Abstract Background: Circulating tumor DNA (ctDNA) is a clinically validated biomarker in breast cancer for predicting the risk of recurrence, monitoring response to neoadjuvant treatment, and detecting molecular relapse after adjuvant therapy. Signatera™ is a tumor-informed, multiplex PCR-NGS ctDNA assay that may be developed from a backbone of whole-exome sequencing (WES) or whole-genome sequencing (WGS). Early clinical validation of Signatera Genome in a pan-cancer cohort demonstrated improved performance in certain clinical scenarios. In this study, we specifically assessed the performance of the Signatera Genome assay in the surveillance setting for breast cancer. Methods: A retrospective analysis was performed using clinically annotated residual patient samples from commercial testing with WES-based Signatera. Treatment decisions and cadence of ctDNA testing were at the provider’s discretion. Signatera Genome assays were designed, consisting of 64 high-quality tumor-specific variants, from matched tumor and normal whole genome sequencing data. These assays were then used to detect ctDNA in the corresponding patients’ plasma. ctDNA levels were quantified as mean tumor molecules per mL of plasma (MTM/mL). Longitudinal blood samples represented time points after surgery/definitive treatment until recurrence or the end of follow-up. The correlation between ctDNA status and distant recurrence-free survival (DRFS) was assessed using Cox regression analysis. Results: Clinical performance of the Signatera Genome assay was assessed in a real-world cohort of 228 patients with breast cancer triple-negative breast cancer (TNBC), n=130; HR+/HER2-, n=85; and HER2+, n=13. In a landmark analysis, patients with ctDNA-positivity within 3 months of surgery were significantly at a higher risk of distant disease and/or molecular recurrence (HR: 8.1, 95% CI: 3.5-8.6, P 0.001). With longitudinal testing, ctDNA-positivity at any time post-definitive treatment was associated with significantly worse DRFS (HR: 22.2, 95% CI: 11.0-44.7, P 0.0001). ctDNA detection in the surveillance setting preceded clinical recurrence as confirmed by imaging with a sensitivity and specificity of 100%. Upon analyzing ctDNA dynamics, patients with unfavorable ctDNA dynamics (persistently/converted to positive) were observed to have significantly shorter DRFS than those with favorable ctDNA dynamics (serially/converted to negative) (HR with Firth’s penalized likelihood hazard model: 97.8, 95% CI: 11.5-12768.9, P 0.001). Conclusions: These data indicate the robust clinical performance of the Signatera Genome in breast cancer for detecting recurrence after surgery or definitive treatment. Prospective clinical trials are warranted to establish the clinical utility of the assay for guiding treatment decisions for patients with molecular non-radiographic recurrence. Citation Format: W. McHayleh, C. Scalise, E. Kalashnikova, B. Sridhar, J. Feeney, W. Tan, A. Hsieh, A. Butskova, J. Ortega, S. Velichko, C. Palsuledesai, H. Sethi, G. Heilek, J. McKenzie, A. Rodriguez, M. Liu, M. George. Clinical performance of Signatera Genome assay for predicting recurrence in patients with breast cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-07-26.
McHayleh et al. (2026) studied this question.