Beyond <i>ESR1</i> : Multi-pathway genomic drivers of endocrine resistance in HR⁺/HER2⁻ breast cancer.

1071 Background: Endocrine resistance in HR⁺/HER2⁻ breast cancer is often associated with acquisition of ESR1 point mutations, but many patients at progression remain ESR1 wild type. Identifying ESR1 -independent, multi-pathway acquired resistance mechanisms is critical for guiding subsequent therapy. Methods: We analyzed 1,429 HR⁺/HER2⁻ breast cancer samples (468 tissue, 1,171 cfDNA), including 210 paired cases, from patients with metastatic breast cancer undergoing molecular testing for treatment guidance or disease monitoring. Next-generation sequencing performed at Datar Cancer Genetics assessed SNVs, CNAs, and gene fusions. Alterations implicated in endocrine resistance were analysed for incidence, specimen type, and co-alteration patterns. Results: ESR1 -wild-type tumors frequently harbored clinically relevant endocrine resistance mechanisms, most commonly involving the PI3K/AKT, FGFR, ERBB2, MAPK , and cell-cycle pathways. At least one resistance-associated alteration was identified in 40.4% (473/1172) of evaluable ESR1 -wild-type cases, while 14.8% (173/1172) harbored alterations across multiple resistance pathways, highlighting substantial ESR1 -independent resistance biology. Among ESR1 -driven mechanisms, ESR1 SNVs were detected slightly more frequently in cfDNA than tissue (13.7% vs 12.8%), despite inclusion of cfDNA samples obtained during clinical surveillance, supporting the sensitivity of liquid biopsy for detecting emergent resistance. ESR1 fusions represented a distinct, non-overlapping resistance mechanism, identified in 7.4% (28/379) of tissue samples, most commonly ESR1–CCDC170 (71%) and ESR1–AKAP12 (14%); 21 fusion-positive cases lacked ESR1 SNVs, underscoring the need for fusion assessment to fully capture ESR1 -driven resistance. Dynamic pathway activation was further supported by acquired PI3K pathway mutations, detected in cfDNA in 3.3% (4/122) of cases initially PIK3CA -wild-type in paired tissue samples. Conclusions: Endocrine resistance in HR⁺/HER2⁻ breast cancer is mediated by multiple actionable genomic pathways beyond ESR1 , including PI3K/AKT, FGFR, ERBB2, MAPK , and cell-cycle alterations. These findings expand opportunities to personalize therapy and guide clinical trial selection by targeting non- ESR1 resistance mechanisms. Incidence of endocrine resistance-associated genomic alterations beyond ESR1 point mutations in tissue and liquid biopsy for HR⁺/HER2⁻ breast cancer. Endocrine Resistance Biomarker ESR1 -Mutant Cases (%) ESR1 Wild-Type Cases (%) ESR1 fusions 11.1 6.7 FGFR1 alterations 13.2 7.0 CCND1 amplification 15.2 7.0 FGFR2/3 alterations 3.7 2.0 HER2 -mutant, non-amplified 2.5 2.6 PIK3CA mutation 43.7 25.1 PTEN mutation 2.0 1.6 AKT1 mutation 5.6 3.6 MAPK Pathway( RAS/RAF/MEK ) 4.6 4.0