Use of multimodal circulating tumor cell assay to detect HER2-ultralow and ER co-expression matched with single-cell chromosomal instability in metastatic breast cancer.

e13024 Background: Breast cancer is the second leading cause of cancer-related mortality among US women, with metastatic breast cancer (MBC) accounting for most deaths. The DefineMBC comprehensive blood biopsy clinical diagnostic combines ctDNA analysis and robust circulating tumor cell (CTC) characterization to provide valuable information for MBC patients when a tissue biopsy is not feasible. Innovation in the DefineMBC 2.0 multiplexed 5-channel immunofluorescent (IF) CTC characterization assay now measures HER2-ultralow (UL), HER2+, and ER+ protein co-expression levels, as well as ERBB2 amplification and chromosomal instability (CI) via single-cell sequencing. Cell-level HER2/ER co-detection enables monitoring of receptor conversion during a patient’s MBC treatment journey. Additionally, given Trastuzumab-Deruxtecan (T-DxD) has become standard of care for HER2-low disease, distinguishing HER2-UL from HER2- can identify additional patients who may benefit from such therapy. Methods: Assay development including analytical validation studies utilized biologically relevant cell lines of epithelial origin and known expression levels of HER2 and ER (MDA-MB-453: HER2+ MCF-7: HER2-low and MCF-7/ ERBB2 knockout) that were spiked into healthy donor blood. Additionally, patients previously tested were reexamined on the 5-channel platform for concordance analysis. For all samples, following red blood cell lysis, nucleated cells were deposited on glass slides at high density. Slides underwent IF staining for cytokeratins (CK), CD31, CD45, HER2, ER, and Hoechst to localize nuclear DNA, followed by scanning and AI-based rare cell detection. CK+, CD31-/CD45- cells were classified as CTC candidates and assessed for HER2/ER expression. Select CTCs were isolated for single-cell whole genome sequencing and analyzed using a proprietary CTC DNA copy number pipeline to detect CI and amplification at the ERBB2 locus. Results: Validation studies demonstrated the limit of detection for HER2-ultralow at 1 CTC per 11.2 million white blood cells (~1.6 mL of blood) with a linear range of 1- 300 CTCs per slide. Sensitivity for HER2-ultralow and HER2+ ( ERBB2 -amplified) was 89.6% and 99.7%, respectively. Specificity was 99.5% and overall accuracy was 93% and 99.6% for HER2-UL and HER2+, respectively. Overall precision for HER2-ultralow and HER2+ was 7.8% and 0.8% CV, respectively. DefineMBC 2.0 patient correlation studies indicated CTC enumeration, HER2, ER, and CI levels similar to previous clinical results. Conclusions: Advancement of our comprehensive cancer profiling now includes ER/HER2/CI co-detection on enumerated CTCs in patients with MBC. Clinical studies will reveal whether HER2-UL/HER2+ detection on CTCs will identify patients better suited for T-DxD or Trastuzumab, respectively.