Analysis of cellular signaling dynamic changes in circulating tumor cells as response to therapy in BRAF mutant melanoma patients

• Grant number: 14/00529-4
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: May 20, 2014
• End date: May 19, 2015
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Rafael Malagoli Rocha
• Grantee: Fernanda Machado de Carvalho
• Supervisor: Shannon Leigh Stott
• Host Institution: A C Camargo Cancer Center. Fundação Antonio Prudente (FAP). São Paulo , SP, Brazil
• Institution abroad: Harvard University, Boston, United States
• Associated to the scholarship: 12/00041-6 - Evaluation of the epithelial mesenchimal transition and the role of transcriptional coactivator PGC-1± in breast carcinomas: study involving circulating tumor cells (CTCs), BP.DR

Abstract

Melanoma accounts for less than 5% of skin cancer cases, but it is the major cause of skin cancer related deaths and its incidence has been increasing over the past decades.More than 50% of melanomas cases present mutations of the BRAF gene, which leads to an increase in BRAF kinase activity and constitutive activation of the MAPK pathway, which are potential targets for development of highly specific inhibitors. These drugs have improved progression-free survival in BRAF-mutant melanoma patients, however most of them present clinical resistance during the treatment. For this reason, the challenge is to define rational strategies for predicting responsiveness to RAF and MEK inhibition in BRAF-mutant melanoma. Circulating tumor cells (CTCs) analysis could represent an important less invasive alternative to the traditional biopsies for monitoring cellular signaling dynamic changes as response to therapy in BRAF mutant melanoma patients. The aim of the proposed project is evaluate expression of signaling markers related to acquisition of resistance to RAF inhibitors (e.g. ps6, pERK and pAKT) in CTCs during treatment of melanoma patients. To achieve this, CTCs will be isolated from BRAF-melanoma patients during the course of treatment using microfluidic technology (CTC-iChip). Optimization of fixation and signal amplification strategies will be performed to preserve signaling epitopes in circulating tumor cells using BRAF-mutant melanoma cell lines and CTCs isolated from patient blood. Dynamic changes in cell signaling in CTCs will be analyzed using high throughput, multi-spectral imaging strategy to quantify multiple immunofluorescent markers on a single cancer cell and comparisons will be made with the primary tumor biopsies. This approach will determine the potential use of CTCs as a less invasive method and real-time biomarker to predict the responsiveness to therapy in BRAF-mutant melanoma. (AU)