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Comparative analysis of molecular profile mutational status of genes BRAF, NRAS and C-Kit in sporadic melanomas, primary and metastatic

Grant number: 12/12582-1
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): November 01, 2012
Effective date (End): March 31, 2016
Field of knowledge:Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
Principal Investigator:João Pedreira Duprat Neto
Grantee:Ana Cláudia Machado Urvanegia
Home Institution: A C Camargo Cancer Center. Fundação Antonio Prudente (FAP). São Paulo , SP, Brazil

Abstract

Abnormal activation of MAPK, via which normally regulates proliferation, differentiation and cell growth is present in more than 80% of the primary melanomas, mutations and proteins in some way along the RAS-RAF-MEK-ERK. These mutations have been documented in all subtypes of melanoma, including the skin, in which mutations in BRAF and NRAS are more common. It was observed that BRAF was mutated in 50% to 70% of patients with melanoma. The most common mutation in BRAF, V600E introduces a conformational change that results in an exchange of valine for glutamic acid, lysine, arginine or aspartic acid in the activation domain of BRAF. In general, mutations in Ras does not occur very often occur in melanoma and other cancers, melanoma is most common mutation in NRAS (15-30%), generally by substitution of a leucine with a glutamine at position 61 (Gln61Leu). The NRAS oncogene mutation has a ratio of 56% in congenital nevi, 33% in primary melanoma and metastatic melanoma in 26%. Mutations in C-Kit showed initiate a series of signaling events that result in cell proliferation and spread of cancer. Several studies have shown that expression of the receptor tyrosine kinase encoded by the protooncogene C-Kit gradually decreases during the growth and invasion of human melanoma. To identify mutations of the genes BRAF and NRAS by pyrosequencing technique and C-Kit by direct sequencing technique and quantification of its protein expression in primary and metastatic sporadic malignant cutaneous melanoma. Through the identification of mutations aims to: establish the relationship between the incidence of mutation of BRAF, NRAS and C-Kit in primary and metastatic cutaneous melanomas and also establish relationships between the mutations found and tumor progression in cutaneous melanoma. Materials and methods: the DNA will be extracted from paraffin embedded tissues using macrodissection technique, when you can not be performed laser microdissection or search a bank of macromolecules. The extracted DNA will be evaluated for the presence of mutations in BRAF and NRAS genes via pyrosequencing technique and C-Kit, by direct sequencing. It will be used for PCR primers purchased commercially and pyrosequencing of BRAF and NRAS, and primers for PCR and sequencing of C-Kit, developed within the institution. C-Kit immunohistochemistry also be performed in the automated system. Outcome: to identify different profiles of expression of BRAF and NRAS genes and protein expression and C-Kit gene in primary tumor and their metastases, and thus, if possible, to establish the relationship between these and the progression of cutaneous melanoma. (AU)