Mutations and molecular signatures in human melanoma
Abstract: Cutaneous melanoma is a disease which results from a complex mixture of various extrinsic and intrinsic factors, with some major players still unknown. Although primary tumors are highly curable with surgical excision, particularly those which are non-ulcerated and have a thickness of less than 1 mm, poor survival rates are observed in advanced disease. This has been associated both with the biological nature of melanoma, as well as with the limited efficacy of current treatments of metastases. With this work, we wanted to obtain further insights into the biological background of skin melanoma in different stages of progression. We focused on commonly altered pathways and genes, NRAS, BRAF and CDKN2A, and their effects on activation and expression of downstream genes in different stages of melanoma development. For the first two studies, we used primary melanoma tumors that had either sporadic or hereditary origin, respectively. Samples used in the analyses were formalin-fixed, paraffin-embedded tissue blocks. For the other two studies we focused on melanoma metastases originating from sporadic melanoma cases. Fresh frozen tumor specimens were used in these two analyses. Results obtained showed that NRAS and BRAF mutations in sporadic primary melanomas were observed at 26% and 39%, respectively. Approximately a two-fold increase in mutation frequencies were observed, for both genes, in the group of patients that had shorter overall survival. The presence of ulceration and lack of cytoplasmic activation of ERK were independent prognostic markers. BRAF mutations were markers of worse survival in the presence of ulceration. In familial primary melanomas, NRAS and BRAF mutations were found in 3 samples (16%) and 7 samples (37%), respectively. Three samples harbored coexisting alterations in both genes on the background of a germline CDKN2A L32P mutation. Metastatic melanoma samples with BRAF mutations had higher expression of genes when compared to tumors with NRAS alterations. The majority of observed genes were found to be located on chromosome 7. Additionally, this overexpression pattern was accompanied with copy number increase of chromosome 7 in BRAF-mutated tumors. Results obtained through the last study showed that the majority of differentially genes show significantly decreased expression which paralleled CDNK2A allelic loss. These differences were more evident in samples with bi-allelic loss, when compared to those with intact CDKN2A. Finally, pathways analysis showed that differentially regulated genes, that showed lower expression in respect to CDKN2A gene dosage, were linked to immune response in metastatic melanoma samples. In conclusions, NRAS and BRAF mutations are observed at similar frequencies in sporadic and familial melanomas. Although both N-Ras and B-Raf convey signals along the same pathway, signatures in our analysis showed differences between these two effectors. In metastatic melanomas with BRAF mutation the great majority of observed changes included overexpression and amplification of genes located on chromosome 7, in samples with BRAF mutation. The differences in gene expression between BRAF and NRAS mutated tumors may be caused both by direct effects on cell signaling by activated BRAF as well as effects related to overexpression and gene copy number gains on chromosome 7, where the BRAF gene is located. Finally, in the last study, loss of the CDNK2A gene correlated mainly with lower expression of genes in metastatic melanomas with a majority of these linked to immune respons
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