1. ¹Department of Dermatology, University of Würzburg, Würzburg, Germany
2. ²Division of General Dermatology, Medical University of Graz, Graz, Austria
3. ³Department of Dermatology, University of Essen, Essen, Germany

Correspondence: Jürgen C. Becker, Division of General Dermatology, Medical University of Graz, Auenbruggerplatz 8, Graz 8036, Austria. E-mail: [email protected]

Abstract

Chemotherapeutic drugs are clinically used to treat cancer because of their cytotoxic activities against tumor cells. Recently, however, evidence is accumulating—including the report of Hervieu et al. (2012) in the current issue of The Journal of Investigative Dermatology—indicating that at least some of these drugs have broader activities and that they should also be considered immunomodulatory agents. Indeed, Hervieu demonstrates that dacarbazine (DTIC) exerts immunostimulatory effects by inducing local activation of natural killer (NK) and T cells, suggesting that upon treatment with DTIC, the tumor participates in the initiation of an immune response: (i) DTIC treatment elicits the expression of ligands of the immunoreceptor NKG2D on melanoma cells; (ii) engagement of the ligands by NKG2D on NK cells leads to their activation, allowing enhanced tumor-cell killing and the release of IFN-γ; and (iii) IFN-γ in turn upregulates major histocompatibility complex class I expression on tumor cells, which favors their recognition by cytotoxic CD8+ T lymphocytes (CTLs).

—————————————————————————————————————————

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 537–544; doi:10.1038/jid.2012.274; published online 6 September 2012

Pathophysiological Characteristics of Melanoma In-Transit Metastasis in a Lymphedema Mouse Model

Kohei Oashi¹, Hiroshi Furukawa¹, Hiroshi Nishihara², Michitaka Ozaki³, Akihiko Oyama¹, Emi Funayama¹, Toshihiko Hayashi¹, Yuji Kuge⁴ and Yuhei Yamamoto¹

Abstract

In-transit metastasis (ITM) is a unique manifestation of intralymphatic tumor dissemination, characterized by the presence of melanoma cells between the primary lesion and the draining regional lymph node basin that is clinically associated with poor prognosis. In this study, we aimed to establish an experimental animal model of melanoma ITM, as research progress in this field has been hampered by a lack of suitable experimental models. We reproduced melanoma ITM in a mouse hind limb by transplanting melanoma cells into the footpad of a mouse with lymphedema (LE). The tumor cells at the ITM site were highly proliferative, and mice with ITMs were more likely than control mice to develop distant lymph node and lung metastases. Peritumoral lymphatic vessels and tumor-associated blood vessels were increased in the primary tumor site of the LE mice. Our established ITM melanoma mouse model enabled us to clarify the molecular determinants and pathophysiology of ITM. This ITM model is also comparable to the unfavorable clinical behavior of melanoma ITM in humans and, moreover, underlined the importance of lymphangiogenic factors in the tumor dissemination through the lymphatic system.

—————————————————————————————————————————————

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 545–552; doi:10.1038/jid.2012.336; published online 27 September 2012

Methods to Improve Adoptive T-Cell Therapy for Melanoma: IFN-γ Enhances Anticancer Responses of Cell Products for Infusion

Marco Donia^1,2, Morten Hansen¹, Sarah L Sendrup¹, Trine Zeeberg Iversen^1,3, Eva Ellebæk^1,3, Mads H Andersen¹, Per thor Straten¹ and Inge Marie Svane^1,3

Abstract

Further development of adoptive T-cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has the potential to markedly change the long-term prognosis of patients with metastatic melanoma, and modifications of the original protocol that can improve its clinical efficacy are highly desirable. In this study, we demonstrated that a high in vitro tumor reactivity of infusion products was associated with clinical responses upon adoptive transfer. In addition, we systematically characterized the responses of a series of TIL products to relevant autologous short term–cultured melanoma cell lines from 12 patients. We provide evidence that antitumor reactivity of both CD8⁺ and CD4⁺ T cells could be enhanced in most TIL products by autologous melanoma sensitization by pretreatment with low-dose IFN-γ. IFN-γ selectively enhanced responses to tumor-associated antigens other than melanoma differentiation antigens. In addition, IFN-γ treatment was invariably associated with restored/increased cancer immunogenicity as demonstrated by upregulation of major histocompatibility complex molecules. These findings suggest a potential synergism between IFN-γ and ACT, and have important implications for clinical development of combination strategies for the treatment of metastatic melanoma.

———————————————————————————————————————-

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 518–527; doi:10.1038/jid.2012.317; published online 30 August 2012

Dual Role of Apoptosis-Associated Speck-Like Protein Containing a CARD (ASC) in Tumorigenesis of Human Melanoma

Weimin Liu¹, Yuchun Luo¹, Jeffrey H Dunn¹, David A Norris^1,2, Charles A Dinarello³ and Mayumi Fujita^1,2

Abstract

Apoptosis-associated Speck-like protein containing a CARD (caspase recruitment domain) (ASC) was originally named because it triggered apoptosis in certain tumors. More recently, however, ASC was found to be a central adaptor protein of inflammasome, which mediates the secretion of protumorigenic inflammation. Here we examined the role of ASC in tumorigenesis of human melanoma. Compared with primary melanoma, ASC protein expression was generally downregulated in metastatic melanoma. Although overexpressing ASC in metastatic melanoma showed no effects on cell viability, silencing ASC with short hairpin RNA induced G1 cell cycle arrest, reduced cell viability, and suppressed tumorigenesis in metastatic melanoma. On the other hand, silencing ASC in primary melanoma reduced cell death, increased cell viability, and enhanced tumorigenesis. In primary and metastatic melanoma cells, ASC knockdown inhibited inflammasome-mediated caspase-1 activity and IL-1β secretion. However, phosphorylated IκB kinase (IKK)α/β expression and NF-κB activity were suppressed in metastatic melanoma and enhanced in primary melanoma after ASC knockdown. These findings suggest stage-dependent dual roles of ASC in tumorigenesis. ASC expression in primary melanoma inhibits tumorigenesis by reducing IKKα/β phosphorylation and inhibiting NF-κB activity. In metastatic melanoma, on the other hand, this inhibitory effect is diminished, and ASC induces tumorigenic pathways through enhanced NF-κB activity and inflammasome-mediated IL-1β secretion.

—————————————————————————————————————————–

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 509–517; doi:10.1038/jid.2012.283; published online 30 August 2012

BRAF Mutation, NRAS Mutation, and the Absence of an Immune-Related Expressed Gene Profile Predict Poor Outcome in Patients with Stage III Melanoma

Graham J Mann^1,2, Gulietta M Pupo^1,2, Anna E Campain³, Candace D Carter², Sarah-Jane Schramm^1,2, Svetlana Pianova^1,2, Sebastien K Gerega⁴, Chitra De Silva^2,5, Kenneth Lai^2,5, James S Wilmott^2,5, Maria Synnott^2,5, Peter Hersey², Richard F Kefford^1,2, John F Thompson^2,6, Yee Hwa Yang³ and Richard A Scolyer^2,5,7

Abstract

Prediction of outcome for melanoma patients with surgically resected macroscopic nodal metastases is very imprecise. We performed a comprehensive clinico-pathologic assessment of fresh-frozen macroscopic nodal metastases and the preceding primary melanoma, somatic mutation profiling, and gene expression profiling to identify determinants of outcome in 79 melanoma patients. In addition to disease stage <II at initial presentation, the following clinical and pathologic factors were independent predictors of improved outcome (odds ratios for survival >4 years, 90% confidence interval): the presence of a nodular component in the primary melanoma (6.8, 0.6–76.0), and small cell size (11.1, 0.8–100.0) or low pigmentation (3.0, 0.8–100.0) in the nodal metastases. Absence of BRAF mutation (20.0, 1.0–1000.0) or NRAS mutation (16.7, 0.6–1000.0) were both favorable prognostic factors. A 46-gene expression signature with strong overrepresentation of immune response genes was predictive of better survival (10.9, 0.4–325.6); in the full cohort, median survival was >100 months in those with the signature, but 10 months in those without. This relationship was validated in two previously published independent stage III melanoma data sets. We conclude that the presence of BRAF mutation, NRAS mutation, and the absence of an immune-related expressed gene profile predict poor outcome in melanoma patients with macroscopic stage III disease.

1. ¹Department of Dermatology, University of Würzburg, Würzburg, Germany
2. ²Division of General Dermatology, Medical University of Graz, Graz, Austria
3. ³Department of Dermatology, University of Essen, Essen, Germany

Correspondence: Jürgen C. Becker, Division of General Dermatology, Medical University of Graz, Auenbruggerplatz 8, Graz 8036, Austria. E-mail: [email protected]

Abstract

Chemotherapeutic drugs are clinically used to treat cancer because of their cytotoxic activities against tumor cells. Recently, however, evidence is accumulating—including the report of Hervieu et al. (2012) in the current issue of The Journal of Investigative Dermatology—indicating that at least some of these drugs have broader activities and that they should also be considered immunomodulatory agents. Indeed, Hervieu demonstrates that dacarbazine (DTIC) exerts immunostimulatory effects by inducing local activation of natural killer (NK) and T cells, suggesting that upon treatment with DTIC, the tumor participates in the initiation of an immune response: (i) DTIC treatment elicits the expression of ligands of the immunoreceptor NKG2D on melanoma cells; (ii) engagement of the ligands by NKG2D on NK cells leads to their activation, allowing enhanced tumor-cell killing and the release of IFN-γ; and (iii) IFN-γ in turn upregulates major histocompatibility complex class I expression on tumor cells, which favors their recognition by cytotoxic CD8+ T lymphocytes (CTLs).

—————————————————————————————————————————

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 537–544; doi:10.1038/jid.2012.274; published online 6 September 2012

Pathophysiological Characteristics of Melanoma In-Transit Metastasis in a Lymphedema Mouse Model

Kohei Oashi¹, Hiroshi Furukawa¹, Hiroshi Nishihara², Michitaka Ozaki³, Akihiko Oyama¹, Emi Funayama¹, Toshihiko Hayashi¹, Yuji Kuge⁴ and Yuhei Yamamoto¹

Abstract

In-transit metastasis (ITM) is a unique manifestation of intralymphatic tumor dissemination, characterized by the presence of melanoma cells between the primary lesion and the draining regional lymph node basin that is clinically associated with poor prognosis. In this study, we aimed to establish an experimental animal model of melanoma ITM, as research progress in this field has been hampered by a lack of suitable experimental models. We reproduced melanoma ITM in a mouse hind limb by transplanting melanoma cells into the footpad of a mouse with lymphedema (LE). The tumor cells at the ITM site were highly proliferative, and mice with ITMs were more likely than control mice to develop distant lymph node and lung metastases. Peritumoral lymphatic vessels and tumor-associated blood vessels were increased in the primary tumor site of the LE mice. Our established ITM melanoma mouse model enabled us to clarify the molecular determinants and pathophysiology of ITM. This ITM model is also comparable to the unfavorable clinical behavior of melanoma ITM in humans and, moreover, underlined the importance of lymphangiogenic factors in the tumor dissemination through the lymphatic system.

—————————————————————————————————————————————

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 545–552; doi:10.1038/jid.2012.336; published online 27 September 2012

Methods to Improve Adoptive T-Cell Therapy for Melanoma: IFN-γ Enhances Anticancer Responses of Cell Products for Infusion

Marco Donia^1,2, Morten Hansen¹, Sarah L Sendrup¹, Trine Zeeberg Iversen^1,3, Eva Ellebæk^1,3, Mads H Andersen¹, Per thor Straten¹ and Inge Marie Svane^1,3

Abstract

Further development of adoptive T-cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has the potential to markedly change the long-term prognosis of patients with metastatic melanoma, and modifications of the original protocol that can improve its clinical efficacy are highly desirable. In this study, we demonstrated that a high in vitro tumor reactivity of infusion products was associated with clinical responses upon adoptive transfer. In addition, we systematically characterized the responses of a series of TIL products to relevant autologous short term–cultured melanoma cell lines from 12 patients. We provide evidence that antitumor reactivity of both CD8⁺ and CD4⁺ T cells could be enhanced in most TIL products by autologous melanoma sensitization by pretreatment with low-dose IFN-γ. IFN-γ selectively enhanced responses to tumor-associated antigens other than melanoma differentiation antigens. In addition, IFN-γ treatment was invariably associated with restored/increased cancer immunogenicity as demonstrated by upregulation of major histocompatibility complex molecules. These findings suggest a potential synergism between IFN-γ and ACT, and have important implications for clinical development of combination strategies for the treatment of metastatic melanoma.

———————————————————————————————————————-

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 518–527; doi:10.1038/jid.2012.317; published online 30 August 2012

Dual Role of Apoptosis-Associated Speck-Like Protein Containing a CARD (ASC) in Tumorigenesis of Human Melanoma

Weimin Liu¹, Yuchun Luo¹, Jeffrey H Dunn¹, David A Norris^1,2, Charles A Dinarello³ and Mayumi Fujita^1,2

Abstract

Apoptosis-associated Speck-like protein containing a CARD (caspase recruitment domain) (ASC) was originally named because it triggered apoptosis in certain tumors. More recently, however, ASC was found to be a central adaptor protein of inflammasome, which mediates the secretion of protumorigenic inflammation. Here we examined the role of ASC in tumorigenesis of human melanoma. Compared with primary melanoma, ASC protein expression was generally downregulated in metastatic melanoma. Although overexpressing ASC in metastatic melanoma showed no effects on cell viability, silencing ASC with short hairpin RNA induced G1 cell cycle arrest, reduced cell viability, and suppressed tumorigenesis in metastatic melanoma. On the other hand, silencing ASC in primary melanoma reduced cell death, increased cell viability, and enhanced tumorigenesis. In primary and metastatic melanoma cells, ASC knockdown inhibited inflammasome-mediated caspase-1 activity and IL-1β secretion. However, phosphorylated IκB kinase (IKK)α/β expression and NF-κB activity were suppressed in metastatic melanoma and enhanced in primary melanoma after ASC knockdown. These findings suggest stage-dependent dual roles of ASC in tumorigenesis. ASC expression in primary melanoma inhibits tumorigenesis by reducing IKKα/β phosphorylation and inhibiting NF-κB activity. In metastatic melanoma, on the other hand, this inhibitory effect is diminished, and ASC induces tumorigenic pathways through enhanced NF-κB activity and inflammasome-mediated IL-1β secretion.

—————————————————————————————————————————–

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 509–517; doi:10.1038/jid.2012.283; published online 30 August 2012

BRAF Mutation, NRAS Mutation, and the Absence of an Immune-Related Expressed Gene Profile Predict Poor Outcome in Patients with Stage III Melanoma

Graham J Mann^1,2, Gulietta M Pupo^1,2, Anna E Campain³, Candace D Carter², Sarah-Jane Schramm^1,2, Svetlana Pianova^1,2, Sebastien K Gerega⁴, Chitra De Silva^2,5, Kenneth Lai^2,5, James S Wilmott^2,5, Maria Synnott^2,5, Peter Hersey², Richard F Kefford^1,2, John F Thompson^2,6, Yee Hwa Yang³ and Richard A Scolyer^2,5,7

Abstract

Prediction of outcome for melanoma patients with surgically resected macroscopic nodal metastases is very imprecise. We performed a comprehensive clinico-pathologic assessment of fresh-frozen macroscopic nodal metastases and the preceding primary melanoma, somatic mutation profiling, and gene expression profiling to identify determinants of outcome in 79 melanoma patients. In addition to disease stage <II at initial presentation, the following clinical and pathologic factors were independent predictors of improved outcome (odds ratios for survival >4 years, 90% confidence interval): the presence of a nodular component in the primary melanoma (6.8, 0.6–76.0), and small cell size (11.1, 0.8–100.0) or low pigmentation (3.0, 0.8–100.0) in the nodal metastases. Absence of BRAF mutation (20.0, 1.0–1000.0) or NRAS mutation (16.7, 0.6–1000.0) were both favorable prognostic factors. A 46-gene expression signature with strong overrepresentation of immune response genes was predictive of better survival (10.9, 0.4–325.6); in the full cohort, median survival was >100 months in those with the signature, but 10 months in those without. This relationship was validated in two previously published independent stage III melanoma data sets. We conclude that the presence of BRAF mutation, NRAS mutation, and the absence of an immune-related expressed gene profile predict poor outcome in melanoma patients with macroscopic stage III disease.

1. ¹Department of Dermatology, University of Würzburg, Würzburg, Germany
2. ²Division of General Dermatology, Medical University of Graz, Graz, Austria
3. ³Department of Dermatology, University of Essen, Essen, Germany

Correspondence: Jürgen C. Becker, Division of General Dermatology, Medical University of Graz, Auenbruggerplatz 8, Graz 8036, Austria. E-mail: [email protected]

Abstract

Chemotherapeutic drugs are clinically used to treat cancer because of their cytotoxic activities against tumor cells. Recently, however, evidence is accumulating—including the report of Hervieu et al. (2012) in the current issue of The Journal of Investigative Dermatology—indicating that at least some of these drugs have broader activities and that they should also be considered immunomodulatory agents. Indeed, Hervieu demonstrates that dacarbazine (DTIC) exerts immunostimulatory effects by inducing local activation of natural killer (NK) and T cells, suggesting that upon treatment with DTIC, the tumor participates in the initiation of an immune response: (i) DTIC treatment elicits the expression of ligands of the immunoreceptor NKG2D on melanoma cells; (ii) engagement of the ligands by NKG2D on NK cells leads to their activation, allowing enhanced tumor-cell killing and the release of IFN-γ; and (iii) IFN-γ in turn upregulates major histocompatibility complex class I expression on tumor cells, which favors their recognition by cytotoxic CD8+ T lymphocytes (CTLs).

—————————————————————————————————————————

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 537–544; doi:10.1038/jid.2012.274; published online 6 September 2012

Pathophysiological Characteristics of Melanoma In-Transit Metastasis in a Lymphedema Mouse Model

Kohei Oashi¹, Hiroshi Furukawa¹, Hiroshi Nishihara², Michitaka Ozaki³, Akihiko Oyama¹, Emi Funayama¹, Toshihiko Hayashi¹, Yuji Kuge⁴ and Yuhei Yamamoto¹

Abstract

In-transit metastasis (ITM) is a unique manifestation of intralymphatic tumor dissemination, characterized by the presence of melanoma cells between the primary lesion and the draining regional lymph node basin that is clinically associated with poor prognosis. In this study, we aimed to establish an experimental animal model of melanoma ITM, as research progress in this field has been hampered by a lack of suitable experimental models. We reproduced melanoma ITM in a mouse hind limb by transplanting melanoma cells into the footpad of a mouse with lymphedema (LE). The tumor cells at the ITM site were highly proliferative, and mice with ITMs were more likely than control mice to develop distant lymph node and lung metastases. Peritumoral lymphatic vessels and tumor-associated blood vessels were increased in the primary tumor site of the LE mice. Our established ITM melanoma mouse model enabled us to clarify the molecular determinants and pathophysiology of ITM. This ITM model is also comparable to the unfavorable clinical behavior of melanoma ITM in humans and, moreover, underlined the importance of lymphangiogenic factors in the tumor dissemination through the lymphatic system.

—————————————————————————————————————————————

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 545–552; doi:10.1038/jid.2012.336; published online 27 September 2012

Methods to Improve Adoptive T-Cell Therapy for Melanoma: IFN-γ Enhances Anticancer Responses of Cell Products for Infusion

Marco Donia^1,2, Morten Hansen¹, Sarah L Sendrup¹, Trine Zeeberg Iversen^1,3, Eva Ellebæk^1,3, Mads H Andersen¹, Per thor Straten¹ and Inge Marie Svane^1,3

Abstract

Further development of adoptive T-cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has the potential to markedly change the long-term prognosis of patients with metastatic melanoma, and modifications of the original protocol that can improve its clinical efficacy are highly desirable. In this study, we demonstrated that a high in vitro tumor reactivity of infusion products was associated with clinical responses upon adoptive transfer. In addition, we systematically characterized the responses of a series of TIL products to relevant autologous short term–cultured melanoma cell lines from 12 patients. We provide evidence that antitumor reactivity of both CD8⁺ and CD4⁺ T cells could be enhanced in most TIL products by autologous melanoma sensitization by pretreatment with low-dose IFN-γ. IFN-γ selectively enhanced responses to tumor-associated antigens other than melanoma differentiation antigens. In addition, IFN-γ treatment was invariably associated with restored/increased cancer immunogenicity as demonstrated by upregulation of major histocompatibility complex molecules. These findings suggest a potential synergism between IFN-γ and ACT, and have important implications for clinical development of combination strategies for the treatment of metastatic melanoma.

———————————————————————————————————————-

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 518–527; doi:10.1038/jid.2012.317; published online 30 August 2012

Dual Role of Apoptosis-Associated Speck-Like Protein Containing a CARD (ASC) in Tumorigenesis of Human Melanoma

Weimin Liu¹, Yuchun Luo¹, Jeffrey H Dunn¹, David A Norris^1,2, Charles A Dinarello³ and Mayumi Fujita^1,2

Abstract

Apoptosis-associated Speck-like protein containing a CARD (caspase recruitment domain) (ASC) was originally named because it triggered apoptosis in certain tumors. More recently, however, ASC was found to be a central adaptor protein of inflammasome, which mediates the secretion of protumorigenic inflammation. Here we examined the role of ASC in tumorigenesis of human melanoma. Compared with primary melanoma, ASC protein expression was generally downregulated in metastatic melanoma. Although overexpressing ASC in metastatic melanoma showed no effects on cell viability, silencing ASC with short hairpin RNA induced G1 cell cycle arrest, reduced cell viability, and suppressed tumorigenesis in metastatic melanoma. On the other hand, silencing ASC in primary melanoma reduced cell death, increased cell viability, and enhanced tumorigenesis. In primary and metastatic melanoma cells, ASC knockdown inhibited inflammasome-mediated caspase-1 activity and IL-1β secretion. However, phosphorylated IκB kinase (IKK)α/β expression and NF-κB activity were suppressed in metastatic melanoma and enhanced in primary melanoma after ASC knockdown. These findings suggest stage-dependent dual roles of ASC in tumorigenesis. ASC expression in primary melanoma inhibits tumorigenesis by reducing IKKα/β phosphorylation and inhibiting NF-κB activity. In metastatic melanoma, on the other hand, this inhibitory effect is diminished, and ASC induces tumorigenic pathways through enhanced NF-κB activity and inflammasome-mediated IL-1β secretion.

—————————————————————————————————————————–

Original Article

Subject Category: Melanocytes/Melanoma

Journal of Investigative Dermatology (2013) 133, 509–517; doi:10.1038/jid.2012.283; published online 30 August 2012

BRAF Mutation, NRAS Mutation, and the Absence of an Immune-Related Expressed Gene Profile Predict Poor Outcome in Patients with Stage III Melanoma

Graham J Mann^1,2, Gulietta M Pupo^1,2, Anna E Campain³, Candace D Carter², Sarah-Jane Schramm^1,2, Svetlana Pianova^1,2, Sebastien K Gerega⁴, Chitra De Silva^2,5, Kenneth Lai^2,5, James S Wilmott^2,5, Maria Synnott^2,5, Peter Hersey², Richard F Kefford^1,2, John F Thompson^2,6, Yee Hwa Yang³ and Richard A Scolyer^2,5,7

Abstract

Prediction of outcome for melanoma patients with surgically resected macroscopic nodal metastases is very imprecise. We performed a comprehensive clinico-pathologic assessment of fresh-frozen macroscopic nodal metastases and the preceding primary melanoma, somatic mutation profiling, and gene expression profiling to identify determinants of outcome in 79 melanoma patients. In addition to disease stage <II at initial presentation, the following clinical and pathologic factors were independent predictors of improved outcome (odds ratios for survival >4 years, 90% confidence interval): the presence of a nodular component in the primary melanoma (6.8, 0.6–76.0), and small cell size (11.1, 0.8–100.0) or low pigmentation (3.0, 0.8–100.0) in the nodal metastases. Absence of BRAF mutation (20.0, 1.0–1000.0) or NRAS mutation (16.7, 0.6–1000.0) were both favorable prognostic factors. A 46-gene expression signature with strong overrepresentation of immune response genes was predictive of better survival (10.9, 0.4–325.6); in the full cohort, median survival was >100 months in those with the signature, but 10 months in those without. This relationship was validated in two previously published independent stage III melanoma data sets. We conclude that the presence of BRAF mutation, NRAS mutation, and the absence of an immune-related expressed gene profile predict poor outcome in melanoma patients with macroscopic stage III disease.