Abstract Background Pre-clinical evidence and retrospective studies suggest that PAM50 HER2-Enriched (HER2-E), hormone receptor-positive (HR+)/HER2-negative tumors have estrogen receptor (ER)-independency and poor prognosis, but seem to have androgen receptor (AR)-addiction (1). Enzalutamide (EZM) is a potent inhibitor of androgen receptor signaling (3). AR expression has been shown to induce resistance to both tamoxifen and aromatase inhibitors in estrogen receptor HR-expressing cell lines (4,5) The hypothesis of the ARIANNA trial is that EZM induces a significant proliferative arrest in PAM50 HER2-E, HR+/HER2-negative advanced breast cancer (BC), leading to clinical benefit in this poor prognosis population. Methods ARIANNA is an exploratory, phase II clinical trial in two independent cohorts evaluating the effect of EZM on proliferation after 2 weeks (+7 days window) on treatment in pre- or post-menopausal female or male patients with endocrine-resistant, locally advanced or metastatic HR+/HER2-negative BC. Cohort A will include 22 patients with PAM50 HER2-E HR+/HER-negative tumors and Cohort B (control group) will include 22 patients with PAM50 Luminal A/B HR+/HER2-negative tumors. Fresh tumor biopsy will be obtained at screening and sent to central laboratory for PAM50 subtyping determination to confirm the molecular subtype status prior to study treatment initiation, and to determine PAM50 11-gene proliferation-related signature. Patients with PAM50 Basal-like or Normal-like tumors will be excluded. Patients will receive EZM 160 mg once a day (QD). After 2 weeks on treatment, a tumor biopsy from the same baseline lesion (or, if not feasible, a lesion in the same organ) will be obtained for the purpose of the primary endpoint analysis. After this on-treatment biopsy, exemestane 50mg QD can be added to EZM at physician’s discretion. Tumor assessment will be performed at screening and every 8 weeks thereafter. Treatment will be continued until disease progression, unacceptable toxicity, investigator’s decision or withdrawal of consent. An optional tumor biopsy will be collected at the end of treatment. The primary objective is to evaluate the anti-proliferative effect of EZM after 2 weeks of treatment in patients with HER2-E HR+/HER2-negative tumors, measured as relative changes in the PAM50 11-gene proliferation-related signature by the PAM50 nCounter-based assay between baseline and on-treatment tumor biopsies. Secondary objectives include: anti-proliferative effect of EZM after 2 weeks of treatment in patients included in Cohort B (control group), safety, overall response rate, progression-free survival, and further correlative molecular analyses both at the tumor tissue (IHC, RNA and DNA) and ctDNA level for both cohorts. The trial will enroll patients in 8 Spanish sites and recruitment period will be 18 months. Funding was granted by Breast Cancer Research Foundation and drug was supplied by Astellas Pharma Global Development, Inc./Pfizer, Inc. Trial identification: NCT04142060 1. Cochrane DR, Bernales S, Jacobsen BM, Cittelly DM, Howe EN, D’Amato NC, et al. Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide. Breast Cancer Res. 2014;16:R7. 3. Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Arora V, et al. Development of a Second-Generation Antiandrogen for Treatment of Advanced Prostate Cancer. Science. 2009;324:787-90. 4. Rechoum Y, Rovito D, Iacopetta D, Barone I, Andò S, Weigel NL, et al. AR collaborates with ERα in aromatase inhibitor-resistant breast cancer. Breast Cancer Res Treat. 2014;147:473-85. 5. De Amicis F, Thirugnansampanthan J, Cui Y, Selever J, Beyer A, Parra I, et al. Androgen receptor overexpression induces tamoxifen resistance in human breast cancer cells. Breast Cancer Res Treat. 2010;121:1-11 Citation Format: Mafalda Oliveira, Sonia Pernas, Mireia Margelí, Salvador Blanch, Barbara Adamo, Javier Salvador Bofill, Diana Moreno, Xavier Gonzalez-Farré, José Rios, Charles M Perou, Aleix Prat, Tomás Pascual, Juan M Ferrero-Cafiero, Patricia Villagrasa, Luis Manso. Solti-1502 aRIANNA: Targeting PAM50 HER2-enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-08.