To search for proteins expressed in human melanocytes and melanoma, we employed an antibody-based proteomics strategy to screen for protein expression in tissue microarrays containing normal tissues, cancer tissues and cell lines. Syntaxin-7 (STX7) was identified as a novel protein, not previously characterized in cells of melanocytic lineage, displaying a cell type-specific protein expression pattern. In tumor tissues, STX7 was expressed in malignant melanoma and lymphoma. The protein was further characterized regarding subcellular localization, specificity, tissue distribution pattern and potential as a diagnostic and prognostic marker using cell lines and tissue microarrays containing normal skin, melanocytic nevi and primary and metastatic melanoma. STX7 was expressed in normal melanocytes, various benign melanocytic nevi, atypical nevi and malignant melanoma. Analysis in two independent melanoma cohorts demonstrated STX7 expression in nearly all investigated tumors, although at varying levels (>90% positive tumors). The expression level of STX7 protein was inversely correlated to tumor stage, suggesting that decreased expression of STX7 is associated with more aggressive tumors. In conclusion, we present protein profiling data for a novel protein showing high sensitivity and specificity for cells of the melanocytic lineage. The presented antibody-based proteomics approach can be used as an effective strategy to identify novel tumor markers and evaluate their potential clinical relevance.

SOX10 is a transcription factor expressed in nerve cells and melanocytes. The aim of this study was to investigate the protein expression pattern of SOX10 in malignant melanoma tumors and to analyze whether the results correlated with clinical parameters and the proliferation marker Ki-67. Furthermore, proliferation and migration were analyzed in three different cell lines employing SOX10 small interfering RNA-mediated silencing. Expression patterns were determined in 106 primary tumors and 39 metastases in addition to 16 normal skin samples and six benign nevi employing immunohistochemistry and tissue microarrays. The immunohistochemical staining was evaluated manually and with an automated algorithm. SOX10 was strongly expressed in the benign tissues, but for the malignant tumors superficial spreading melanomas stained stronger than nodular malignant melanomas (P=0.008). The staining intensity was also inversely correlated with T-stage (Spearman's ρ=-0.261, P=0.008). Overall survival and time to recurrence were significantly correlated with SOX10 intensity, but not in multivariate analysis including T-stage. With the automated algorithm there was an inverse correlation between the SOX10 staining intensity and the proliferation marker, Ki-67 (ρ=-0.173, P=0.02) and a significant difference in the intensity signal between the benign tissues, the primary tumors and the metastases where the metastases stained the weakest (P≤0.001). SOX10 downregulation resulted in variable effects on proliferation and migration rates in the melanoma cell lines. In conclusion, the SOX10 intensity level differed depending on the tissue studied and SOX10 might have a role in survival. No conclusion regarding the role of SOX10 for in-vitro proliferation and migration could be drawn.

Background: Microphthalmia associated transcription factor (MITF) protein has a central role in the differentiation and survival of melanocytes. The aim of the study was to investigate whether MITF can be employed as a prognostic marker in patients operated on for cutaneous malignant melanoma.

Methods: A cohort study design based on information collected from population-based registers. For included patients tissue microarrays and immunohistochemistry were employed to study the protein expression of MITF in the primary malignant melanoma tumors by estimating the fraction of positive tumor cells and the staining intensity.

Results: The vast majority of tumors expressed MITF in >25% of the tumor cells with a strong staining intensity and looking at these factors individually these patients had a better prognosis. When cell fraction and intensity were combined a high-risk group dying of malignant melanoma was identified as those with 25% -75% of tumor cells staining with weak intensity and those with <25% of tumor cells staining with strong intensity. However, the majority of the deaths occurred in the lower risk groups.

Conclusions: Although a high-risk group for death in malignant melanoma was identified we conclude that MITF is not useful as a prognostic marker because of the distribution of that particular expression in the population.

Impact: Our results indicate a bi-phasic pattern of MITF expression and although not useful as a prognostic marker these results are in line with other experimental studies and are relevant to explore further.

The thickness of a primary malignant melanoma tumor is the most important prognostic indicator for a patient with primary cutaneous malignant melanoma. To optimize the management and treatment of melanoma patients there is an unmet need to identify characteristics that can further stratify melanoma patients into high or low risk for progressive disease. Despite numerous studies no single marker has yet been shown to add significant prognostic information. An algorithmic approach, combining data from several markers provides an attractive model to identify patients of increased risk of dying from malignant melanoma. The primary aim of the present study was to analyze the correlation between clinical outcome and protein expression patterns of multiple proteins in malignant melanoma tumors using immunohistochemistry and tissue microarrays. Candidate proteins were identified based on a selective and differential expression pattern in melanoma tumors and tested in a cohort of 143 melanoma patients. Protein expression was analyzed using both manual scoring and automated image analysis-based algorithms. We found no single marker of prognosis that was independent of tumor thickness. When combining potential prognostic markers we could define a prognostic index, based on RBM3, MITF, SOX10 and Ki-67, that was independent of tumor thickness in multivariate analysis. Our findings suggest that a good prognosis signature can be identified in melanoma patients with tumors showing a low fraction of Ki-67 positive tumor cells and a high fraction of RBM3 positive tumor cells combined with low intensity levels of SOX10 and MITF.