AGK-BRAF in pediatric papillary thyroid carcinoma: investigation of the mechanism associated with the high rate and functional analysis

• Grant number: 18/09911-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: September 01, 2018
• End date: September 30, 2022
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Janete Maria Cerutti
• Grantee: Luiza de Mello Oliveira Sisdelli
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 14/06570-6 - Comprehensive whole exome, paired-end RNA and genome sequencing: new insights into genetic bases of thyroid carcinoma in pediatric and adult ages and applications in clinical practice, AP.TEM
• Associated scholarship(s): 18/26395-5 - 3D Analysis of AGK-BRAF fusion of Paraffin-Embedded Sections in Pediatric Thyroid Carcinomas and High-Throughput Imaging to Study Cellular Phenotype of Cells transformed with AGK-BRAF, BE.EP.DR

Abstract

In papillary thyroid carcinoma (PTC), the BRAF V600E mutation is the most prevalent alteration in adults (40%). On the other hand, in the pediatric PTC (d 18 years old), the BRAF V600E mutation is rare, and fusions involving BRAF have been identified in both radiation-exposed and sporadic cases. Recently, our group identified the AGK-BRAF rearrangement in 19% (15/80) of the PTC pediatric cases, predominantly sporadic. In addition, with the FISH break-apart technique, we have identified two cases with BRAF breakage and unknown partner fusion gene. Unlike the V600E mutation, the AGK-BRAF fusion is rarely identified in adult PTC. Importantly, the presence of mutation or fusion in the BRAF gene is specific to PTC and was not identified in benign lesions or other subtypes of thyroid cancer and has been associated with a worse prognosis in both adult and pediatric populations. Thus, this project aims to deepen the knowledge about the AGK-BRAF fusion, investigating the possible mechanisms associated with its high prevalence and worse prognosis in the pediatric population. To evaluate the mechanism associated with the AGK-BRAF high prevalence, we will investigate the distance between these two genes in normal thyroid tissue nucleus, in pediatric and adult populations, using the DNA 3D-FISH technique. In addition, in order to understand the role of AGK-BRAF fusion in the thyroid tumor pathogenesis, a normal thyroid cell line will be transfected with plasmid containing the AGK-BRAF rearrangement, as well as BRAF-AGK, and we will compare with the effects caused by the RET/PTC1, RET/PTC3, ETV6-NTRK3 and BRAF V600E alterations, also identified in thyroid tumors, and BRAF wild-type. By Western Blot, we will evaluate the MAPK-pathway activation and the presence of epithelial-mesenchymal transition markers, as well as migration and invasion assays. Thus, the results from this project will help in understanding the generation mechanisms and the consequences of the AGK-BRAF rearrangement.