Further examining each ESR1 mutation in the breast cancer samples (n = 405, 129 from MSK, 262 from FMI, and 14 from MET500), we found that the liver metastasis enrichment is in fact primarily contributed by four hotspot positions located at the ligand-binding domain (LBD): D538, Y537, L538 and E380 (n > 20, Fig. 5c), all of which are spatially close to each other and have been demonstrated to give rise to estrogen-independent activation of downstream signaling and promote cellular proliferation43. Here, ESR1 is linked to breast cancer.