Desarrollo de nuevas herramientas en biopsia líquida aplicadas a cáncer de mama y gliomas

Abstract

Breast cancer and gliomas are two of the tumors with the greatest impact on today’s society. Breast cancer is the most prevalent tumor and has the highest incidence, and gliomas for its difficulty in detection and its high mortality rate. Despite advances in the field of clinical oncology, diagnosis, prognosis, monitoring and therapy prediction, there is still a long way to go to achieve personalized medicine. The rise of new tools such as liquid biopsy (LB), is presented as an opportunity to reinforce early detection; monitoring of tumor evolution; the evaluation of the response to a treatment; the detection of actionable mutation changes and the discovery of new biomarkers. The great challenge of LB is the detection of those compounds that have truly been released by the tumor. The technologies that, with a sufficient detection limit, make it possible to find genetic alterations derived from the tumour, represent a huge economic investment by laboratories. In this way, extracellular vesicles (EVs) are one of the most investigated sources of biomarkers since their isolation can increase the representation of tumor nucleic acids. However, its isolation and detection entail technical requirements that are not available to all laboratories. In this thesis we present a protocol for isolating EVs with a higher yield than the most used reference methods. We also analysed two types of genetic biomarkers extracted from isolated EVs: miRNA in breast tumors and DNA in glioma. Out of all the molecules contained in the EVs, miRNAs are the ones that are attracting the most interest within the scientific community. Here, we propose for the first time the analysis of relative expression of miRNAs in VEs for the stratification of response to neoadjuvant chemotherapy in patients with most aggressive breast cancer types: HER2 overexpressed, luminal B with HER2 overexpressed and triple negative. Which we call them extreme phenotypes. In addition, we present a cost-effective alternative for the detection of the IDH1R132H mutation in DNA from EVs from glioma patients. The technology described here is independent of both classical methods (sequencing and RT-q-PCR) and cutting-edge technologies (digital PCR and next-generation sequencing), so it can be installed in any minimally equipped laboratory.