The goal of these experiments was to determine the number and distribution of brain cells that contain progestin receptors (PR) and to determine the effect of estrogen and estrogen plus progesterone on PR content of those cells. Ovariectomized adult female guinea pigs were treated with oil (control), or estrogen followed by oil, or estrogen followed by progesterone. As expected, only those animals treated with estrogen plus progesterone became sexually receptive. The cellular content of PR was determined using a monoclonal antibody to the receptor, and standard immunocytochemical techniques. Analysis of the PR-immunoreactive (PR-IR) cells consisted of: (1) mapping the anatomical distribution of PR-IR cells; (2) analyzing the effect of steroid hormones on PR-IR cell number, and (3) determining the effect of steroid hormones on PR immunoreactivity per cell. PR immunoreactivity was located exclusively in the nuclei of cells in the preoptic area and hypothalamus. The most dense collections of PR-IR cells were found in the preoptic area, ventrolateral nucleus of the hypothalamus, and infundibular nucleus. Estrogen caused a dramatic increase in the number of PR-IR cells in these cell groups. Sequential treatment with estrogen plus progesterone further increased PR-IR cell number, in the preoptic area by 65%, in the ventrolateral nucleus by 38%, and in the infundibular nucleus by 49%. A cell-by-cell rating of the PR immunoreactivity was carried out in these three cell groups. We found that the staining intensity across the populations of PR-IR cells was increased by estrogen and further increased by sequential estrogen plus progesterone. Alterations in cellular PR content may contribute importantly to the ability of progesterone target cell groups to perform their specialized roles in steroid-regulated activity.

ESR1 hotspot mutations have been identified in 30-40% of patients with ER+ MBC and promote resistance to aromatase inhibitors (AIs). Identification of these mutations has been aided by the use of plasma DNA for their detection, however many such tests only survey for hotspot mutations. In this study, we examined the prevalence, biologic and clinical significance of mutations in ESR1 that lie outside previously described hotspots (E380Q, Y537, D538G). Using next generation sequencing of tumor DNA from over 4000 patients with breast cancer, we have identified numerous somatic alterations in ESR1. Among the somatic alterations were mutations detected in the transcription activation function-1 (AF-1), DNA binding domain, dimerization interface and C-terminus of ER. We characterized the functional significance of these non-canonical mutations alongside hotspot mutations, starting with assays of ER driven transcription and deduced several classes of mutations: (1) mutations that weakly promote ligand-independent activity, (2) mutations that led to ligand-independent activity comparable to estradiol stimulation, and (3) mutations that resulted in impaired transcriptional activity. Class 2 mutations remain localized at amino acids 536-538, while the class 1 mutations are observed in various domains of ESR1, including the DNA binding domain and dimerization interface. Several Class 3 mutations were found in proximal to Helix 12, highlighting the critical role of this region. Clinically, non-canonical mutations were not exclusively observed among patients treated with AI, as there were several mutations from SERM/SERD treated patients. We thus examined the effects of different mutants on their sensitivity to ER antagonists, such as fulvestrant or tamoxifen. The data revealed key differences between the different classes of mutants; with majority of the class 2 mutants exhibiting reduced sensitivity to the antagonists compared to wild type. This also correlated with the relative binding affinities (RBA) of the mutants to fulvestrant and 4-hydroxytamoxifen, in which the RBA of class 2 mutants (Y537S and D538G) were significantly lower than wild type, perhaps accounting for their reduced sensitivities. Nevertheless, it appeared that all mutants could be effectively inhibited either by higher concentrations or more potent ER antagonists, implying a continued ability to distort ER into the antagonist conformation. Interestingly, several Class 1 mutants showed weak agonism in response to specific antagonists raising the possibility of their role in SERM/SERD resistance. Taken together, the data demonstrated that not all ESR1 mutations affect receptor function or respond to antiestrogen therapies similarly. These data also imply the importance of more broad sequencing coverage of ESR1 in the clinic to effectively capture the spectrum of biologically relevant alleles. Citation Format: Toy W, Carlson KE, Martin TA, Razavi P, Berger M, Baselga J, Greene G, Katzenellenbogen J, Chandarlapaty S. Non-canonical, clinical ESR1 mutations promote resistance to antiestrogen therapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-11.

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Endocrine based therapy is the standard treatment for estrogen receptor positive (ER+) MBC. Agents targeting the ER pathway including aromatase inhibitors (AIs), fulvestrant and tamoxifen along with CDK 4/6 inhibitors are considered standard for first and 2nd line treatment. However, endocrine resistance develops in nearly all patients and the optimal systemic therapy after progression on a CDK 4/6 inhibitor is unknown. Lasofoxifene is a third generation SERM previously investigated for the treatment of osteoporosis and vulvo-vaginal atrophy (VVA). In a large phase 3 trial evaluating the efficacy of lasofoxifene for the postmenopausal treatment of osteoporosis, lasofoxifene significantly reduced the incidence of ER+ breast cancer. Further unpublished preclinical data have demonstrated significant in vitro and in vivo efficacy in non-clinical breast cancer models including models with and without ESR1 mutants. Moreover, lasofoxifene significantly reduced metastases in ESR1 mutated models. These non-clinical and clinical data provide a strong rationale to pursue a phase 2 clinical trial in women with ER+, ESR1 mutated MBC. This open-label, multi-center study will compare the efficacy and tolerability of lasofoxifene (5 mg orally daily) to fulvestrant (IM 500 mg D1,15,29 and then q30 D) in a 1:1 randomization. Inclusion criteria include postmenopausal women with ER+ advanced breast cancer; progression on a non-steroidal AI in combination with a CDK 4/6 inhibitor; and a known ESR1 mutation. Approximately 90 patients with measurable or evaluable disease (i.e. bone only) will be recruited to have at least 40 patients per treatment arm. The primary endpoint will be progression free survival (PFS) with secondary endpoints of objective response rate (ORR), clinical benefit rate (CBR), duration of response (DoR) and time to response (TTR). It is assumed that lasofoxifene will double the median PFS compared to fulvestrant in this ESR1 mutation patient population for a hazard ratio 0.5 and a power of 89% to reach a 1-sided p of <0.05. The study will commence in 4Q2018 and will complete recruitment in 1 year. It is anticipated that 25-30 centers in the US will be participating. Citation Format: Plourde PV, Schwartzberg LS, Greene GL, Portman DJ, Komm BS, Jenkins SN, Liu P-Y, Portman MD, Goetz MP. An open-label, randomized, multi-center phase 2 study evaluating the activity of lasofoxifene relative to fulvestrant for the treatment of postmenopausal women with locally advanced or metastatic ER+/HER2 - breast cancer (MBC) with an ESR1 mutation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-01-02.