RESUMO
OBJECTIVE: Heat shock protein 60 (HSP60) plays an essential role in malignant cell survival. We evaluated the prognostic and treatment predictive value of HSP60 in advanced ovarian cancer. METHODS: Fresh tumor samples were prospectively collected from 123 patients undergoing primary surgery for suspected advanced ovarian cancer. Of these, 57 fulfilled the eligibility criteria, that is, International Federation of Gynecology and Obstetrics stage IIC-IV, serous/endometrioid tumors, platinum-based chemotherapy, and specimens with 50% tumor cells or greater. Heat shock protein 60 mRNA and protein expression was determined by real-time polymerase chain reaction and immunohistochemistry. We estimated the association between HSP60 and overall survival (OS) and platinum-free interval (PFI) by Cox proportional hazards models and its relationship with treatment response by Fisher's exact test. Median follow-up was 60 months. RESULTS: High HSP60 mRNA expression was associated with shorter OS (hazard ratio [HR], 3.4; 95% confidence interval [CI], 1.3-8.5) and PFI (HR, 3.3; 95% CI, 1.5-7.2). Likewise, high HSP60 protein expression was associated with shorter OS (HR, 3.2; 95% CI, 1.5-7.1) and PFI (HR, 2.6; 95% CI, 1.3-5.3). Median survival for patients with high HSP60 protein expression was 31 months compared with 55 months for low expression cases (P = 0.016). The impact on OS and PFI was even stronger in the subgroup of grade 3 serous tumors. All patients with low HSP60 levels responded to first-line chemotherapy. CONCLUSION: Heat shock protein 60 may identify groups of advanced serous ovarian cancer with different prognosis and treatment response.
Assuntos
Chaperonina 60/metabolismo , Cistadenocarcinoma Seroso/mortalidade , Neoplasias do Endométrio/mortalidade , Neoplasias das Tubas Uterinas/mortalidade , Proteínas Mitocondriais/metabolismo , Neoplasias Ovarianas/mortalidade , Neoplasias Peritoneais/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Chaperonina 60/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Neoplasias das Tubas Uterinas/metabolismo , Neoplasias das Tubas Uterinas/patologia , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/patologia , Prognóstico , Estudos Prospectivos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
PURPOSE: New prognostic markers are needed for malignant melanoma. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. The BRAF/NRAS mutation status and expression of COX-2 and iNOS were examined to compare their prognostic value for overall survival (OS) in stage III malignant cutaneous melanoma. EXPERIMENTAL DESIGN: The expression of iNOS and COX-2 in metastatic lymph nodes from 21 rapidly progressing (OS from date of diagnosis of stage III disease < or =14 months) and 17 slowly progressing (OS > or =60 months) stage III cutaneous melanoma patients was examined by immunohistochemistry. The presence of BRAF/NRAS mutations was analyzed using direct DNA sequencing. Chi2 exact trend test and logistic regression analysis were used for statistical analysis. RESULTS: Both iNOS (P = 0.002) and COX-2 (P = 0.048) alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly (P = 0.013) correlated with BRAF mutation frequency. Furthermore, the odds ratio (OR) with respect to OS of iNOS (OR = 10.4) was higher than that of COX-2 (OR = 5.6) and was stable in the multivariate analysis of OS together with disease stage IIIB/C, ulceration, number of metastatic lymph nodes, and Breslow tumor thickness. CONCLUSION: Our data show that iNOS is an independent and stronger prognostic factor for OS in stage III malignant cutaneous melanoma than COX-2.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Melanoma/mortalidade , Melanoma/patologia , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Proto-Oncogênicas B-raf/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Modelos Logísticos , Metástase Linfática , Masculino , Melanoma/enzimologia , Pessoa de Meia-Idade , Mutação/genética , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologiaRESUMO
Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E-mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600E-mutated melanomas, particularly when found in the context of a CDK4 mutation/overexpression.
Assuntos
Ciclina D1/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imidazóis/farmacologia , Melanoma/genética , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Substituição de Aminoácidos/efeitos dos fármacos , Sequência de Bases , Linhagem Celular Tumoral , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Análise Mutacional de DNA , Amplificação de Genes/efeitos dos fármacos , Ácido Glutâmico/genética , Humanos , Melanoma/metabolismo , Dados de Sequência Molecular , Valina/genéticaRESUMO
We have previously demonstrated the use of pyrosequencing to investigate NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog] mutations in melanoma biopsies. Here, we expanded the analysis to include BRAF (V-raf murine sarcoma viral oncogene homolog B1), another member of the Ras-Raf-mitogen-activated protein kinase (MAPK) signalling pathway, and analysed a total of 294 melanoma tumours from 219 patients. Mutations in BRAF exons 11 and 15 were identified in 156 (53%) tumours and NRAS exon 2 mutations in 86 (29%) tumours. Overall, mutations in NRAS or BRAF were found in 242 of 294 tumours (82%) and were found to be mutually exclusive in all but two cases (0.7%). Multiple metastases were analysed in 57 of the cases and mutations were identical in all except three, indicating that BRAF and NRAS mutations occur before metastasis. Association with preexisting nevi was significantly higher in BRAF mutated tumours (P=0.014). In addition, tumours with BRAF mutations showed a significantly more frequent moderate to pronounced infiltration of lymphocytes (P=0.013). NRAS mutations were associated with a significantly higher Clark level of invasion (P=0.022) than BRAF mutations. Age at diagnosis was significantly higher in tumours with NRAS mutations than in those with BRAF mutations (P=0.019). NRAS and BRAF mutations, however, did not influence the overall survival from time of diagnosis (P=0.7). In conclusion, the separate genotypes were associated with differences in several key clinical and pathological parameters, indicating differences in the biology of melanoma tumours with different proto-oncogene mutations.
Assuntos
Genes ras/genética , Melanoma/genética , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Genótipo , Humanos , Masculino , Melanoma/classificação , Melanoma/patologia , Pessoa de Meia-Idade , Nevo Pigmentado/patologia , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proto-Oncogene Mas , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/secundário , Taxa de SobrevidaRESUMO
PURPOSE: Both the retinoblastoma and p53 pathways are often genetically altered in human cancers and their complex regulation is in part mediated by the three gene products p16, p14(ARF), and p15 of the INK4 locus on chromosome 9p21. Partial or complete biallelic deletions of the INK4 locus have been recognized in a variety of malignant tumors, including malignant melanoma. We have in the present study measured the frequency of INK4 deletions in a large number of melanoma metastases and determined their association with clinicopathologic variables and survival data. EXPERIMENTAL DESIGN: Quantitative real-time PCR, as well as fluorescence-based fragment analysis, has been used to perform measurements of the relative allelic concentrations of the INK4 genes in 112 human melanoma tumor samples from 86 patients. RESULTS: Thirty-eight of 86 melanoma patients (44%) had metastases with biallelic losses in INK4. Ten of 20 patients with multiple metastases showed similar deletion patterns in all analyzed tumors. There was no significant association between any of the clinicopathologic variables and loss of INK4. However, loss of INK4 had an adverse effect on median survival from time of diagnosis. Patients with tumors with diploid INK4 had a median survival of 142 months, whereas those with monoallelic or biallelic loss in INK4 had a median survival of only 47 months (P = 0.006). CONCLUSIONS: Our results point to homozygous deletions in the INK4 region as being one of the most common genetic alterations in malignant cutaneous melanoma. INK4 deletions are associated with an adverse prognosis.
Assuntos
Deleção de Genes , Melanoma/patologia , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Análise de Variância , Proteínas de Ciclo Celular/genética , Distribuição de Qui-Quadrado , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina/genética , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Feminino , Genótipo , Homozigoto , Humanos , Perda de Heterozigosidade , Masculino , Melanoma/genética , Pessoa de Meia-Idade , Metástase Neoplásica , Reação em Cadeia da Polimerase/métodos , Neoplasias Cutâneas/genética , Análise de Sobrevida , Proteína Supressora de Tumor p14ARF/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
INTRODUCTION: Adjuvant breast cancer therapy significantly improves survival, but overtreatment and undertreatment are major problems. Breast cancer expression profiling has so far mainly been used to identify women with a poor prognosis as candidates for adjuvant therapy but without demonstrated value for therapy prediction. METHODS: We obtained the gene expression profiles of 159 population-derived breast cancer patients, and used hierarchical clustering to identify the signature associated with prognosis and impact of adjuvant therapies, defined as distant metastasis or death within 5 years. Independent datasets of 76 treated population-derived Swedish patients, 135 untreated population-derived Swedish patients and 78 Dutch patients were used for validation. The inclusion and exclusion criteria for the studies of population-derived Swedish patients were defined. RESULTS: Among the 159 patients, a subset of 64 genes was found to give an optimal separation of patients with good and poor outcomes. Hierarchical clustering revealed three subgroups: patients who did well with therapy, patients who did well without therapy, and patients that failed to benefit from given therapy. The expression profile gave significantly better prognostication (odds ratio, 4.19; P = 0.007) (breast cancer end-points odds ratio, 10.64) compared with the Elston-Ellis histological grading (odds ratio of grade 2 vs 1 and grade 3 vs 1, 2.81 and 3.32 respectively; P = 0.24 and 0.16), tumor stage (odds ratio of stage 2 vs 1 and stage 3 vs 1, 1.11 and 1.28; P = 0.83 and 0.68) and age (odds ratio, 0.11; P = 0.55). The risk groups were consistent and validated in the independent Swedish and Dutch data sets used with 211 and 78 patients, respectively. CONCLUSION: We have identified discriminatory gene expression signatures working both on untreated and systematically treated primary breast cancer patients with the potential to spare them from adjuvant therapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Genes Neoplásicos , Adulto , Idoso , Neoplasias da Mama/cirurgia , Quimioterapia Adjuvante , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Países Baixos , Razão de Chances , Prognóstico , Análise de Sobrevida , Suécia , Resultado do TratamentoRESUMO
DNA mismatch repair (MMR) deficiency and increased O6-methylguanine-DNA methyltransferase (MGMT) activity have been related to resistance to O6-guanine methylating agents in tumour cell lines. However, the clinical relevance of MMR and MGMT as drug resistance factors is still unclear. In a retrospective study, the expression levels of the MMR proteins, hMSH2, hMSH6 and hMLH1, were analysed by immunohistochemistry in melanoma metastases from 64 patients, who had received dacarbazine (DTIC) based chemotherapy. More than half of the melanoma patients had tumours with no nuclear staining for either hMSH2 or hMSH6 or both, while all tumours showed positive nuclear staining for hMLH1. The response rates were similar in patients with hMSH2 and/or hMSH6 positive tumours to these in patients with negative tumours. By combination of MMR with previously obtained MGMT data, only 2 of 12 responders had tumours with low MGMT and positive MMR expression. Still all except 3 of the non-responders were identified by having either high MGMT expression or absent staining for hMSH2 or hMSH6 or both in their tumours. However, there was no significant correlation of MMR expression alone or combined with MGMT levels with clinical response to DTIC-based chemotherapy in metastatic melanoma.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Ligação a DNA/biossíntese , Dacarbazina/uso terapêutico , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Neoplasias/biossíntese , O(6)-Metilguanina-DNA Metiltransferase/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Pareamento Incorreto de Bases , Proteínas de Transporte , Reparo do DNA , Feminino , Humanos , Imuno-Histoquímica , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Metástase Neoplásica , Proteínas NuclearesRESUMO
The p16(INK4A) tumor suppressor is often deleted, or otherwise inactivated, in malignant melanoma. To investigate the loss of p16(INK4A) in greater detail, we analyzed 77 cutaneous melanoma metastases. Of these 56 retained at least one p16(INK4A) allele, and 21 had biallelic deletions. Using methylation-specific PCR, direct sequencing, and immunohistochemical methods, we analyzed p16(INK4A) promoter methylation, mutations, and protein expression, respectively. In addition, 14 corresponding primary tumors were analyzed for protein expression. Results were compared to clinicopathological parameters and previously obtained data regarding mutations in proto-oncogenes NRAS and BRAF. Results revealed that p16(INK4A) promoter methylation was present in 15 of 59 (25%) metastases; nonsynonymous mutations in 9 of 56 (16%) metastases; and protein expression in 12 of 67 (18%) metastases. Protein expression was lost during progression from primary to metastatic tumors, 71% (10 of 14) and 43% (6 of 14) being positive, respectively. However, the genetic and epigenetic alterations of p16(INK4A) observed could not explain the lack of p16(INK4A) protein in 27 metastases, indicating the presence of additional inactivating mechanisms for p16(INK4A). Interestingly, p16(INK4A) promoter methylation was significantly overrepresented in NRAS-mutated samples compared to NRAS wild-type samples (P=0.0004), indicating an association between these two events.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA/fisiologia , Genes ras/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Biópsia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Melanoma/secundário , Metástase Neoplásica/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/patologiaRESUMO
Malignant cutaneous melanoma is a highly aggressive form of skin cancer. Despite improvements in early melanoma diagnosis, the 5-year survival rate remains low in advanced disease. Therefore, novel biomarkers are urgently needed to devise new means of detection and treatment. In this study, we aimed to improve our understanding of microRNA (miRNA) deregulation in melanoma development and their impact on patient survival. Global miRNA expression profiles of a set of melanoma lymph node metastases, melanoma cell lines, and melanocyte cultures were determined using Agilent array. Deregulated miRNAs were evaluated in relation with clinical characteristics, patient survival, and mutational status for BRAF and NRAS. Several miRNAs were differentially expressed between melanocytes and melanomas as well as melanoma cell lines. In melanomas, miR-193a, miR-338, and miR-565 were underexpressed in cases with a BRAF mutation. Furthermore, low expression of miR-191 and high expression of miR-193b were associated with poor melanoma-specific survival. In conclusion, our findings show miRNA dysregulation in malignant melanoma and its relation to established molecular backgrounds of BRAF and NRAS oncogenic mutations. The identification of an miRNA classifier for poor survival may lead to the development of miRNA detection as a complementary prognostic tool in clinical practice.
Assuntos
Perfilação da Expressão Gênica , Melanoma/genética , Melanoma/mortalidade , MicroRNAs/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Genes ras/genética , Estudo de Associação Genômica Ampla , Humanos , Metástase Linfática , Masculino , Melanócitos/fisiologia , Melanoma/secundário , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/patologia , Adulto JovemRESUMO
The genetic background of cutaneous malignant melanoma (CMM) includes both germ line aberrations in high-penetrance genes, like CDKN2A, and allelic variation in low-penetrance genes like the melanocortin-1 receptor gene, MC1R. Red-hair colour associated MC1R alleles (RHC) have been associated with red hair, fair skin and risk of CMM. We investigated MC1R and CDKN2A variation in relation to phenotype, clinical factors and CMM risk in the Swedish population. The study cohort consisted of sporadic primary melanoma patients, familial melanoma patients and a control group. An allele-dose dependent increase in melanoma risk for carriers of variant MC1R alleles (after adjusting for phenotype), with an elevated risk among familial CMM patients, was observed. This elevated risk was found to be significantly associated with an increased frequency of dysplastic nevi (DN) among familial patients compared to sporadic patients. MC1R variation was found to be less frequent among acral lentiginous melanomas (ALM) and dependent on tumour localisation. No association was found between CDKN2A gene variants and general melanoma risk. Two new variants in the POMC gene were identified in red haired individuals without RHC alleles.
Assuntos
Predisposição Genética para Doença , Melanoma/genética , Melanoma/patologia , Mutação/genética , Receptor Tipo 1 de Melanocortina/genética , População Branca/genética , Adulto , Idoso , Alelos , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Fenótipo , Suécia , Proteína Supressora de Tumor p14ARF/genéticaRESUMO
PURPOSE: This phase II study evaluated the efficacy and safety of ixabepilone as neoadjuvant therapy for invasive breast cancer not amenable to breast conservation surgery. Gene expression studies were undertaken using genes that were identified as potentially associated with sensitivity/resistance to ixabepilone in prior preclinical investigations. PATIENTS AND METHODS: Patients with invasive breast cancer >or= 3 cm were eligible. Ixabepilone 40 mg/m(2) was administered as a 3-hour intravenous infusion on day 1 of a 21-day cycle for four or fewer cycles. RESULTS: One hundred sixty-one patients were treated. The overall complete pathologic response (pCR) rate was 18% in breast and 29% in estrogen receptor (ER) -negative patients. Gene expression data were available for 134 patients. ER gene expression (ER1) was inversely related to pCR in breast and had a positive predictive value (PPV) of 37% and negative predictive value (NPV) of 92%. A 10-gene penalized logistic regression (PLR) model developed from 200 genes predictive of ixabepilone sensitivity in preclinical experiments included ER and tau and had higher PPV (45%) and comparable NPV (89%) to ER1. Grade 3 to 4 adverse events (AEs) were reported for 32% of patients. Except for neutropenia and leukopenia, all grade 3 to 4 AEs occurred in Assuntos
Adenocarcinoma/tratamento farmacológico
, Antineoplásicos/uso terapêutico
, Neoplasias da Mama/tratamento farmacológico
, Epotilonas/uso terapêutico
, Adenocarcinoma/genética
, Adenocarcinoma/patologia
, Adenocarcinoma/cirurgia
, Antineoplásicos/administração & dosagem
, Antineoplásicos/efeitos adversos
, Neoplasias da Mama/genética
, Neoplasias da Mama/patologia
, Neoplasias da Mama/cirurgia
, Resistencia a Medicamentos Antineoplásicos/genética
, Epotilonas/administração & dosagem
, Epotilonas/efeitos adversos
, Feminino
, Perfilação da Expressão Gênica
, Regulação Neoplásica da Expressão Gênica
, Humanos
, Linfonodos/patologia
, Terapia Neoadjuvante
, Valor Preditivo dos Testes
, RNA Mensageiro/análise
, Receptores de Estrogênio/análise
RESUMO
A majority of cutaneous melanomas show activating mutations in the NRAS or BRAF proto-oncogenes, components of the Ras-Raf-Mek-Erk signal transduction pathway. Consistent data demonstrate the early appearance, in a mutually exclusive manner, of these mutations. The purpose of this paper is to summarize the literature on NRAS and BRAF activating mutations in melanoma tumors with respect to available data on histogenetic classification as well as body site and presumed UV-exposure. Common alterations of the signal transducing network seem to represent molecular hallmarks of cutaneous melanomas and therefore should continue to strongly stimulate design and testing of targeted molecular interventions.
Assuntos
Genes ras/genética , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Genes ras/efeitos da radiação , Humanos , Melanoma/classificação , Melanoma/patologia , Mutação , Proteínas Proto-Oncogênicas B-raf/efeitos da radiação , Transdução de Sinais , Neoplasias Cutâneas/classificação , Neoplasias Cutâneas/patologia , Raios Ultravioleta/efeitos adversos , Proteínas ras/genéticaRESUMO
The aim of this study was to estimate the impact on survival of NRAS and BRAF mutations and activation of Akt and extracellular signal-regulated kinase (ERK) in primary melanomas. A cohort of 57 primary cutaneous T1-2 melanoma tumors was analyzed. Mutation frequency for both genes was 61% (NRAS 26% and BRAF 39%). In a univariate analysis, shorter overall survival was associated with the presence of ulceration (P=0.001) and BRAF exon 15 mutations (P=0.005) as well as the absence of nuclear activation of Akt (P=0.022) and of cytoplasmic activation of ERK (P=0.003). Unexpectedly, ulceration was a significant adverse prognostic factor only in melanomas with BRAF mutations, whereas there was no effect of ulceration on overall survival in tumors with wild-type BRAF. A multivariate analysis showed that significant independent adverse survival prognostic markers were absence of cytoplasmic activation of ERK (P=0.007) and ulceration (P=0.008), whereas BRAF exon 15 mutation status showed a nonsignificant trend (P=0.066). The absence of cytoplasmic ERK activation in poor prognosis T1-2 melanomas may be associated with activation of some other uncharacterized pathway leading to tumor progression and adverse outcome. Immunohistochemical analysis of cytoplasmic phosphorylated ERK could be used as a prognostic marker in primary melanomas if confirmed in another data set.