ERBB2 amplification is a late event in the pathogenesis of high-grade endometrial carcinomas with heterogeneous HER2 expression.

Intratumor heterogeneity of ERBB2 amplification/HER2 overexpression is frequently observed in ERBB2-amplified high-grade endometrial carcinoma (HG-EC) and contributes to anti-HER2 therapy resistance. To elucidate the molecular pathogenesis and evolutionary trajectory of HER2-heterogeneous HG-ECs, we performed next-generation sequencing of spatially distinct HER2-negative (HER2-) and HER2-positive (HER2+) tumor areas from nine tumors (whole exome, n = 7; targeted panel, n = 2). HER2- and HER2+ components shared a high proportion of somatic mutations, particularly clonal mutations, including known EC driver genetic alterations. The 17q12 amplicon, containing the ERBB2 gene, was the only significant recurrent copy number alteration that differed between HER2- and HER2+ components. By unsupervised hierarchical clustering of genome-wide copy number alterations, samples clustered together at the patient level rather than by HER2 status. Intra- and intertumor heterogeneity in ERBB2 amplification/HER2 expression was also observed in metastatic lesions, which likely originated from different tumor subpopulations within the primary tumor. Exploratory spatial transcriptomics analyses revealed gene expression differences associated with HER2 status, including a shift from 'mesenchymal-like' toward epithelial differentiation in HER2+ components for a subset of cases, a finding that warrants further investigation. Our results suggest that HER2 heterogeneity in HG-EC reflects late acquisition of ERBB2 amplification during tumor evolution. ERBB2 does not appear to drive tumor initiation in HER2-heterogeneous HG-EC but likely serves a context-dependent role in the progression of established tumors. © 2026 The Pathological Society of Great Britain and Ireland.