Combining circulating tumor cell and circulating cell free DNA analyses enhances liquid biopsy sensitivity in detecting high grade serous tubo-ovarian carcinoma

Background: Liquid biopsy is a promising strategy for detecting and monitoring neoplastic diseases, with circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) being the most common objects of investigation. Most analyses have focused on these biomarkers separately, and simultaneous detection has not yet been attempted in high grade serous tubo-ovarian carcinoma (HGSOC). The aim of our study was to assess whether the tandem CTC/ctDNA approach increases HGSOC detection efficiency of peripheral blood liquid biopsy. Methods: For CTC detection, by using healthy donor samples spiked with known cancer cell numbers, we tested gene expression assays and TP53 next-generation sequencing (NGS). The latter was also applied for ctDNA detection where analytical validity was ensured by calculating the optimal variant allele frequency (VAF) threshold for mutation calling. The clinical validity of the assays was then verified on two HGSOC cohorts and respective controls. Standard 2x2 contingency tables and Wilson method were used to evaluate clinical validity, by calculating specificity, sensitivity, and accuracy with 95% confidence intervals (CI). Results: High analytical sensitivity and specificity were found for both gene expression and TP53 NGS based CTC detection, as these assays specifically detected as few as five cancer cells spiked in control sample. Regarding clinical validity, the gene expression-based CTC detection showed 0.48 accuracy, 13.3% sensitivity, and 100% specificity, whereas TP53 sequencing demonstrated better assay performance (0.77 accuracy, 46.7% sensitivity, 100% specificity). For circulating cell-free DNA (cfDNA) analysis, we first identified 0.31% VAF cut-off for accurate ctDNA TP53 mutation calling. Subsequent clinical validity assessment showed solid performance efficiency of the ctDNA based liquid biopsy (0.71 accuracy, 60% sensitivity, and 100% specificity), outperforming the CTC detection methods. Importantly, the tandem ctDNA/CTC analysis improved disease detection rate in both HGSOC cohorts, allowing to achieve, respectively, 73.3% and 93.3% sensitivity. Interestingly, TP53 NGS revealed CTC private variants, and shared ctDNA/CTC mutations undetected in the primary tissue, highlighting the ability of the dual-analyte approach to capture tumor heterogeneity and allow mutation cross-validation. Conclusions: Our study reveals the complementary value of simultaneous CTC and cfDNA analysis in HGSOC, advancing the translational potential of liquid biopsy integration for the management of this disease.