HER2 phenotyping of CTCs in breast cancer: a comparison across the spectrum of tissue HER2 expression level

Background: Assessing and trackinghuman epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) is essential for selecting the right targeted therapies, but conventional tissue biopsies fall short because they cannot fully reflect tumor heterogeneity or provide ongoing, real-time monitoring. Circulating tumor cells (CTCs) provide a minimally invasive way to dynamically assess HER2 status, but they still require further validation and standardization before routine clinical use. Methods: We compared the HER2 expression on CTCs through a quantitative assessment using the ACCEPT tool across patients with BC with different HER2 status on tissue (positive, low and null). The proportion of HER2-expressing CTCs in individual samples (CTC-HER2ratio) was calculated and correlation with tissue HER2 status was analyzed in a cohort of patients with metastatic BC (MBC) to define an optimal cutoff value for discrimination by receiver operating characteristic (ROC) analysis. Results: A total of 143 patients were included. The median CTC-HER2ratio was significantly higher in patients with HER2-positive (HER2+) tumors compared to HER2-negative (HER2-)tumors (30% vs 9%, p = 0.0003). The ROC curve yielded an area under the curve (AUC) of 0.7417 for distinguishing HER2+ from HER2- disease, with an exploratory optimal cutoff of 0.22. In contrast, the CTC-HER2ratio did not significantly differentiate HER2-low from HER2-null tumors (p = 0.327; AUC 0.5646), highlighting limited resolution in the low-expression range. Conclusions: This proof-of-concept study suggests that a ratio-based assessment of HER2 expression on CTCs may capture biological heterogeneity and correlate with tissue HER2 status. However, the modest discriminative performance and limited resolution in the HER2-low range indicate that further prospective validation is necessary before clinical integration.