<i>ESR1</i> mutations and associated alterations in patients with estrogen receptor (ER+), HER2-positive (HER2+) breast cancers at diagnosis of metastatic disease (mBC).

e13035 Background: Activating mutations in the ER, encoded by the ESR1 gene (ESR1 mt ), result in a constitutively active receptor and emerge as a resistance mechanism following endocrine therapy in ER+, HER2-negative mBC. The mutant ER is a key driver of metastasis, and its inhibition represents a promising strategy in endocrine-resistant mBC. However, the role of ESR1 mt in ER+, HER2+ tumors remains poorly understood. In this study, we aim to characterize the prevalence and mutational landscape of ESR1 mt in ER+, HER2+ mBC. Methods: We identified patients from the Tempus database with an immunohistochemically defined ER+ HER2+ mBC that underwent Tempus xT (648 gene panel) and xR whole transcriptome analysis within 6 months from diagnosis of mBC. For patients with multiple samples, the closest to the primary diagnosis was selected. Germline alterations were identified in normal-matched DNA. ESR1 mt in this analysis were limited to pathogenic/likely pathogenic short variants. Categorical variables were analyzed using the Wilcoxon rank sum test or Fisher's exact test. Significance level was defined at p &lt; = 0.05 or q &lt; = 0.2 for multiple testing correction. Results: We identified a total of 421 patients, including 403 with wild-type ESR1 (ESR1 wt ) and 18 with ESR1 mt , representing 4.3% of the entire cohort. Among the ESR1 mt , 17/18 (94%) occurred at the 536–538 hotspot. No significant demographic differences were observed between the ESR1 wt and ESR1 mt groups. In terms of biopsy site, 34% (n = 136) of ESR1 wt samples were obtained from breast tissue, compared to only 6% (n = 1) in the ESR1 mt group. ESR1 mt tumors were significantly enriched for alterations in GATA3 (39% vs 13%, p = 0.007, q = 0.2) and KMT2C (22% vs 8%, p = 0.05, q = 0.2). Additionally, ESR1 mt tumors showed a higher prevalence of amplifications in MYC (22% vs 8%, p = 0.05, q = 0.2) and RNF139 (22% vs 7%, p = 0.03, q = 0.2). Conversely, ESR1 wt tumors exhibited significantly higher prevalence of amplifications in ERBB2 (76% vs 44%, p = 0.005, q = 0.2) and CDK12 (51% vs 11%, p &lt; 0.001, q = 0.1) as well as higher prevalence of TP53 alterations (52% vs 33%, p = 0.12, q = 0.2). Interestingly, 11% of the study cohort harbored a pathogenic germline variant, with CHEK2 being the most frequently observed alteration (26% of cases, 7/27). Transcriptome analysis identified no differences in major pathways irrespective of the presence of ESR1 mt ; ER and ERBB2 pathways, cell cycle pathway, and inflammatory response. Conclusions: In our cohort of ER+, HER2+ mBC, ESR1 mt were uncommon and comparable with HER2-negative mBC at diagnosis, in keeping with these mutations as emerging resistance mechanisms during therapy. Further research is required to study prevalence of these mutations during patients' journey in the metastatic setting (e.g. after exposure to aromatase inhibitor maintenance therapy), and their potential impact on outcomes.