RESUMO
BACKGROUND: Public health sun safety campaigns introduced during the 1980s have successfully reduced skin cancer rates in Australia. Despite this success, high rates of sunburn continue to be reported by youth and young adults. As such, new strategies to reinforce sun protection approaches in this demographic are needed. OBJECTIVE: This study aims to develop a virtual reality (VR) game containing preventive skin cancer messaging and to assess the safety and satisfaction of the design based on end user feedback. METHODS: Using a two-phase design approach, we created a prototype VR game that immersed the player inside the human body while being confronted with growing cancer cells. The first design phase involved defining the problem, identifying stakeholders, choosing the technology platform, brainstorming, and designing esthetic elements. In the second design phase, we tested the prototype VR experience with stakeholders and end users in focus groups and interviews, with feedback incorporated into refining and improving the design. RESULTS: The focus groups and interviews were conducted with 18 participants. Qualitative feedback indicated high levels of satisfaction, with all participants reporting the VR game as engaging. A total of 11% (2/8) of participants reported a side effect of feeling nauseous during the experience. The end user feedback identified game improvements, suggesting an extended multistage experience with visual transitions to other environments and interactions involving cancer causation. The implementation of the VR game identified challenges in sharing VR equipment and hygiene issues. CONCLUSIONS: This study presents key findings highlighting the design and implementation approaches for a VR health intervention primarily aimed at improving sun protection behaviors. This design approach can be applied to other health prevention programs in the future.
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The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti-apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra-terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti-apoptotic targets regulated by NF-kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro-apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I-BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3-only protein, BIM, and downregulated anti-apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT-199 or ABT-263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I-BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance.
Assuntos
Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pirimidinas/farmacologia , Tiofenos/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.
Assuntos
Genoma Humano/genética , Melanoma/genética , Mutação/genética , DNA Helicases/genética , GTP Fosfo-Hidrolases/genética , Genes p16 , Humanos , Melanoma/classificação , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Neurofibromatose 1/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Processamento de RNA/genética , Transdução de Sinais/efeitos dos fármacos , Telomerase/genética , Telômero/genética , Proteína Supressora de Tumor p53/genética , Raios Ultravioleta/efeitos adversos , Proteína Nuclear Ligada ao XRESUMO
To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.
Assuntos
Melanoma/genética , Melanoma/secundário , Neoplasias Cutâneas/patologia , Microambiente Tumoral , Sequência de Bases , Comunicação Celular , Ciclo Celular , Resistencia a Medicamentos Antineoplásicos/genética , Células Endoteliais/patologia , Genômica , Humanos , Imunoterapia , Ativação Linfocitária , Melanoma/terapia , Fator de Transcrição Associado à Microftalmia/metabolismo , Metástase Neoplásica , RNA/genética , Análise de Sequência de RNA , Análise de Célula Única , Células Estromais/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , TranscriptomaRESUMO
Somatic mutation analysis of melanoma has been performed at the single gene level extensively over the past several decades. This has provided considerable insight into the critical pathways controlling melanoma initiation and progression. During the last 5 yr, next-generation sequencing (NGS) has enabled even more comprehensive mutational screening at the level of multigene panels, exomes and genomes. These studies have uncovered many new and unexpected players in melanoma development. The recent landmark study from The Cancer Genome Atlas (TCGA) consortium describing the genomic architecture of 333 cutaneous melanomas provides the largest and broadest analysis to date on the somatic aberrations underlying melanoma genesis. It thus seems timely to review the mutational landscape of melanoma and highlight the key genes and cellular pathways that appear to drive this cancer.
Assuntos
Genoma Humano , Melanoma/genética , Animais , Genes Neoplásicos , Humanos , Taxa de Mutação , Regiões Promotoras Genéticas/genética , Neoplasias Cutâneas , Raios Ultravioleta , Melanoma Maligno CutâneoRESUMO
Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.
Assuntos
Biomarcadores Tumorais/genética , Exoma/genética , Melanoma/genética , Mutação/genética , Neoplasias Cutâneas/genética , Proteínas Ativadoras de ras GTPase/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Melanoma/mortalidade , Melanoma/patologia , Prognóstico , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Taxa de SobrevidaRESUMO
The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma.
Assuntos
Genes Supressores de Tumor , Melanoma/genética , Melanoma/metabolismo , Complexo Repressor Polycomb 2/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Fator 3 Ativador da Transcrição/genética , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste , Epigenômica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genótipo , Histonas/química , Humanos , Indóis/química , Concentração Inibidora 50 , Mutação , Piridonas/química , Regulação para CimaRESUMO
BACKGROUND: The shelterin complex protects chromosomal ends by regulating how the telomerase complex interacts with telomeres. Following the recent finding in familial melanoma of inactivating germline mutations in POT1, encoding a member of the shelterin complex, we searched for mutations in the other five components of the shelterin complex in melanoma families. METHODS: Next-generation sequencing techniques were used to screen 510 melanoma families (with unknown genetic etiology) and control cohorts for mutations in shelterin complex encoding genes: ACD, TERF2IP, TERF1, TERF2, and TINF 2. Maximum likelihood and LOD [logarithm (base 10) of odds] analyses were used. Mutation clustering was assessed with χ(2) and Fisher's exact tests. P values under .05 were considered statistically significant (one-tailed with Yates' correction). RESULTS: Six families had mutations in ACD and four families carried TERF2IP variants, which included nonsense mutations in both genes (p.Q320X and p.R364X, respectively) and point mutations that cosegregated with melanoma. Of five distinct mutations in ACD, four clustered in the POT1 binding domain, including p.Q320X. This clustering of novel mutations in the POT1 binding domain of ACD was statistically higher (P = .005) in melanoma probands compared with population control individuals (n = 6785), as were all novel and rare variants in both ACD (P = .040) and TERF2IP (P = .022). Families carrying ACD and TERF2IP mutations were also enriched with other cancer types, suggesting that these variants also predispose to a broader spectrum of cancers than just melanoma. Novel mutations were also observed in TERF1, TERF2, and TINF2, but these were not convincingly associated with melanoma. CONCLUSIONS: Our findings add to the growing support for telomere dysregulation as a key process associated with melanoma susceptibility.
Assuntos
Códon sem Sentido , Melanoma/genética , Neoplasias Cutâneas/genética , Proteínas de Ligação a Telômeros/genética , Adulto , Idoso , DNA de Neoplasias/análise , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Mutação Puntual , Análise de Sequência de DNA , Complexo Shelterina , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/genéticaAssuntos
GTP Fosfo-Hidrolases/metabolismo , Melanoma/tratamento farmacológico , Proteínas de Membrana/metabolismo , Mutação/genética , Neurofibromina 1/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , Linhagem Celular Tumoral , Análise Mutacional de DNA , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melanoma/genética , Piridonas/farmacologia , Pirimidinonas/farmacologiaRESUMO
BACKGROUND: 5-year survival for melanoma metastasis to regional lymph nodes (American Joint Committee on Cancer stage III) is <50%. Knowledge of outcomes following therapeutic lymphadenectomy for stage III melanoma related to BRAF status may guide adjuvant use of BRAF/MEK inhibitors along with established and future therapies. AIMS: To determine patterns of melanoma recurrence and survival following therapeutic lymph node dissection (TLND) associated with oncogenic mutations. METHODS: DNA was obtained from patients who underwent TLND and had ⩾2 positive nodes, largest node >3cm or extracapsular invasion. Mutations were detected using an extended Sequenom MelaCARTA panel. RESULTS: Mutations were most commonly detected in BRAF (57/124 [46%] patients) and NRAS (26/124 [21%] patients). Patients with BRAF mutations had higher 3-year recurrence rate (77%) versus 54% for BRAF wild-type patients (hazard ratio (HR) 1.8, p=0.008). The only prognostically significant mutations occurred in BRAF: median recurrence-free (RFS) and disease-specific survival (DSS) for BRAF mutation patients was 7 months and 16 months, versus 19 months and not reached for BRAF wild-type patients, respectively. Multivariate analysis identified BRAF mutant status and number of positive lymph nodes as the only independent prognostic factors for RFS and DSS. CONCLUSIONS: Patients with BRAF mutations experienced rapid progression of metastatic disease with locoregional recurrence rarely seen in isolation, supporting incorporation of BRAF status into melanoma staging and use of BRAF/MEK inhibitors post-TLND.
Assuntos
Melanoma/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Análise Mutacional de DNA , Feminino , Seguimentos , Humanos , Imunoterapia , Estimativa de Kaplan-Meier , Excisão de Linfonodo , Metástase Linfática , Masculino , Melanoma/cirurgia , Melanoma/terapia , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Prognóstico , Modelos de Riscos Proporcionais , Proto-Oncogene Mas , Radioterapia Adjuvante , Neoplasias Cutâneas/cirurgia , Neoplasias Cutâneas/terapiaRESUMO
The incidence of melanoma continues to rise globally and is increasing at a rate greater than any other cancer. To systematically search for new genes involved in melanomagenesis, we collated exome sequencing data from independent melanoma cohort datasets, including those in the public domain. We identified recurrent mutations that may drive melanoma growth, survival or metastasis, and which may hold promise for the design of novel therapies to treat melanoma. These included a frequent recurrent (i.e. hotspot) mutation in the 5' untranslated region of RPS27 in ~10% of samples. We show that the mutation expands the 5'TOP element, a motif known to regulate the expression of most of the ribosomal protein family, to which RPS27 belongs, and thus might sensitize the mutated transcript to growth-mediated regulation. This finding highlights not only the important role of non-protein coding genetic aberrations in cancer development but also their potential as novel therapeutic targets.
Assuntos
Regiões 5' não Traduzidas , Melanoma/genética , Metaloproteínas/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Neoplasias Cutâneas/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/biossíntese , Quinase 6 Dependente de Ciclina/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Invasividade NeoplásicaRESUMO
Patients with advanced metastatic melanoma have poor prognosis and the genetics underlying its pathogenesis are poorly understood. High-throughput sequencing has allowed comprehensive discovery of somatic mutations in cancer samples. Here, on analysis of our whole-genome and whole-exome sequencing data of 29 melanoma samples, we identified several genes that harbor recurrent nonsynonymous mutations. These included MAP3K5 (mitogen-activated protein kinase kinase kinase-5), which in a prevalence screen of 288 melanomas was found to harbor a R256C substitution in 5 cases. All MAP3K5-mutated samples were wild type for BRAF, suggesting a mutual exclusivity for these mutations. Functional analysis of the MAP3K5 R256C mutation revealed attenuation of MKK4 (mitogen-activated protein kinase kinase 4) activation through increased binding of the inhibitory protein thioredoxin (TXN/TRX-1/Trx), resulting in increased proliferation and anchorage-independent growth of melanoma cells. This mutation represents a potential target for the design of new therapies to treat melanoma.
Assuntos
MAP Quinase Quinase Quinase 5/genética , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Tiorredoxinas/metabolismo , Apoptose/fisiologia , Proliferação de Células , Células HEK293 , Humanos , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Melanoma/metabolismo , Modelos Genéticos , Mutação Puntual , Ligação Proteica , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/metabolismo , Células Tumorais CultivadasRESUMO
The individuals carrying melanocortin-1 receptor (MC1R) variants, especially those associated with red hair color, fair skin, and poor tanning ability (RHC trait), are more prone to melanoma; however, the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers phosphatase and tensin homolog (PTEN) interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAF(V600E), MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes' response to UVB exposure and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis.
Assuntos
Regulação da Expressão Gênica/efeitos da radiação , Melanócitos/metabolismo , Melanoma Experimental/patologia , PTEN Fosfo-Hidrolase/metabolismo , Receptor Tipo 1 de Melanocortina/metabolismo , Pigmentação da Pele/fisiologia , Raios Ultravioleta , Animais , Western Blotting , Células Cultivadas , Humanos , Técnicas Imunoenzimáticas , Melanócitos/efeitos da radiação , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Mutação/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Melanocortina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Pigmentação da Pele/efeitos da radiação , alfa-MSH/genética , alfa-MSH/metabolismoRESUMO
Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683-691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671-5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12, and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies.
Assuntos
Apoptose/genética , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Melanoma/genética , Proteínas Musculares/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sequência de Bases , Western Blotting , Primers do DNA/genética , Exoma/genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Imunoprecipitação , Lentivirus , MicroRNAs/genética , Dados de Sequência Molecular , Proteínas Musculares/metabolismo , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/metabolismoRESUMO
Melanoma of unknown primary (MUP) is an uncommon phenomenon whereby patients present with metastatic disease without an evident primary site. To determine their likely site of origin, we combined exome sequencing from 33 MUPs to assess the total rate of somatic mutations and degree of UV mutagenesis. An independent cohort of 91 archival MUPs was also screened for 46 hot spot mutations highly prevalent in melanoma including BRAF, NRAS, KIT, GNAQ, and GNA11. Results showed that the majority of MUPs exhibited high somatic mutation rates, high ratios of C>T/G>A transitions, and a high rate of BRAF (45 of 101, 45%) and NRAS (32 of 101, 32%) mutations, collectively indicating a mutation profile consistent with cutaneous sun-exposed melanomas. These data suggest that a significant proportion of MUPs arise from regressed or unrecognized primary cutaneous melanomas or arise de novo in lymph nodes from nevus cells that have migrated from the skin.
Assuntos
Melanoma/genética , Melanoma/patologia , Mutação/genética , Neoplasias Primárias Desconhecidas/genética , Neoplasias Primárias Desconhecidas/patologia , Neoplasias Cutâneas/genética , Luz Solar , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Estudos de Coortes , Análise Mutacional de DNA , Exoma/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Cutâneas/patologia , Melanoma Maligno CutâneoRESUMO
Melanoma has historically been refractive to traditional therapeutic approaches. As such, the development of novel drug strategies has been needed to improve rates of overall survival in patients with melanoma, particularly those with late stage or disseminated disease. Recent success with molecularly based targeted drugs, such as Vemurafenib in BRAF-mutant melanomas, has now made "personalized medicine" a reality within some oncology clinics. In this sense, tailored drugs can be administered to patients according to their tumor "mutation profiles." The success of these drug strategies, in part, can be attributed to the identification of the genetic mechanisms responsible for the development and progression of metastatic melanoma. Recently, the advances in sequencing technology have allowed for comprehensive mutation analysis of tumors and have led to the identification of a number of genes involved in the etiology of metastatic melanoma. As the methodology and costs associated with next-generation sequencing continue to improve, this technology will be rapidly adopted into routine clinical oncology practices and will significantly impact on personalized therapy. This review summarizes current and emerging molecular targets in metastatic melanoma, discusses the potential application of next-generation sequencing within the paradigm of personalized medicine, and describes the current limitations for the adoption of this technology within the clinic.
Assuntos
Exoma/genética , Genoma Humano/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Terapia de Alvo Molecular , Medicina de Precisão , Análise de Sequência de DNA , HumanosRESUMO
We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1(P29S)) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1(P29S) showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.
Assuntos
Exoma/genética , Melanoma/genética , Mutação , Neoplasias Cutâneas/genética , Proteínas rac1 de Ligação ao GTP/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Melanoma/epidemiologia , Pessoa de Meia-Idade , Modelos Moleculares , Mutação/fisiologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Neoplasias Cutâneas/epidemiologia , Neoplasias Uveais/epidemiologia , Neoplasias Uveais/genética , Proteínas rac1 de Ligação ao GTP/químicaRESUMO
Success with molecular-based targeted drugs in the treatment of cancer has ignited extensive research efforts within the field of personalized therapeutics. However, successful application of such therapies is dependent on the presence or absence of mutations within the patient's tumor that can confer clinical efficacy or drug resistance. Building on these findings, we developed a high-throughput mutation panel for the identification of frequently occurring and clinically relevant mutations in melanoma. An extensive literature search and interrogation of the Catalogue of Somatic Mutations in Cancer database identified more than 1,000 melanoma mutations. Applying a filtering strategy to focus on mutations amenable to the development of targeted drugs, we initially screened 120 known mutations in 271 samples using the Sequenom MassARRAY system. A total of 252 mutations were detected in 17 genes, the highest frequency occurred in BRAF (n = 154, 57%), NRAS (n = 55, 20%), CDK4 (n = 8, 3%), PTK2B (n = 7, 2.5%), and ERBB4 (n = 5, 2%). Based on this initial discovery screen, a total of 46 assays interrogating 39 mutations in 20 genes were designed to develop a melanoma-specific panel. These assays were distributed in multiplexes over 8 wells using strict assay design parameters optimized for sensitive mutation detection. The final melanoma-specific mutation panel is a cost effective, sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular-based therapeutics for the treatment of melanoma. When used in a clinical research setting, the panel may rapidly and accurately identify potentially effective treatment strategies using novel or existing molecularly targeted drugs.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Melanoma/genética , Mutação , Neoplasias Cutâneas/genética , Alelos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Estudos de Coortes , Humanos , Metástase Linfática , Melanoma/patologia , Dados de Sequência Molecular , Neoplasias Cutâneas/patologiaRESUMO
Metastatic melanoma has traditionally been difficult to treat, and although molecularly based targeted therapies have shown promising results, they have yet to show consistent improvements in overall survival rates. Thus, identifying the key mutation events underlying the etiology of metastatic melanoma will no doubt lead to the improvement of existing therapeutic approaches and the development of new treatment strategies. Significant advances toward understanding the complexity of the melanoma genome have recently been achieved using next-generation sequencing (NGS) technologies. However, identifying those mutations driving tumorigenesis will continue to be a challenge for researchers, in part because of the high rates of mutation compared to other cancers. This article will review the catalog of mutations identified in melanoma through a variety of approaches, including the use of unbiased exome and whole-genome NGS platforms, as well discuss complementary strategies for identifying driver mutations. The promise of personalized medicine afforded by better understanding these mutation events should provide impetus for increased activity and rapid advances in this field.