Background: Prior to use of cell cycle inhibitor drugs such as palbociclib, mutations in the retinoblastoma cell cycle inhibitory gene (RB1) were extremely uncommon. To assess recent descriptions of RB1 mutations in MBC as treatment related, we compared primary untreated ER+ breast cancer samples from patients who developed MBC with ER+ MBC metastasis biopsies in post-primary treatment patients. Methods: Extracted DNA from 15 untreated primary breast tumors (PBx) and 36 treated metastatic MBC tumors (MBx) were sequenced by hybrid capture genomic profiling to evaluate all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA. PD-L1 expression was determined by IHC (Dako 22C3). Results: The age distribution, GA per tumor, CDH1 and ERBB2 GA frequencies were similar in PBx and MBx. GA in ESR1 (P < 0.0008), classically associated with acquired hormonal therapy resistance, and PIK3CA (p < 0.11) were increased in the MBx vs the PBx samples. RB1 GA were more frequent in the MBx samples (11%) than the pre-treatment PBx samples (0%, P < 0.31). GA not associated with targeted therapy programs were similar in both groups. In addition to low frequencies of ERBB2 amplification in the ER+ tumors, the most frequent potentially targetable GA identified in both PBx and MBx included GA in FGFR1 and NF1. BRCA1 GA were identified in the MBx group only. Biomarkers of potential immunotherapy benefit including MSI status, TMB levels and PD-L1 IHC staining were at similarly low levels in both groups. Additional cases are being identified to expand the cohort sizes for final presentation. Conclusions: In addition to the well-known acquisition of hormonal therapy resistance mutations in ESR1 and PIK3CA in ER+ MBC, this study also supports the acquisition of RB1 mutations when post-treatment metastasis biopsies are sequenced rather than pre-treatment primary tumors. This increase in RB1 mutation identification is associated with exposure of the tumors to anti-cell cycle drugs such as palbociclib in the treated but not the untreated cohort.