Epigenetic markers of tumor progression in estrogen receptor–positive breast cancer patients with high nodal involvement

Background Management of axillary lymph node (LN) disease in breast cancer (BC) is evolving, with declining use of axillary lymph node dissection. Consequently, prognostic information from nodal assessment is becoming limited. Molecular determinants of primary tumors associated with greater nodal burden could provide complementary prognostic value and mechanistic insights into progression. Methods We retrospectively analyzed 47 patients with estrogen receptor–positive (ER+), HER2–negative (HER2–) BC with nodal metastases at diagnosis, excluding patients treated with neoadjuvant therapy. Patients were stratified by nodal stage (pN1: 1–3 positive nodes, n = 29; >pN1: ≥4 positive nodes, n = 18). Genome-wide DNA methylation profiling was performed. Findings were validated in TCGA, SCAN-B, AURORA-US, and additional datasets integrating DNA methylation and transcriptomic data. Results We identified 7,794 differentially methylated sites, with promoter hypermethylation enriched in CpG islands and hypomethylation in open sea regions of > pN1 primary BC tumors. Cross-cohort integration with TCGA (n = 148) revealed 95 promoter-associated genes with consistent methylation differences, enriched in developmental and adhesion gene pathways. Among these, seven genes also showed concordant differential expression. From five clinically relevant genes ( TTC23, ARL10, RIC3, CXCL14, KCNH2 ), we derived the Lymph-node Involvement Outcome Numerator (LION) Score. Lower LION Scores were associated with higher nodal burden and poorer distant metastasis–free survival across multiple cohorts. Conclusions Epigenetic alterations in ER+/HER2– BC are associated with higher LN involvement and metastatic risk. The LION Score, while not a predictive classifier, represents a biologically informed metric with potential to complement prognostic staging in an era of reduced axillary surgery.