Abstract
BACKGROUND: CDK4/6 inhibitors (CDK4/6i) in combination with endocrine therapy (ET) represent the standard of care for hormone receptor-positive, HER2-negative (HR+/HER2-) metastatic breast cancer (MBC) patients. However, no head-to-head randomized trials have directly compared palbociclib, ribociclib, and abemaciclib. Moreover, predictive biomarkers of resistance to CDK4/6i remain largely undefined. This study aimed to evaluate circulating tumor DNA (ctDNA)-based epigenetic and fragmentomic biomarkers as potential predictors of response and resistance in patients receiving CDK4/6i. METHODS: We conducted a biomarker-driven analysis within the prospective, multicenter MAGNETIC.1 study, enrolling 149 patients with HR+/HER2- MBC treated with first-line endocrine therapy and a CDK4/6 inhibitor. Plasma samples were collected at baseline and during treatment (3 and 6 months). Droplet digital PCR was used to assess ESR1 promoter methylation and ACTB fragmentomic profiles. Progression-free survival (PFS) and overall survival (OS) were evaluated, and molecular dynamics were compared between treatment groups. RESULTS: After a median follow-up of 34.8 months, no statistically significant differences in PFS or OS were observed between ribociclib and palbociclib treated patients, although ribociclib was associated with numerically longer PFS and higher survival rates. At the molecular level, palbociclib treatment was characterized by transient increases in ESR1 promoter methylation at the first evaluation and a rebound in ACTB(short) fragment levels at six months relative to baseline. These dynamic patterns were not observed among patients receiving ribociclib. CONCLUSIONS: ctDNA-based methylation and fragmentomic profiling revealed exploratory, treatment specific molecular dynamics, highlighting biological differences between CDK4/6 inhibitors. These findings support the feasibility of liquid biopsy-based biomarker studies in this setting, although their potential clinical relevance remains preliminary and requires validation in larger cohorts with earlier and more granular on-treatment timepoints.