De Leo, A. | Search bioRxiv

bioRxiv2025

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

BackgroundLiquid 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. MethodsFor 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). ResultsHigh 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. ConclusionsOur 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.

cancer biology↗

bioRxiv2021

A Focused Small-Molecule Screen Identifies PHA-680626 as an Amphosteric Inhibitor Disrupting the Interaction between Aurora-A and N-Myc

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcriptional factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation, by directly binding to a highly conserved N-Myc region, i.e. Myc Box I. As a result, elevated levels of N-Myc, which are required for the growth of MYCN amplified cells, are observed. During the last years, it has been demonstrated that the ATP competitive inhibitors of AURKA CD532, MLN8054 and Alisertib also cause essential conformational changes in the structure of the activation loop of the kinase that prevent N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complex with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results, identifying PHA-680626 as an amphosteric inhibitor both in vitro and MYCN overexpressing cell lines, expand the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex, and confirm that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

cancer biology↗