Kerkar, Z. Yu, et al. 2011 Improving Adoptive T Cell Therapy by Targeting and Controlling Il-12 Expression to the Tumor Environment, Mol Ther, 19 4 751759 , 1525-0024
152 - M. Zheng, D. Bocangel, R. Ramesh, et al. 2008 Interleukin-24 Overcomes Temozolomide Resistance and Enhances Cell Death by Down-Regulation of O6-Methylguanine-DNA Methyltransferase in Human Melanoma Cells, Mol Cancer Ther, 7 12 38423851 , 1535-7163
153 - L. Zitvogel, L. Apetoh, F. Ghiringhelli, et al. 2008 Immunological Aspects of Cancer Chemotherapy, Nat Rev Immunol, 8 1 5973 , 1474-1741
154 - M.B Atkins, S. Lee, L.E. Flaherty, et al. 2003 A Prospective Randomized Phase III Trial of Concurrent Biochemotherapy (BCT) with Cisplatin, Vinblastine, Dacarbazine (CVD), IL-2, and Interferon α-2b (IFN) Versus CVD Alone in Patients with Metastatic Melanoma (E3695): An ECOG-Coordinated Intergroup Trial. In: Program and abstracts of the American Society of Clinical Oncology Annual Meeting, May 31June 3 , 708a