2272P Minimal DNA-methylation (DNAm) signatures for non-invasive tumor-agnostic estimation of tumor content (TC) and molecular subtype in patients (pts) with metastatic breast cancer (mBC)

Non-invasive detection and quantification of circulating tumor DNA by DNAm signatures is emerging as powerful tool for monitoring disease status in pts with metastatic cancer. We recently developed MIMESIS, a computational workflow to design tumor agnostic DNAm signatures of few dozen sites for TC and molecular subtype estimation in liquid biopsy. Here we applied a MIMESIS informed assay to a retrospective cohort of mBC pts (MIMESIS-BC). 72 mBC pts (42 ER+/HER2-, 2 ER+/HER2 N/A, 8 ER+/HER2+, 6 ER-/HER2+, 14 ER-/HER2-, up to 6 previous lines of treatment) and 11 healthy individuals (ctrl) were included. Plasma from mBC pts was collected before starting treatment (T0, n=58) and after the first cycle (T1, n=52) (n=38 pts T0 and T1). MIMESIS-BC for TC, PAM50 molecular subtypes and ER status (n=139 sites) was applied to circulating free DNA as described in the MIMESIS study. Samples were defined as TC+ when TC>0. TC values as continuous variable and TC classes (+/-) were associated with OS and PFS. Concordance of subtype and ER status by MIMESIS-BC with IHC-based from primary tumor was estimated by chi-square and percent agreement. TC was not detected in ctrl. A significantly higher median TC was observed at T0 compared to T1 (5.6% (range 0-73%) vs 0% (range 0-47%); p=0.02). 39 TC+ samples at T0 showed 61% agreement for molecular subtype (p=0.002) and 86% for ER status (p<0.001). In the entire population, PFS and OS were significantly associated with TC values both at T0 and T1 (max p=0.002). TC+ pts had significantly worse OS compared to TC- at T0 (HR=2.1 CI=1.1-3.9 p=0.02) and T1 (HR=2.0 CI=1.1-3.6 p=0.03) and worse PFS at T1 (HR=2.9 CI=1.5-5.4 p<0.001). In ER+ pts, TC+ at T0 and T1 associated with worse PFS (HR=2.6 CI=1.2-5.3 p=0.008, and HR=3.8 CI=1.7-8.2 p<0.001) and OS (HR=3.5 CI=1.6-7.6 p=0.001, HR=3.0 CI=1.4-6.1 p=0.002), while in ER- pts TC classes showed no association with outcome. In a small and heterogenous retrospective cohort of mBC pts, MIMESIS-BC showed promising capabilities for prognostic stratification and subtype estimation from liquid biopsy. Validation in an extended prospective cohort is ongoing.