CellSearch-Based Isolation of Circulating Tumor Cells from Ascitic Fluid Enables Molecular Characterization of Ovarian Cancer

Background High-grade serous tubo-ovarian cancer (HGSOC) frequently presents with malignant ascites, a biologically relevant compartment rich in tumor cells. While circulating tumor cells (CTCs) in peripheral blood (PB) are often scarce, ascitic fluid (AF) may represent a more accessible and abundant source of tumor-derived material. This study evaluated the feasibility of isolating AF-derived CTCs using the CellSearch platform for subsequent molecular characterization. Methods Paired PB and AF samples from 12 HGSOC patients were collected at surgery and analyzed using the CellSearch system. After CTC enumeration, DNA was extracted from CellSearch cartridges containing AF-derived CTC-enriched fractions and subjected to next-generation sequencing (NGS) using a panel clinically validated for solid tumors. Associations between AF-derived CTC burden and clinical variables were explored. Results CTCs were detected in 100% of AF samples and in 50% of PB samples. AF contained markedly higher numbers of CTCs than PB, whereas the latter showed low or undetectable levels. AF-derived CTCs frequently formed multicellular aggregates, potentially leading to underestimation by automated counting. An optimized protocol for DNA extraction from post-processing CellSearch cartridges enabled successful downstream NGS analysis from CTC-enriched AF fractions. Tumor-specific TP53 mutations identified in AF-derived CTC-enriched fractions showed complete concordance with matched tumor tissue in all analyzed cases, supporting the feasibility of using these fractions for molecular characterization of the tumor. No significant associations were observed between AF-derived CTC burden and CA125, PCI, progression-free survival (PFS), or overall survival (OS), although higher CTC levels were associated with a trend toward shorter PFS. Conclusions Compared to PB, AF is a richer source of CTCs in HGSOC. Importantly, AF-derived CellSearch fractions enabled reliable molecular profiling through downstream NGS analysis, suggesting that this approach may be exploited to enrich for tumor-poor peritoneal washings usually considered for staging IC HGSOC. The complete mutational concordance between AF-derived CTCs and matched tumor tissue supports the biological validity of this approach and highlights its potential as a minimally invasive liquid biopsy for tumor characterization and longitudinal disease monitoring in advanced HGSOC.