A workflow for assessing antibody-drug conjugate target expression on circulating tumour cells from triple-negative breast cancer and epithelial ovarian cancer patients

Background: Antibody-drug conjugates (ADCs) are transforming the therapeutic landscape of solid tumours. Both patient selection and ADC efficacy in triple-negative breast cancer (TNBC) and epithelial ovarian cancer (EOC) are impacted by target antigen expression, which is often heterogeneous. Single tissue biopsies cannot capture this spatial heterogeneity, creating a need for real-time assessment to guide treatment. Circulating tumour cells (CTCs) may serve as minimally invasive biomarkers that can reflect ADC target expression. The aim of this study was to assess the detectability of clinically relevant ADC targets on CTCs in TNBC and EOC. Methods: ADC targets TROP-2 (TNBC) and FRα (EOC) were selected, along with emerging targets PD-L1 and CLDN6. Flow cytometry was used to characterise surface target antigen expression in three TNBC (MDA-MB-231, MDA-MB-468, HCC1937) and three EOC (SKOV3, OVCAR3, Kuramochi) cell lines. Cell spike-in assays using the Parsortix® system were performed to optimise in-cassette fixation and staining conditions, which were then applied to patient-derived samples for CTC enrichment and immunostaining. Results: Flow cytometry revealed heterogeneous antigen expression across cell lines. In TNBC, TROP-2 was uniformly expressed including in EpCAM-low MDA-MB-231 cells, whereas FRα was minimal or absent. PD-L1 was highly expressed in MDA-MB-231 and HCC1937 but low in MDA-MB-468. In EOC, FRα and TROP-2 were detectable in SKOV3 and OVCAR3 but low or absent in Kuramochi cells. Spike-in assays confirmed in-cassette detection of TROP-2, FRα, and PD-L1 following Parsortix® enrichment. In patient samples, TROP-2+ CTCs were identified in TNBC and FRα+ CTCs in EOC. Conclusion: This study demonstrates the feasibility of real-time ADC target assessment on CTCs using epitope-independent enrichment. TROP-2 shows additional utility for detecting CTCs with low EpCAM expression in TNBC. Incorporating ADC target markers into CTC workflows may enable monitoring of target expression, with potential to guide therapeutic decision-making in TNBC and EOC.