Detection of BRAF V600E mutation in ctDNA by ddPCR: from laboratory to market

• Autor(a) principal: Oliveira, Inês Domingues
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/53897
• topic: liquid biopsy; ctDNA; melanoma; BRAF V600E; ddPCR

Cutaneous melanoma is the most aggressive form of skin cancer and has a high mortality rate. The current methods used to stage melanoma and detect metastases are imaging techniques and sentinel lymph node biopsies (SLNB). However, these have limited sensitivity for the detection of early stage melanoma and often can’t provide timely clinical evidence of disease recurrence or for monitoring therapies. Cell-free tumour DNA (ctDNA) could be a specific biomarker in melanoma patients which present V600E mutation in BRAF gene. ctDNA is released by cancer cells into the bloodstream and then excreted in urine, allowing it to be analysed by liquid biopsy. This analysis provides a real-time snapshot of tumour burden and represents a non-invasive, cost-effective alternative that can be performed repeatedly. In this work, a surveillance tool was implemented to help patients with melanoma through the detection of BRAF V600E mutation in ctDNA by Droplet Digital PCR (ddPCR). Firstly, we focused on design and optimization of the assay to ensure a low limit of detection and a distinction between false-positives and true-positives. Secondly, the assay was tested in order to show the possibility to detect V600E mutation in BRAF gene in plasma and midstream urine samples from melanoma patients. In addition to the test implementation, a go-to-market strategy was explored for the commercialization of the test through a collection kit. This test is directed to laboratories, clinics and hospitals aiming to help doctors in the diagnosis, prognosis and treatment of their patients. After a market analysis, we concluded that the new test has advantages over existing competitors in the market and has a potential to improve the melanoma management.