Analysis of cell cycle proteins and genes in the p16/pRb, p53, PTEN and RAS/RAF signaling pathways

• Grant number: 07/00324-0
• Support Opportunities: Regular Research Grants
• Duration: April 01, 2007 - September 30, 2009
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Gilles Landman
• Grantee: Gilles Landman
• Host Institution: Hospital A C Camargo. Fundação Antonio Prudente (FAP). São Paulo , SP, Brazil

Abstract

Melanoma is a melanocyte-derived malignant neoplasia. The acral lentiginous melanoma, as compared to other types has different clinical and histopathological features. It is less frequent in the caucasians and more often seen in asian, hispanics and afro-descendents. They occur in extremities, non-sun exposed areas, such as palms, soles and sub-ungual regions. Its behaviour is agressive and very treacherous, because its visualization is impaired by the location. Brazilian frequency varies in south and southeast regions from 5 to 13%, whereas in northeast regions, higher rates are seen (18% of all cutaneous melanomas). In South Africa, rates are around 32,5%, whereas in Taiwan it is 58% of all cutaneous melanomas. Median five-years survival rate is aproximately 40%. In this type of melanoma, few molecular studies are found, though it has been found that genomic amplifications of chromosome 11q13, 22q11-13 e 5p15, inactivation of the p16 signaling pathway, as well as different expressions of pRb, cyclin D1 e p16. The involvement of p53 has shown controversial results. Imbalance of genes B-RAF, K-RAS, e PTEN were evaluated. Genetic alterations were used to propose a new molecular classification for cutaneous melanomas, based upon differentially expressed proteins and genes such as B-RAF, K-RAS, cyclin D1 e CDKN2A. General objective: To evaluate the cell-cycle proteins, the supressor genes and oncogenes in acral lentiginous melanomas. Specific aims: 1. Establish a clinical and pathological data bank for acral lentiginous melanoma and correlate histopathological prognostic factors to clinical variables. 2. Evaluate the immunohistochemical expression of cell-cycle proteins (p53, cyclin D1, p16,pRb,CDK4 and p21) in tissue microarray (for > 1 mm thick melanomas) and in conventional sections (less than or equal to 1 mm thick melanomas). 3. Evaluate the genes p16INK4A, CCDN1 e PTEN for amplification or deletion, through FISH. 4. Evaluate the expression of p16, PTEN and CCDN1 mRNA in parafin embedded tissue, obtained by laser microdissection. 5. Correlate immunohistochemical expression, mRNA e gene amplification or deletion of genes p16, CCDN1 e PTEN. 6. Identify the mutation status of genes Kras and BRAF in laser microdissected parafin embedded melanomas and epidermal melanocytes adjacent to the lesion. 7. Correlate the immunohistochemical, FISH and mRNA expression of cyclin D1, p16, CCDN1e PTEN, K-RAS and B-RAF mutations with histopathology and clinical behaviour. Materials and methods: This is a retrospective study of acral lentiginous melanomas, obtained from the Hospital do A C Camargo Anatomic Pathology Department. Demographic, clinical and follow-up data will be obtained from the patient charts. Immunohistochemistry for cell-cycle proteins (p53, p21, ciclina D1, pRb, CDK4, p16). will be performed in available paraffin blocks, through tissue microarray. Fluorescent in situ hybridization (FISH) will allow quantification of p16, CCDN1 e PTEN genes. Pyrosequencing in will be used to evaluate mutation status of K-RAS (códons 12 and 13) and of BRAF (códon 600) genes. Polymerase chain reaction (PCR) for PTEN, p16 and CCND1 mRNA will be used to analyze laser microdissected melanoma cells. (AU)