Liquid biopsy in ovarian cancer: from clinical optimization to development of innovative eco-friendly technologies

High Grade Serous Ovarian Cancer (HGSOC) management represents a significant clinical challenge. Liquid biopsy, in terms of searching for circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in peripheral blood, has emerged as a promising, minimally invasive, and more sustainable strategy for detecting and monitoring neoplastic diseases. However, standardization and optimization are needed to enhance its clinical utility in ovarian cancer (OC). The aim of this study was to lay the groundwork for an efficient liquid biopsy for HGSOC through (i) clinical optimization of OC CTC and ctDNA detection and (ii) the development of sustainable technologies for liquid biopsy workflows. For the first task, CTC detection was assessed using marker agnostic enrichment and downstream WT1-GPX8-AGR2 gene expression analysis, demonstrating high specificity and high sensitivity of the assay. However, clinical validation on liquid biopsies from HGSOC patients (N=15) resulted in low disease detection rate (13%). In parallel, ctDNA analysis via deep Next-Generation Sequencing (NGS) of TP53 coding region in plasma cell-free DNA (cfDNA) provided a 0.45% variant allele frequency (VAF) threshold for cancer-specific variant calling, achieving 53% disease detection rate. Importantly, for the first time in the context of OC, applying TP53 deep sequencing to both CTC fractions and plasma cfDNA in an additional cohort (N=6) raised disease detection to 83%, confirming the complementarity of the two approaches and addressing false positives from clonal hematopoiesis. To enhance sustainability a label-free CTC identification system using single cell tomography flow cytometry was evaluated, achieving 97.4% accuracy in distinguishing OC cells from monocyte models. Additionally, in collaboration with Cyanagen s.r.l., an efficient eco-friendly prototype kit for cfDNA extraction was developed, reducing plastic use by 50% without compromising performance. Overall, this study demonstrates the feasibility of a tandem CTC/ctDNA liquid biopsy strategy for HGSOC and introduces sustainable innovations for clinical and research applications.