Abstract 2252: Septin 6 in malignant melanoma

Melanoma describes a malignancy of epidermal melanocytes located predominantly in the skin. Melanoma accounts for only 4% of all skin cancers; however, it causes the greatest number of skin cancer-related deaths worldwide. Malignant melanoma is now the seventh most common cancer in the USA. The incidence of melanoma is increasing faster than that of any other human cancer. The sequence of events in which normal melanocytes transform into melanoma cells, referred to as melanomagenesis, is poorly understood. It likely involves a multistep process of progressive genetic mutations which alter cell proliferation and differentiation, and thereby reduce host susceptibility to environmental carcinogens such as ultraviolet radiation. Septins are an evolutionary conserved family of genes which encode guanosine-5′-triphosphate binding proteins and are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. A total of 14 septin genes have been identified in humans. Septin gene expression is extremely complex, and has been noted to be altered in human neoplasia, infectious diseases and neurological conditions. Septin 6 (SEPT6) is encoded by a gene on Xq24 and has been implicated in human leukaemia as a fusion partner of MLL. Our laboratory has experimentally shown that the 12 exons of SEPT6 span 85 kbase of Xq24 and undergo complex splicing events such that 7 transcripts (v1-v4***) are produced which encode 5 distinct polypeptides. Of note are 3 discrete transcripts, named v4* to v4***, encoding the same 427 amino acid polypeptide: a phenomenon also seen with SEPT9. Recent cDNA microarray studies have demonstrated up-regulation of SEPT6 in metastatic melanoma versus primary melanoma, but have only documented pan-SEPT6 expression. But the complexity of septin gene splicing and the limitations of microarray analysis do not allow definition of which splice variants are altered during progression. We hypothesize that specific SEPT6 splice variants may be associated with the metastatic phenotype of melanoma. We received ethical approval for the extraction of RNA from 300 formalin-fixed, paraffin-embedded skin samples, ranging from benign naevi to both primary and metastatic melanomas. However, RNA retrieved from FFPE tissue is usually of poor quality as it highly degraded. To overcome this phenomenon, we have optimized specific real time quantitative PCR assays, amplifying amplicons of less than 80bp, allowing us to detect each