IntroductionAccording 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 preneoplastic 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 www.intechopen.com

The immune system includes a subpopulation of CD8(+) T cells equipped to inhibit the expansion of follicular T helper (T(FH)) cells, resulting in suppression of autoantibody production and associated lupus-like disease. These CD8(+) T regulatory (Treg) cells recognize Qa-1/peptide complexes on target T(FH) cells and depend on the IL-15 cytokine for development and function. Here we show that these CD8(+) Treg cells express a triad of surface receptors--CD44, CD122, and the class I MHC receptor Ly49--and account for <5% of CD8(+) T cells. Moreover, the development of systemic lupus erythematosus-like disease in B6-Yaa mutant mice is associated with a pronounced defect in CD8(+) Treg cell activity, suggesting that this regulatory subset may represent an effective therapeutic approach to systemic lupus erythematosus-like autoimmune disease.

Background: Approaches that enhance radiation effect may lead to improved clinical outcome and decrease toxicity. Here we investigated whether activated CD4+ T cells (aCD4) can serve as an effective radiosensitizer.Methods: CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs. Hela cells were presensitized with aCD4 or conditioned supernatant (aCD4S) or recombinant cytokines for 2 days, followed gamma-irradiation. The treated cells were cultured for an additional 2 to 5 days for cell proliferation, cell cycle, and western blot assays. For confirmation, other cancer cell lines were also used.Results: Presensitization of tumor cells with aCD4 greatly increased tumor cell growth inhibition. Soluble factors secreted from activated CD4(+) T cells were primarily responsible for the observed effect. IFN-gamma seemed to play a major role. TNF-alpha, though inactive by itself, significantly augmented the radiosensitizing activity of IFN-gamma. aCD4S, but not IFN-gamma or IFN-gamma/TNF-alpha combination, was found to enhance the gamma-irradiation-induced G2/M phase arrest. Bax expression was highly upregulated in Hela cells presensitized with aCD4S followed by gamma-irradiation. The radiosensitizing activity of aCD4 is not uniquely observed with Hela cell line, but also seen with other cancer cell lines of various histology.Conclusions: Our findings suggest possible molecular and cellular mechanisms that may help explain the radio-sensitization effect of activated lymphocytes, and may provide an improved strategy in the treatment of cancer with radiotherapy.

Background: Melanoma incidence is on the rise and advanced melanoma carries an extremely poor prognosis. Treatment options, including chemotherapy and immunotherapy, are limited and offer low response rates and transient efficacy. Thus, identification of new melanocyte/melanoma antigens that serve as potential novel candidate biomarkers in melanoma is an important area for investigation.Methods: Full length MITF-M and its splice variant cDNA were cloned from human melanoma cell line 624 mel by reverse transcription polymerase chain reaction (RT-PCR). Expression was investigated using regular and quantitative RT-PCR in three normal melanocytes (NHEM), 31 melanoma cell lines, 21 frozen melanoma tissue samples, 18 blood samples (pheripheral blood mononuclear cell; PBMC) from healthy donors and 12 non-melanoma cancer cell lines, including three breast, five glioma, one sarcoma, two kidney and one ovarian cancer cell lines.Results: A novel splice variant of MITF-M, which we named MITF-Mdel, was identified. The predicted MITF-Mdel protein contains two in frame deletions, 56- and 6-amino acid deletions in exon 2 (from V32 to E87) and exon 6 (from A187 to T192), respectively. MITF-Mdel was widely expressed in melanocytes, melanoma cell lines and tissues, but almost undetectable in non-melanoma cell lines or PBMC from healthy donors. Both isoforms were expressed significantly higher in melanoma tissues than in cell lines. Two of 31 melanoma cell lines expressed only one isoform or the other.Conclusion: MITF-Mdel, a novel melanocyte/melanoma-specific isoform of MITF-M, may serve as a potential candidate biomarker for diagnostic and follow-up purposes in melanoma.

OCT4 is a transcription factor critical for the pluripotency of human embryonal stem (ES) and induced pluipotency stem (IPS) cells. OCT4 is commonly expressed in germ-cell tumors as well as putative cancer stem cells in several tumors, and is a key determinant of oncogenic fate in germ-cell tumors. The capacity of the human immune system to recognize this critical stem-cell gene is not known, but has implications for preventing tumors with ES/IPS-based therapies and targeting stem-cell pathways in cancer. Here we show that OCT4-specific T cells can be readily detected in freshly isolated T cells from most (>80%) healthy donors. The reactivity to OCT4-derived peptides resides primarily in the CD45RO + memory T-cell compartment and consists predominantly of CD4 + T cells. T cells reactive against OCT4-derived peptides can be readily expanded in culture using peptide-loaded dendritic cells. In contrast to healthy donors, immunity to OCT4 was detected in only 35% of patients with newly diagnosed germ-cell tumors. However, chemotherapy of germ-cell tumors led to the induction of anti-OCT4 immunity in vivo in patients lacking such responses at baseline. These data demonstrate the surprising lack of immune tolerance to this critical pluripotency antigen in humans. Harnessing natural immunity to this antigen may allow immune-based targeting of pluripotency-related pathways for prevention of cancers, including those in the setting of ES/IPS-based therapies.

Chemoimmunotherapy has been widely studied in melanoma, with various degrees of success. One of the most common approaches is the so-called biochemotherapy, which is associated with increased toxicities, but without overall survival benefit. Another conventional strategy is the use of 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 resulted in an impressive response rate. However, the development of immunotherapy for the treatment of a broad range of cancer type is still lacking. In this study, we report the development of a simple yet universal approach termed "chemocentric chemoimmunotherapy" that has potential application in the treatment of all cancer types. This technique uses nonspecifically activated CD4(+) T cells as a chemosensitizer before the administration of chemotherapy. Dramatic enhancement of the cytotoxic effect of chemotherapeutic drugs, either active or nonactive as single agents, was observed both in in vitro and in vivo human tumor xenograft models. Soluble factors secreted from activated CD4(+) T cells, likely acting on the tumor and its microenvironment, were responsible for the observed effect. Although IFN-gamma played a major role in the therapeutic outcome, it was consistently found to be inferior to the use of activated CD4(+) T cells in tumor chemosensitization. Our model may provide a plausible mechanism to facilitate further understanding, design and development of improved chemoimmunotherapy in the treatment of cancer.

Lytic bone disease in myeloma is characterized by an increase in multinucleate osteoclasts in close proximity to tumor cells. However, the nature of osteoclast precursors and the mechanisms underlying multinuclearity are less understood. Here we show that culture of myeloma cell lines as well as primary myeloma cells with human dendritic cells (DCs) but not monocytes or macrophages leads to spontaneous cell-cell fusion, which then leads to the facile formation of multinucleate bone-resorbing giant cells. Osteoclastogenesis is cell contact dependent, leading to up-regulation of thrombospondin-1 (TSP-1) in DCs. Disruption of CD47-TSP-1 interaction by TSP-1-blocking antibodies or down-regulation of CD47 on tumor cells by RNA interference abrogates tumor-induced osteoclast formation. Blockade of CD47-TSP-1 interactions also inhibits receptor activator for nuclear factor kappaB ligand- and macrophage colony-stimulating factor-induced formation of osteoclasts from human monocytes. Further, TSP-1 blockade attenuates hypercalcemia induced by parathyroid hormone in vivo. These data point to a role for CD47-TSP-1 interactions in regulating cell-fusion events involved in human osteoclast formation. They also suggest that DCs, known to be enriched in myeloma tumors, may be direct precursors for tumor-associated osteoclasts. Disruption of CD47-TSP-1 interactions or preventing the recruitment of DCs to tumors may provide novel approaches to therapy of myeloma bone disease and osteoporosis.

3116 Background: Radiotherapy is one of the standard modalities for the treatment of cancer. Irradiation causes not only tumor cell death, but also can up-regulate tumor antigen and MHC class I molecule expression, thereby promoting the accumulation and activation of immune cells in tumor tissue. We hypothesize that activated CD4+ T cells (aCD4) can presensitize tumor cells and enhance gamma-irradiation -induced cytotoxicity. The rationale is that aCD4 release multiple cytokines, which may regulate gene-expression in tumor cells, thereby affecting their proliferation and apoptosis. Methods: Human cervical carcinoma cell line Hela was used as our initial tumor model. Hela cells were irradiated with 4 Gy after presensitization with aCD4 for 48 hours. The cells were cultured for 2 or 5 more days for apoptosis and cell proliferation assays, respectively. Transwell system was used to determine whether cell contact, soluble factors released by aCD4, or both were essential in this model. Several cytokines including IFN- γ were used to replace aCD4 to determine the contribution of each cytokine. Other cancer cell lines used to validate the strategy included cervical carcinoma cell line Caski, glioma cell lines LN229 and U373, and prostate carcinoma cell line DU145. Results: For Hela cells, growth inhibition was 25.8 ±3.3% in group treated with irradiation alone, 31.5±11.3% in group treated with aCD4 alone, and 60.5±1.3% in the combination group; while the percentage of apoptotic cells as determined by flow cytometry assay using Annexin V was 7.15±0.6%, 9.25±0.4% and 15.65±4.6%, respectively. Similar results were also obtained with other cancer cell lines. For example, for DU145, cell growth inhibition was 35.4±4.0 % in group treated with irradiation alone, 32.65±2.7% in group treated with aCD4 alone, and 70.3±0.14% in the combination group. This effect was aCD4 and irradiation dose dependent. Transwell experiments showed that soluble factors secreted by aCD4 were responsible for the observed activity. Of the cytokines tested, IFN-γ was found to play major role in our model. Growth inhibition was 10.15±1.0% in group treated with IFN- γ alone and 57.03±1.9% in the IFN- γ and gamma-irradiation combination group. Conclusion: We have demonstrated that aCD4 can act as a radiosensitizer to enhance the cytotoxic effect of gamma irradiation in multiple cancer cell lines. Soluble factors released by aCD4 were essential in this effect. IFN- γ was found to play a major role. We are currently working on the mechanism underlying the observed activity. In a separate project, we have also demonstrated great synergism when chemotherapy was given after tumor cells had been presensitized with activated immune cells.

Advanced melanoma is a devastating disease with a very poor overall prognosis. There are only two agents that are approved by the FDA for use in patients with metastatic melanoma: dacarbazine and IL-2. Both agents have an overall response rate well below 20%, with only rare long-term responders noted. Metastatic melanoma is known to be one of the most resistant cancers to a plethora of treatment modalities, such as single-agent and combination chemotherapy, chemoimmunotherapy and immunotherapy with a host of immune stimulators. Indeed, researchers worldwide have recognized the lack of effective therapies and have refocused their efforts on developing novel and cutting-edge strategies of treatment. This is based on an improved understanding of the complex interactions that occur within the tumor microenvironment, and the central role that the host immune system plays in the surveillance of cancer. This review summarizes the recent results of novel immunotherapeutic regimens and focuses on cutting-edge modalities of treatment that encompass new lines of thinking in the war against cancer and, in particular, melanoma.

956 Background: Most patients with metastatic cancer will eventually succumb to their disease despite an initial response to chemotherapy. To enhance treatment efficacy, chemotherapy has been combined with cytokines, often with disappointing results. Another example is “Immunocentric Chemoimmunotherapy,” with primary focus placed on immunotherapy, with chemotherapy as an immunomodulator. We have developed a novel strategy termed “Chemocentric Chemoimmunotherapy,” where chemotherapy plays the central role, with immunotherapy as a chemosensitizer. The rationale is the ability of activated T cells to secret multiple cytokines, which may regulate gene expression in tumor cells. Methods: Human CD4+ T cells were isolated and activated with anti-CD3/anti-CD28 mAb. Tumor cells were presensitized with activated CD4+ T cells (aCD4), followed by treatment with various chemotherapy. Cell viability and apoptosis were determined. Transwell system was used to assess contribution of cell-cell contact vs. soluble factor(s). Results: Presensitization of A375 melanoma cells with aCD4 synergistically enhanced the cytotoxic effects of chemotherapy. Cell viability (CV) was 75% (+SD: 0.11) and 90% (+SD: 0.26) after treatment with temozolomide (TMZ) alone or with aCD4 alone, respectively. However, CV in the experimental group was 7% (+SD: 0.02). This synergistic effect was also observed with other melanoma cell lines, nonmelanoma tumor cell lines, and other chemotherapy. Two breast, one prostate, and one colon cancer cell lines were presensitized with aCD4, followed by treatment with paclitaxel, carboplatin, or 5-FU. Consistent synergistic effect was seen in each case. For example, CV in MDA-MB-231 breast cancer cells treated with paclitaxel alone or aCD4 alone was 93% (+SD: 0.16) and 52% (+SD: 0.10), respectively. However, CV in the experimental group was 8% (+SD: 0.18). The same synergism was observed in DU145 prostate cancer cell line treated with carboplatin. CV in the carboplatin-treated group and aCD4-treated group was 100% (+ SD: 0.13) and 75% (+SD: 0.14), respectively; while CV of the experimental group was 5% (+SD: 0.19). The rate of apoptosis in cells treated with both TMZ and aCD4 was 20 times higher than in cells treated with only TMZ or aCD4. This effect was due entirely to soluble factors. The aCD4 was found to secret large amounts of multiple cytokines. Preliminary data showed that IFN-γ might not be the major cytokine mediating the observed effect. Conclusion: Presensitization of tumor cells with aCD4 synergistically enhanced the cytotoxic effect of chemotherapy. This effect was completely dependent on soluble factors, most likely cytokines. We are in the process of elucidating the mechanisms underlying this dramatic synergy.

Recent studies have shown the involvement of the Fas system (Fas receptor and its ligand FasL) in cancerous processes. The absence or downregulation of Fas, reported in the majority of human tumors, conflicts with its presence in cancerous cells from the same tumors but maintained in vitro. Recently, the eventual role of environmental factors in the loss of Fas expression, or in the in vivo selection of a Fas-negative cell population has been suggested. We determined the Fas expression and function in the Capan-1 human cancerous pancreatic duct cells over 2 successive passages in vivo separated by a period of 10-20 passages in vitro. We showed that Capan-1 cells express Fas and are sensitive to Fas-mediated apoptosis when maintained in vitro. When these cells were xenografted into nude mice the expression of Fas was lost in the majority of the tumors. Culture of tumor-derived cells exhibited that they became Fas-positive and sensitive to Fas-mediated apoptosis after a short period in vitro. The loss/gain of Fas was reproduced after re-explantation and re-culture of these Fas-expressing cells. Furthermore, RT-PCR evidenced a strong inhibition of Fas, FLICE and FADD mRNAs expression in the xeno-grafts. Our observations indicate that the expression of Fas and its function could depend to factors in the tumoral environment. The in vivo loss of Fas may thus play an important role in the tumor formation and in the evasion of tumor cells from immune surveillance. (c) 2005 Wiley-Liss, Inc.