Endocrine therapy resistance occurs in 50% of estrogen receptor positive (ER+) luminal breast cancers but the underlying mechanisms are poorly understood. To gain insight, we have taken advantage of whole-genome and RNA sequencing data of five late-stage hormone-resistant luminal breast tumors all of which have been successfully established as patient-derived mouse xenograft (PDX) models. Here we describe genetic alterations in ESR1-the gene coding for ERa-in three of these five tumors. They include an ESR1 point mutation (Y537S), a gene translocation causing an in-frame fusion between N-terminal ER and C-terminal Yes-associated protein 1 (YAP1), and ESR1 gene amplification. Functional characterization of the ESR1(Y537S) mutant and ESR1-YAP1 fusion in ER+ cell lines indicated that they both possess constitutive transcriptional activity and drive hormone-independent cell proliferation, mirroring the endocrine resistance of the originating tumors and the estradiol-independent growth of the PDX tumors. ESR1 (Y537S), a known gain-of-function experimental mutation in the ligand-binding domain of ER is not seen in primary breast cancer (TCGA data), suggesting it is a mutation associated with acquired resistance. Regarding the ESR1-YAP1 fusion gene, the truncated N-terminal ER fragment lacks the hormone-dependent transactivation domain (AF2) and the ligand binding domain but retains the hormone-independent transactivation domain (AF1) and therefore drives resistance to all endocrine approaches. As for ESR1 gene amplification, the tumor of origin, though resistant to aromatase inhibition, paradoxically responded to estradiol treatment, and this was recapitulated in the PDX model. Interestingly, acquired ESR1 gene amplification also occurred in long-term estrogen-deprived breast cancer MCF-7 cells which similarly regress upon estradiol exposure. Thus, ESR1 amplification may be a biomarker for paradoxical therapy with estradiol. Together, our focused study of advanced endocrine resistant luminal breast tumors revealed three distinct mutational mechanisms affecting the ESR1 gene that drive endocrine therapy resistance. Prevalence studies using RNAseq are underway to determine the frequency of somatic changes in the ESR1 gene in advanced breast cancer samples and additional ER+ PDX models. These additional data will be presented at the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S3-05.