Applications of liquid biopsy in oncology for finding circulating biomarkers: two case studies

Precision medicine in oncology has changed the therapeutic approach, moving away from one-size-fits-all strategies towards personalized treatments based on the specific molecular and clinico-pathological characteristics. In this context, the analysis of plasma samples (liquid biopsy), is establishing itself as a useful, minimally invasive tool for characterizing the molecular tumor profile, assessing prognostic and predictive circulating biomarkers and monitoring the clonal evolution of the tumor. It enables the analysis of various biological components, including circulating DNA and proteins. This thesis focuses on two case studies in which liquid biopsy was applied with different approaches and purposes. First, the CHAMBER study, aimed at characterizing the mutational profile of patients with HR-positive HER2-negative metastatic breast cancer using the NGS method on cfDNA. For this purpose, a customized NGS panel was developed comprising 45 genes of interest covering the entire exonic region of the genes. The experiments performed for the technical evaluation of the customized panel showed that the panel was able to reliably detect mutations with a VAF of 0.50% (sensitivity of 90.60%), whereas the sensitivity for mutations with a VAF of 0.10% was significantly reduced (sensitivity of 43.6%). The analysis of the first 149 patients showed that the most frequently mutated genes were TP53 (43.6%), PIK3CA (29.53%), ESR1 (24.83%) and GATA3 (13.42%). In addition, we reported the detection of rare PIK3CA mutations such as Gly1049Asp and Arg357Gln, which require further functional studies to clarify their biological role and potential clinical impact. Finally, a second analysis focused on evaluating the prognostic significance of the PIK3CA mutation in the endocrine-resistant setting. The data showed that the PIK3CA mutation is associated with a worse prognosis for endocrine-resistant patients in terms of OS and PFS. The second case is a pilot study to assess the feasibility of 2D proteomic analysis in EGFR-mutated (ex19del) aNSCLC patients treated with osimertinib. The analysis was performed on pre-treatment (T0) and 28 days post-treatment (T2) samples to identify potential predictive biomarkers and investigate the biological mechanisms underlying response to treatment. The comparison of the two conditions revealed proteins involved in the coagulation and fibrinolysis process, such as fibrinogen, kininogen and plasminogen. Although the results of the plasminogen variation were not confirmed by the ELISA test, the study still has potential and an innovative character that is worth exploring further. Therefore, optimizing the methods and expanding the sample pool could be starting points for further investigations and obtaining more robust data.