Introduction Metastatic breast cancer (MBC) remains an incurable disease with a 5-year overall survival rate below 25%. Metastases often emerge from subclinical, disseminated tumor cells that persist despite systemic therapy of primary disease – referred to as minimal residual disease (MRD). Detecting MRD is critical for identifying patients at high risk of recurrence and enabling timely intervention.

Methods In this study, we developed MammaTrace, a plasma-only cell-free DNA (cfDNA) methylation-based MRD assay, informed by differentially methylated regions (DMRs) identified in MBC using whole genome bisulfite sequencing. MammaTrace was evaluated in an independent longitudinal cohort of early-stage breast cancer patients treated with curative intent.

Results MammaTrace achieved a sensitivity of 91% and specificity of 83%, with a median follow-up of 12.4 months. A positive MammaTrace score, indicative of MRD, preceded clinical or radiologic recurrence by a median of 457 days, providing a substantial lead time for therapeutic intervention to prevent progression to metastatic disease.

Conclusions MammaTrace enables detection of minimal residual disease in breast cancer patients, offering a substantial lead time before clinical recurrence. This approach may improve risk stratification and guide early therapeutic strategies to delay or prevent metastatic progression.

B. Salhia is founder of CpG Diagnostics and reports other support from CpG Diagnostics during the conduct of the study, as well as personal fees from AstraZeneca outside the submitted work; in addition, B. Salhia and D. Buckley have a patent Cell-Free DNA Methylation Test for breast cancer # PCT/US2023/081012 pending and licensed to CpG Diagnostics Inc. G. Gooden is an advisor to CpG Diagnostics.

This project was funded by grants from the National Institutes of Health (R01CA201352 - B.S., P30CA014089 - USC Norris Comprehensive Cancer Center).

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