ER-positive Patients Research Articles

The prevalence of ER-low-positive breast cancer and its relation to tumor characteristics in Syria

Abstract Background: Breast cancer is a heterogeneous disease with varying clinical behaviors and responses to endocrine therapy. In particular, estrogen receptor (ER)-positive breast cancer can present with various subtypes, and the effectiveness of anti-hormone therapy on ER-low-positive tumors is unclear. This study aimed to evaluate and characterize ER-positive subtypes in patients admitted to the Surgical Oncology Clinic in the National Hospital of Jableh in Syria between 2020 and 2022. Patients and Methods: This study included 120 patients diagnosed with breast cancer; the data from each patient’s report were collected to classify them according to grade, stage, LNR, tumor size, and nodal stage. Results: The prevalence of ER-low-positive subtype was 23.33%. There was a significant difference between ER-negative and ER-positive subtypes concerning age, grade, stage, and LNR. Additionally, we confirmed a variation between ER-low-positive and ER-high-positive tumors, which may explain the difference in therapeutic response in ER-positive tumor patients taking anti-ER drugs. Conclusion: Further research is necessary to study the association between therapeutic response and ER-staining intensity breast cancer subtypes.

Abstract A004: Validation of the PREDICT breast version 3.0 prognostic tool in the US breast cancer patients

Abstract PREDICT Breast v3 is the latest version of a prognostic tool designed to predict the treatment responses for early invasive breast cancer patients. It was developed using data from approximately 35,000 women diagnosed between 2000 and 2018 in the United Kingdom. However, its performance in the United States (US) population is unknown. This study aims to validate PREDICT Breast v3 using the newly released Surveillance, Epidemiology, and End Results (SEER) outcome data for US breast cancer patients and to address potential health disparities. We selected over 860,000 female patients diagnosed with primary breast cancer between 2000 and 2021 and followed for at least 5 years from the SEER database. The model’s performance was measured by calibration and discrimination; comparing predicted and observed 5-, 10- and 15-year breast cancer-specific survival outcomes and determining discrimination through the area under the receiver-operator curves (AUC). PREDICT Breast v3 demonstrated good calibration and discrimination for both short- and long-term up breast cancer-specific mortality in US populations. The predicted 10-year breast cancer-specific mortality differed from observed by 11% in ER-positive and 6 % in ER-negative patients. The AUCs for 15-year breast cancer-specific mortality were 0.81 for ER-positive patients and 0.76 for ER-negative patients. When stratified by race and ethnicity, the model showed strong performance in Non-Hispanic Asians ER-positive patients, despite being originally developed based on the UK population. These results are consistent with previous validations in Malaysia, Japan, and other Asian countries for PREDICT Breast v2. However, the model poorly estimated the number of deaths in Non-Hispanic Black individuals for both ER-positive and ER-negative. In conclusion, this study marks the first validation of PREDICT Breast v3 in the US and demonstrates that it is a reliable and generalizable tool, capable of providing longer term follow-up time up to 15 years for a wide and diverse population. Citation Format: Yi-Wen Hsiao, Paul Pharaoh, Pei-Chen Peng. Validation of the PREDICT breast version 3.0 prognostic tool in the US breast cancer patients [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr A004.

Association of CYP7B1 expression with the prognosis of endometrial cancer: a retrospective study

BackgroundEndometrial cancer (EC) tissues express CYP7B1, but its association with prognosis needs to be investigated.MethodsImmunohistochemistry and image analysis software were used to assess CYP7B1 protein expression in paraffin-embedded endometrial tumor sections. Associations between CYP7B1 and clinical factors were tested with the Wilcoxon rank-sum test. Kaplan-Meier curves were employed to describe survival, and differences were assessed using the log-rank test. Cox regression analysis was used to assess the association between CYP7B1 expression and the prognosis of patients with EC.ResultsA total of 307 patients were enrolled with an average age of 52.6 ± 8.0 years at diagnosis. During the period of follow-up, 46 patients (15.0%) died, and 29 (9.4%) suffered recurrence. The expression of CYP7B1 protein is significantly higher in the cytoplasm than in the nucleus (P < 0.001). Patients aged < 55 years (P = 0.040), ER-positive patients (P = 0.028) and PR-positive patients (P < 0.001) report higher levels of CYP7B1 protein. Both univariate (HR = 0.41, 95% CI: 0.18–0.90, P = 0.025) and multivariate (HR = 0.35, 95%CI:0.16–0.79, P = 0.011) Cox regression analyses demonstrate that high CYP7B1 protein expression predicts longer overall survival (OS). When considering only ER-positive patients (n = 265), CYP7B1 protein expression is more strongly associated with OS (HR = 0.20,95%CI:0.08–0.52, P = 0.001). The 3-year OS and 5-year OS in the low-CYP7B1 subgroup are 81.6% and 76.8%, respectively; while in the high-CYP7B1 subgroup are 93.0% and 92.0%, respectively (P = 0.021).ConclusionsHigh CYP7B1 protein expression predicted longer OS, suggesting that it may serve as an important molecular marker for EC prognosis.

Long non-coding RNA TMEM147 antisense RNA 1/microRNA-124/signal transducer and activator of transcription 3 axis in estrogen receptor-positive breast cancer.

This research aimed to probe the expression of long noncoding RNA TMEM147 antisense RNA 1 (TMEM147-AS1)/micro-RNA (miR)-124/signal transducer and activator of transcription 3 (STAT3) axis in estrogen receptor (ER)-positive breast cancer (BC). Sixty ER-positive BC patients undergoing surgical treatment were gathered. TMEM147-AS1, miR-124, and STAT3 expression levels in BC cells and tissues were measured. The binding sites of TMEM147-AS1 and miR-124, miR-124, and STAT3 were analyzed and validated. The miR-124, STAT3 overexpression (oe) sequences, TMEM147-AS1 oe, and interference sequences and their control sequences were planned and cells were transfected to assess their functions in BC cells biological functions. TMEM147-AS1, as well as STAT3 was extremely expressed and miR-124 was lowly expressed in BC cells and tissues. Interference with TMEM147-AS1 restrained ER-positive BC cell malignant activities. Mechanistically, TMEM147-AS1 could competitively bind miR-124 in refraining miR-124 expression, and STAT3 was a target gene of miR-124. Oe of miR-124 effectively reversed the enhancement of BC cell proliferation and invasion induced by TMEM147-AS1 upregulation. Oe of STAT3 could reverse the inhibitory effect of miR-124 on BC cell malignant behaviors. TMEM147-AS1 has oncogenic activity in ER-positive BC, which may be a result of the altered miR-124/STAT3 axis. Therefore, targeting the TMEM147-AS1/miR-124/STAT3 axis may be a target for ER-positive BC therapy.

Immuno-hormonal regulation of tumor proliferation in breast cancer patients

It is well known that tumor cells proliferation is regulated by sex steroid hormones, estradiol and progesterone (E2 and Pg) in breast cancer patients (BCP). Moreover, specific auto-antibodies to estrogen receptor (ERα) were detected in blood serum of BCP. Their levels positively correlated with the percentage of Ki-67 expressing breast cancer cells. We proposed that antiidiotypic auto-antibodies to E2 and Pg (IgG2-E2 and IgG2-Pg) could act as antibodies against membrane ER and progesterone receptor (PR). Our study aimed for research of IgG2-E2 and IgG2-Pg according to E2 and Pg serum levels in association with Ki-67 positive tumors in BCP. Antiidiotypic antibodies were studied in ER-positive patients with breast cancer (stage I, n = 374, and stage II-IV, n = 379,) using ELISA technique with monoclonal antibodies against E2 and Pg as adsorbed antigens. Blood serum concentrations of E2 and Pg were measured using “ImmunoEA-Estradiol” and “ImmunoEA-Progesterone” test-systems (“Immunotech”, Russia). Tumor Ki-67 was studied by standard immunohistochemical technique at the Kemerovo Oncological Hospital. Higher percentage of Ki-67 positive breast cancer cells (Ki-67 30) was increased in BCP II-IV stages compared with stage I patients (50.7% vs 29.8%, p 0,001). Such increased values were detected for the BCP with low levels of both IgG2-E2 and IgG2-Pg antibodies in the following subgroups: 1) at low serum E2 concentration of ≤ 200 pmol/L (50.9% vs 21.3%, р 0.001); 2) at the E2 exceeding 200 pmol/L (74.2% vs 34.1%, р = 0.003); 3) at the Pg levels under 600 pmol/L (60.6% vs 22.2%, р 0.001); 4) at Pg values exceeding 600 pmol/L (58.5% vs 30.8%, р = 0.02). Similar differences were not revealed between stage II-IV and stage I BCP with low levels of both IgG2-E2 and IgG2-Pg. Corresponding Ki-67 30 indices were as follows: 1) 36.9% vs 22.0% (р = 0.14); 2) 48.4% vs 34.5% (р = 0.08); 3) 34.0% vs 27.7% (р = 0.75). Significant differences were detected in BCP with Pg 600 pmol/L and high IgG2-E2 and IgG2-Pg levels only: 48.1% vs 26.6%, (р = 0.01). Hence, antiidiotypic auto-antibodies to steroid hormones may participate in regulation of tumor proliferation in BCP. Immunoassay of IgG2-E2 and IgG2-Pg may be used for prognosis of tumor proliferation upon breast cancer progression.

EstroGene2.0: A multi-omic database of response to estrogens, ER-modulators, and resistance to endocrine therapies in breast cancer.

Endocrine therapies targeting the estrogen receptor (ER/ESR1) are the cornerstone to treat ER-positive breast cancers patients, but resistance often limits their effectiveness. Understanding the molecular mechanisms is thus key to optimize the existing drugs and to develop new ER-modulators. Notable progress has been made although the fragmented way data is reported has reduced their potential impact. Here, we introduce EstroGene2.0, an expanded database of its precursor 1.0 version. EstroGene2.0 focusses on response and resistance to endocrine therapies in breast cancer models. Incorporating multi-omic profiling of 361 experiments from 212 studies across 28 cell lines, a user-friendly browser offers comprehensive data visualization and metadata mining capabilities (https://estrogeneii.web.app/). Taking advantage of the harmonized data collection, our follow-up meta-analysis revealed substantial diversity in response to different classes of ER-modulators including SERMs, SERDs, SERCA and LDD/PROTAC. Notably, endocrine resistant models exhibit a spectrum of transcriptomic alterations including a contra-directional shift in ER and interferon signaling, which is recapitulated clinically. Furthermore, dissecting multiple ESR1-mutant cell models revealed the different clinical relevance of genome-edited versus ectopic overexpression model engineering and identified high-confidence mutant-ER targets, such as NPY1R. These examples demonstrate how EstroGene2.0 helps investigate breast cancer's response to endocrine therapies and explore resistance mechanisms.

Insights into the performance of PREDICT tool in a large Mainland Chinese breast cancer cohort: a comparative analysis of versions 3.0 and 2.2.

PREDICT is a web-based tool for forecasting breast cancer outcomes. PREDICT version 3.0 was recently released. This study aimed to validate this tool for a large population in mainland China and compare v3.0 with v2.2. Women who underwent surgery for nonmetastatic primary invasive breast cancer between 2010 and 2020 from the First Affiliated Hospital of Wenzhou Medical University were selected. Predicted and observed 5-year overall survival (OS) for both v3.0 and v2.2 were compared. Discrimination was compared using receiver-operator curves and DeLong test. Calibration was evaluated using calibration plots and chi-squared test. A difference greater than 5% was deemed clinically relevant. A total of 5424 patients were included, with median follow-up time of 58 months (IQR 38-89 months). Compared to v2.2, v3.0 did not show improved discriminatory accuracy for 5-year OS (AUC: 0.756 vs 0.771), same as ER-positive and ER-negative patients. However, calibration was significantly improved in v3.0, with predicted 5-year OS deviated from observed by -2.0% for the entire cohort, -2.9% for ER-positive and -0.0% for ER-negative patients, compared to -7.3%, -4.7% and -13.7% in v2.2. In v3.0, 5-year OS was underestimated by 9.0% for patients older than 75 years, and 5.8% for patients with micrometastases. Patients with distant metastases postdiagnosis was overestimated by 10.6%. PREDICT v3.0 reliably predicts 5-year OS for the majority of Chinese patients with breast cancer. PREDICT v3.0 significantly improved the predictive accuracy for ER-negative groups. Furthermore, caution is advised when interpreting 5-year OS for patients aged over 70, those with micrometastases or metastases postdiagnosis.

Biomarkers in breast cancer 2024: an updated consensus statement by the Spanish Society of Medical Oncology and the Spanish Society of Pathology.

This revised consensus statement of the Spanish Society of Medical Oncology (SEOM) and the Spanish Society of Pathological Anatomy (SEAP) updates the recommendations for biomarkers use in the diagnosis and treatment of breast cancer that we first published in 2018. The expert group recommends determining in early breast cancer the estrogen receptor (ER), progesterone receptor (PR), Ki-67, and Human Epidermal growth factor Receptor 2 (HER2), as well as BReast CAncer (BRCA) genes in high-risk HER2-negative breast cancer, to assist prognosis and help in indicating the therapeutic options, including hormone therapy, chemotherapy, anti-HER2 therapy, and other targeted therapies. One of the four available genetic prognostic platforms (Oncotype DX®, MammaPrint®, Prosigna®, or EndoPredict®) may be used in ER-positive patients with early breast cancer to establish a prognostic category and help decide with the patient whether adjuvant treatment may be limited to hormonal therapy. In second-line advanced breast cancer, in addition, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and estrogen receptor 1 (ESR1) should be tested in hormone-sensitive cases, BRCA gene mutations in HER2-negative cancers, and in triple-negative breast cancer (TNBC), programmed cell death-1 ligand (PD-L1). Newer biomarkers and technologies, including tumor-infiltrating lymphocytes (TILs), homologous recombination deficiency (HRD) testing, serine/threonine kinase (AKT) pathway activation, and next-generation sequencing (NGS), are at this point investigational.

Comparison of clinical characteristics and outcomes in primary neuroendocrine breast carcinoma versus invasive ductal carcinoma.

Neuroendocrine breast carcinoma (NECB) is a rare, special histologic type of breast cancer. There are some small sample studies on the clinical outcomes of NECB patients, which are worthy of further discussion. We conducted a retrospective case-control study of clinical characteristics and outcomes among patients with primary NECB versus invasive carcinoma of no special type (NST) between November 2004 and November 2017 in the Peking Union Medical College Hospital, Beijing. NST patients were strictly matched 1:4 during the same period based on the TNM stage. Statistical comparisons were performed to determine the differences in survival between NST and NECB patients and to identify clinical factors that correlate with prognosis. A total of 121 participants affected by primary NECB were included in our analysis from November 2004 to November 2017. Elderly persons (>60 years of age) were more likely to have primary NECB than young persons (p=0.001). In addition, primary NECB patients had significantly higher odds of having tumors 2-5cm (36.5%) and >5cm (6.1%) in size than NST patients. Despite a significant difference in tumor size, the proportion of patients with lymph node metastases showed no difference between the two groups (p=0.021). In addition, the rate of patients with ER-negative tumors in the NECB group (4.2%) was significantly lower than that in the primary NST group (29.8%). Significant differences were noted in the PR-negative (13.3% versus 36.6%, P<0.001) and HER2-negative (90.5% versus 76.4%, P=0.001) expression statuses among these patients. Of 121 primary NECB patients, 11 (9.1%) experienced relapses during the follow-up period. We found that tumor size was an independent risk factor for relapse. For hormone receptors on tumor cells, ER-positive breast cancer patients had significantly lower odds of relapse than receptor-negative patients. Our data demonstrate no significant difference in mortality and relapse between the primary NECB and NST groups. The tumor size in the primary NECB group was significantly larger than that in the NST group. In addition, the absence of ER independently increased the relapse rate for breast carcinoma patients.

Abstract PS15-04: A Phase 1b/2 study of palazestrant (OP-1250), an oral complete estrogen receptor antagonist (CERAN) and selective ER degrader (SERD), with palbociclib in ER-positive, HER2-negative, advanced or metastatic breast cancer patients

Abstract Background: Palazestrant (OP-1250) is a small molecule oral complete estrogen receptor (ER) antagonist (CERAN) and selective ER degrader (SERD) that binds the ligand binding domain of ER and completely blocks ER-driven transcriptional activity in both wild-type (ESR1-wt) and mutant (ESR1-mut) forms of ER. In preclinical studies, palazestrant demonstrated improved tumor growth inhibition and shrinkage when combined with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. In a Phase 1/2 monotherapy clinical study in patients with ER-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer, palazestrant was well tolerated with demonstrated antitumor efficacy and favorable pharmacokinetics (PK) supporting once a day (qd) dosing at the recommended Phase 2 dose of 120 mg. In this study, pts received palazestrant at escalating doses of 30, 60, 90, and 120 mg qd in combination with palbociclib (125 mg qd) and no dose-limiting toxicities were observed. Palazestrant did not affect palbociclib PK and no drug–drug interactions (DDIs) occurred. Here we report updates on safety, efficacy, and PK from the ongoing study of palazestrant in combination with palbociclib. Methods: Patients with advanced or metastatic breast cancer with progression on or after ≤1 line of endocrine therapy (prior CDK4/6 inhibitor therapy and ≤1 chemotherapy allowed) were enrolled to receive oral palazestrant 120 mg qd with oral palbociclib 125 mg qd for 21 of 28 days (NCT0526610). Results: As of May 12, 2023, 33 patients have been treated with palazestrant in combination with palbociclib; 12 pts were dosed in the dose-escalation phase and 21 patients were dosed in the dose-expansion phase of the study. Twenty-two patients (67%) received prior CDK4/6 inhibitor therapy; 15 patients received prior palbociclib. Thirteen of 25 patients (52%) with available baseline ctDNA had an ESR1 mutation. The most common (≥20%) treatment-emergent adverse events (TEAEs) were neutropenia, nausea, vomiting, constipation, and diarrhea. Most of these events were grade 1 or 2. The most common grade 3 or 4 related AE was neutropenia (18/33 [55%] were grade 3; 3/33 [9%] were grade 4). The steady-state exposure of palazestrant when dosed with 125 mg palbociclib was consistent with the monotherapy study. Palbociclib exposure at steady-state was comparable to published monotherapy data when combined with palazestrant at all dose levels tested. As of the data cut-off, there were 4 partial responses (2 confirmed) out of 19 response-evaluable patients, with a clinical benefit rate (CBR) of 42% (8/19) across all patients and 67% (6/9) in patients with ESR1 mutations. Conclusions: Palazestrant and palbociclib dosed in combination were well tolerated with a safety profile consistent with the individual profiles of each drug as monotherapy, and there were no DDIs. Tumor responses and clinical benefit were observed in this population of patients, including those who received prior CDK4/6 inhibitors. Expanding on our previous report, these data provide the rationale to continue advancing the clinical development of palazestrant with the approved dose of palbociclib. Updated data will be presented. Citation Format: Arlene Chan, Daphne Day, Phuong Dinh, Michael Slancar, Janine Lombard, Vinod Ganju, Nicole McCarthy, Rosalind Wilson, Demiana Faltaos, Gurpreet Mathauda-Sahota, Caitlin Murphy. A Phase 1b/2 study of palazestrant (OP-1250), an oral complete estrogen receptor antagonist (CERAN) and selective ER degrader (SERD), with palbociclib in ER-positive, HER2-negative, advanced or metastatic breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS15-04.

Abstract PO4-19-03: FES-PET/MRI for Tailored treatment of luminal A and lobular breast cancer: a prospective cohort study

Abstract Background: Breast cancer (BC) staging is essential to planning the most appropriate treatment pathway. Currently, BC management has become more tailored to the tumor’s and patient’s characteristics. However, BC imaging shows varying performance according to tumor subtypes. In particular, routine imaging may pose some challenges when evaluating luminal A BC (LumA) and lobular BC (Lob). LumA, due to low grade and proliferation index (&amp;lt; 20%) shows lower sensitivity on axillary US, reduced MRI enhancement, and low FDG-avidity on PET. Similarly, Lob, due to its peculiar single-cell growth pattern, exhibits inferior sensitivity on axillary MRI and FDG-avidity. LumA and Lob account for &amp;gt;50% of all BCs. Despite their favourable prognosis, metastases and recurrences still occur; this translates into a higher absolute number of events than in other subtypes. As a result, a concrete risk of disease underestimation and under-treatment exists. Two ongoing studies on FDG-PET/MRI imaging in our institution have already demonstrated that its sensitivity also decreases in LumA and Lob. Based on these premises, our hypothesis states that by combining the advantages of hybrid PET/MRI with the high accuracy of 16α -18F-fluoro-17-beta-estradiol(FES), a radiolabeled form of estrogen binding to functionally active ER, we could obtain a reliable, non-invasive, operator-independent, one-stage imaging method for staging LumA and ER-positive Lob. Trial design: This is a prospective cohort study where patients with LumA and ER-positive Lob will be enrolled in four cohorts undergoing: A) primary surgery; B) induction endocrine therapy; C) neoadjuvant chemotherapy, and D) systemic therapy for metastatic disease. FES PET/MRI examinations will be performed at baseline for local and systemic staging in all cohorts and a second exam after systemic therapy in cohorts C-D. Correlations between the FES PET/MRI parameters and pathology, gene expression, and FDG PET parameters, when available, will be investigated. Study aims will evaluate: 1) the performance of the FES PET/MRI in axillary staging compared with axillary surgery; 2) potential correlations between changes in FES uptake and changes in proliferation index after three weeks of endocrine therapy (ET); 3) the performance of the FES PET/MRI in systemic staging of patients undergoing systemic therapy in comparison with standard imaging; 4) biological determinants of tumor heterogeneity on pathological, imaging, and genomic levels. Statistics: The primary analysis in cohort A will test the sensitivity of the FES PET/MRI in detecting macrometastatic axillary nodes. Assuming that the probability that a patient has positive nodes is 33%, 119 patients (10% drop-out included) will provide 80% power to reject the null hypothesis about sensitivity at a 5% significance level if the true sensitivity is at least 50%. Results from the FES PET/MRI will be also compared with standard imaging in terms of sensitivity and number of lesions detected using the Mc-Nemar test for paired proportions and the Wilcoxon signed-rank test for paired data. The primary analysis in Cohort B will test the association between the FES-SUV change from pre-ET to post-ET and the Ki-67 change from core biopsy to post-ET in ER positive BC patients with a Ki-67 &amp;gt;10%. The association between these changes will be measured by the Pearson correlation index ρ. Α total of 52 patients (10% drop-out included) from Cohort B will provide 80% power to reject the null hypothesis about ρ at a 5% significance level if the true ρ is at least 0.4. No formal sample size was calculated for cohort C and D: results will be considered descriptive in nature, informing sample size considerations for future trials. We intent to recruit 20 and 30 patients, respectively. Accrual has not started yet. Project funded by AIRC (Associazione Italiana Ricerca sul Cancro) Next Gen Clinician Scientist ID 28378 Contact: Dr. Rosa Di Micco, dimicco.rosa@hsr.it Citation Format: Rosa Di Micco, Carla Canevari, Francesca Gallivanone, Nicole Rotmensz, Veronica Zuber, Sara Baleri, Giovanni Cisternino, Mario Rampa, Stefania Zambelli, Giulia Viale, Patrizia Zucchinelli, Manuela Morgante, Paola Scifo, Elena Venturini, Paola Magnani, Isabella Sassi, Vincenzo Bagnardi, Pietro Panizza, Giampaolo Bianchini, Arturo Chiti, Oreste Davide Gentilini. FES-PET/MRI for Tailored treatment of luminal A and lobular breast cancer: a prospective cohort study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-19-03.

Abstract PO2-23-11: SMAD4 depletion contributes to endocrine therapy resistance by ERBB signaling in HR+HER2- breast cancer

Abstract Objectives: Endocrine therapy resistance is a significant clinical challenge for patients with estrogen receptor (ER)-positive breast cancer. Dysregulation of the ER and ERBB signaling pathways plays a key role in endocrine therapy resistance. However, it is unclear how these pathways are integrated during resistance development. SMAD4 is involved in multiple stages of tumorigenesis, but its role in endocrine resistance development remains elusive. Here, we aimed to investigate the role of SMAD4 in the development of acquired endocrine therapy resistance in ER-positive breast cancer. Methods: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) screening was conducted to identify genes involved in regulating the sensitivity of T47D cells to 4-hydroxytamoxifen (OHT). Bioinformatics analysis was performed to explore the clinical significance of SMAD4. The differential expression of SMAD4 in cells before and after endocrine treatment was assessed by RT-qPCR and immunoblotting. Loss-/gain-of-function assays were conducted to validate the phenotype. RNA-seq and phenotype rescue experiments were used to explore the underlying mechanisms. Drug combination experiments were performed to assess the therapeutic effect on SMAD4-depleted cells. Results: CRISPR screening identified SMAD4 as a key determinant of endocrine therapy resistance. Bioinformatic analysis showed that SMAD4 expression was downregulated in breast cancer tissues and that low expression was associated with poor patient prognosis. Differential expression analysis after endocrine therapy treatment showed that endocrine therapy downregulated the expression of SMAD4. In vitro and in vivo models further demonstrated that SMAD4 downregulation leads to endocrine therapy resistance. Transcriptome sequencing analysis identified that the ER, ERBB and PI3K/Akt/mTOR signaling pathways were aberrantly activated upon SMAD4 depletion. Further analysis revealed that the PI3K/Akt/mTOR pathway may contribute to the regulation of ERBB on ER signaling to some extent. The aberrant activation of autophagy in phenotype rescue experiments was found to reduce the rescue effect of the combination of 4-hydroxytamoxifen (OHT) and lapatinib (LAPA). Finally, drug combination experiments verified that the combined use of OHT, LAPA and hydroxychloroquine (CQ) produced a synergistic effect in SMAD4-depleted cells. Conclusions: Taken together, our findings demonstrate that SMAD4 plays a crucial role in endocrine therapy resistance and suggest a reasonable treatment strategy for ER-positive breast cancer patients who are resistant to endocrine therapy. Citation Format: Kang Li, Dan Shu, Han Li. SMAD4 depletion contributes to endocrine therapy resistance by ERBB signaling in HR+HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-23-11.

Abstract PO4-20-03: HIPEx: Adjuvant palbociclib plus endocrine therapy in ER-positive HER2-negative early breast cancer with high recurrence score according to GenesWellTM BCT; a multi-center single arm phase II clinical trial (KCSG BR 19-13)

Abstract Background: In estrogen receptor (ER)-positive human epidermal growth factor receptor2 (HER2)-negative early breast cancer (EBC) patients, NCCN treatment guideline recommend 21-gene tests for decision of adjuvant chemotherapy if tumor size is over 0.5cm or node positive disease. In addition, GenesWellTM BCT is a prognostic assay that predict the risk of recurrence in patients with ER-positive HER2-negative and nodal stage 0-1 EBC. According to gene tests, EBC patients with high recurrence risk would have help from adjuvant chemotherapy to increase the rate of disease free survival but toxicities of chemotherapy are not negligible. Therefore, the effort for escaping adjuvant chemotherapy has been performed and currently CDK4/6 inhibitor would be considered as the substitute for cytotoxic chemotherapy in ER-positive HER2-negative EBC patients. Methods: HIPEx trial is a multi-center, phase II clinical trial to determine the efficacy of adjuvant palbociclib plus endocrine therapy in ER-positive HER2-negative EBC with high recurrence score according to GenesWellTM BCT (NCT04247633). Eligible patients are women who received curative surgery and diagnosed as ER-positive, HER2-negative and pathologic stage T1b-T2 and/or N0-1. Clinical recurrence risk is high according to the modified Adjuvant! Online guideline and GenesWellTM BCT score is 4 or more which being genomic high risk. Other criteria include ECOG performance status ≤1, adequate organ functions and availability of an archival surgical specimens. Patients receive palbociclib 125mg once daily for 21 days of 28-day cycle plus tamoxifen or aromatase inhibitor (anastrozole, letrozole or exemestane) once daily for every day. Palbociclib would be taken for two years and endocrine therapy for five years or more. If adjuvant radiotherapy is required, palbociclib should be taken two weeks after the end date of radiotherapy. If patients are premenopausal status, GnRH agonist is allowed. Primary endpoint is 3-year event free survival (EFS) rate of high clinical and genetic risk ER-positive EBC. Secondary endpoints include 3-year overall survival, quality of life, safety and tolerability of palbociclib with endocrine therapy as an adjuvant setting, exploratory analysis of predictive and prognostic biomarkers and the performance of GenesWellTM BCT for 3-year EFS rate. In total, 578 patients would be enrolled in this trial. Based on the previous results of GenesWell™ BCT in Korea, the 3-year EFS rate for the past control group (chemotherapy followed by endocrine therapy) is 87.0% and the treatment group in this clinical trial is assumed to be 90.5%.(hazard ratio for EFS = 0.717). Null hypothesis is the 3year-EFS rate in patients received adjuvant palbociclib and endocrine therapy is not less than that of the past control group. Alternative hypothesis is the 3year-EFS rate of this trial is less than that of the past control group (one-sided). Significance level is 0.025, power is 0.80. Considering 10% drop out rate, 4-years of accrual patient enrollment and 3-years of follow up period, 578 patients were required in this clinical trial. Citation Format: Ji-Yeon Kim, Sung Hoon Sim, Jieun Lee, In Hae Park, Kyong Hwa Park, Seock-Ah Im, Jee Hung Kim, Kyoung Eun Lee, Jeong Eon Lee, Jai Min Ryu, Young Kee Shin, Yeon Hee Park. HIPEx: Adjuvant palbociclib plus endocrine therapy in ER-positive HER2-negative early breast cancer with high recurrence score according to GenesWellTM BCT; a multi-center single arm phase II clinical trial (KCSG BR 19-13) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-03.

Abstract PO3-25-03: Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？

Abstract Background: MicroRNA-125a (miR-125a) has been shown to function as tumor suppressive miRNA by pre-clinical in vitro studies. However, to the best of our knowledge, there has been no study that investigated the clinical relevance of miR-125a in breast cancer patients. We hypothesized that miR-125a expression in breast cancer associate with its aggressiveness and can be a prognostic biomarker. Material and Methods: Non-tumorigenic epithelial cell line, MCF-10A and breast cancer cell lines, MCF-7, T47D, and MDA-MB231, were used to analyze the expression levels of miR-125a as well as cell viability. The data of 2042 patients were extracted from publicly available databases, The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC), The Cancer Genome Atlas (TCGA), and GSE57897. These databases included clinicopathological and survival data as well as transcriptome data. The survival analysis, gene set enrichment analysis (GSEA) were conducted comparing miR-125a high expressing and low expressing tumors, divided by the median cutoff. The association between the miR-125a expression and tumor immune microenvironment was assessed by xCell. Results: The expression levels of miR-125a were suppressed in breast cancer cell lines, MCF-7, T47D, and MDA-MB231compared with MCF-10A. This was concordant with human samples. The expression levels of miR-125a were lower in tumors compared with normal breast tissues in both TCGA and GSE57897 cohorts. These results were in agreement with the notion that it is a tumor suppressive miRNA (p&amp;lt; 0.001 and p&amp;lt; 0.001, respectively). ER-positive/HER2-negative (ER+/HER2-) showed the highest miR-125a expression among the subtypes in TCGA (p&amp;lt; 0.001). MiR-125a expression was not associated with cancer staging in any of the subtypes in neither TCGA nor METABRIC cohorts. To our surprise, high miR-125a tumors demonstrated worse disease free survival (DFS), disease specific survival (DSS), and overall survival (OS) compared with low expressing in ER+/HER2- breast cancer patients (p=0.008, p=0.005, and p=0.037) which was not the case for the other subtypes in METABRIC cohort. Interestingly, miR-125a expression was associated with Nottingham histological grade only in ER+/HER2- among the subtypes (p&amp;lt; 0.001). Also, high miR-125a tumors significantly demonstrated higher expression of MKI67, one of the most commonly used parameters for cell proliferation, correlated with miR-125a expression in ER+/HER2- (p=0.02), but not in other subtypes. Furthermore, high miR-125a tumors enriched four out of five cell proliferation-related gene sets in Hallmark collection, such as E2F Targets, G2M Checkpoint, Mitotic Spindle, and MYC Targets V2 in ER+/HER2- subtype, but not in TNBC. This was also the case in immune-related gene sets; interferon-alpha response and interferon-gamma response that enriched to high miR-125a tumors in ER+/HER2-, but not in TNBC. Anti-tumor immune cells, CD8 cells, T-helper type 1 cells, and M1 macrophages, were all elevated in high miR-125a tumors in ER+/HER2- subtype (all p&amp;lt; 0.03), but none in TNBC. Also, pro-cancerous immune cell such as T-helper type 2 cells were similarly highly infiltrated with high miR-125a tumors only in ER+/HER2- subtype (p=0.026). Given its association with the survival outcome, we cannot help but speculate that miR-125a expression is parallel with highly proliferative ER+/HER2- breast cancer, but its tumor suppressive effect is not enough to improve survival outcome. Conclusion: High miR-125a levels in tumors were associated with aggressive biology and worse survival, thus it can be a candidate for a prognostic biomarker in ER+/HER2- patients. Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Mai Okawa, Saki Ando, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？ [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-03.

Abstract PO4-27-05: A phase I open-label dose escalation trial of FWD1802 as monotherapy and in combination with palbociclib in patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations

Abstract Background: Estrogen receptor (ER) signaling sits at center of the tumor biology in ER+/HER2- breast cancers. Current standard of care involves hormone therapy with agents including selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), and aromatase inhibitors (AIs). Unfortunately, most patients eventually develop resistance to these therapies. Mutations in estrogen receptor 1 (ESR1) are partially responsible for the acquired resistance. Mutated estrogen receptors are still sensitive to fulvestrant but with a much less sensitivity. In January 2023, elacestrant received its first approval for the treatment of postmenopausal women or adult men with breast cancer harboring ESR1 mutations in US. However, there is still great unmet medical need for this population. FWD1802 is a third-generation oral SERD for ER-positive breast cancer patients. It binds competitively to ER, with a much higher affinity than that of fulvestrant. Preclinically, FWD1802 alone and in combination with palbociclib exhibits significant anti-tumor activity in the ER+/HER2- models and in ESR1-mutated models in vitro and in vivo. This ongoing phase I study is to assess the safety, tolerability, pharmacokinetics (PK) and clinical activity of FWD1802 as monotherapy and in combination with palbociclib in ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations. Methods: This clinical study (CTR20232130) consists of three parts: monotherapy dose escalation (Part A), dose escalation in combination with palbociclib (Part B) and monotherapy dose expansion in ESR1-mutated patients (Part C). A conventional “3+3” design is used for this study. Approximately a total of 129 patients will be enrolled. FWD1802 is administered orally daily. In monotherapy dose escalation study (Part A), at each dose level, a single ascending dose (SAD) is followed by a 28-day continuous multiple ascending dose (MAD) period (defined as 1 cycle). In Part B dose, palbociclib (125 mg daily) is administered on a 28-day treatment cycle with 21-day on treatment and 7-day off treatment, along with the 28-day treatment cycle of FWD1802. The primary objectives include dose limiting toxicity (DLT), maximum tolerated dose (MTD), safety, tolerability, and the recommended phase 2 dose (RP2D) of FWD1802. Secondary objectives include pharmacokinetics characteristics of FWD1802 and its major metabolite as monotherapy and in combination with palbociclib. Preliminary anti-tumor activity including objective response rate (ORR), duration of response (DoR), disease control rate (DCR) and progression-free survival (PFS) will also be evaluated. DLT evaluation will be conducted after the first cycle of multi-dose period, and tumor assessment will be conducted every 8 weeks by investigators according to RECIST version 1.1. Patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutation are eligible for the enrollment. Enrolled patients should have progressed disease, or failed from previous therapy, or intolerable or inaccessible to standard therapy. In addition, patients with ECOG score ≤1 and with adequate renal, cardiac, hepatic, and hematologic function are eligible for enrollment. The monotherapy dose escalation (Part A) is currently ongoing. The dose selection for Part B and Part C will be based on the data from the ongoing Part A. Clinical trial information: CTR20232130. Research Sponsor: Shenzhen Forward Pharmaceuticals Co., Ltd. (FORWARD PHARMA). Citation Format: Ling Zeng, Jin Liu, Dan Wu, Huiwen Ge, Zirui Bian, Chenggang Zhu. A phase I open-label dose escalation trial of FWD1802 as monotherapy and in combination with palbociclib in patients with ER+/HER2- unresectable locally advanced or metastatic breast cancer with or without ESR1 mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-27-05.

Clinical and Analytical Validation of Two Methods for Ki-67 Scoring in Formalin Fixed and Paraffin Embedded Tissue Sections of Early Breast Cancer.

Proliferation determined by Ki-67 immunohistochemistry has been proposed as a useful prognostic and predictive marker in breast cancer. However, the clinical validity of Ki-67 is questionable. In this study, Ki-67 was retrospectively evaluated by three pathologists using two methods: a visual assessment of the entire slide and a quantitative assessment of the tumour margin in 411 early-stage breast cancer patients with a median follow-up of 26.8 years. We found excellent agreement between the three pathologists for both methods. The risk of recurrence for Ki-67 was time-dependent, as the high proliferation group (Ki-67 ≥ 30%) had a higher risk of recurrence initially, but after 4.5 years the risk was higher in the low proliferation group. In estrogen receptor (ER)-positive patients, the intermediate Ki-67 group initially followed the high Ki-67 group, but eventually followed the low Ki-67 group. ER-positive pN0-1 patients with intermediate Ki-67 treated with endocrine therapy alone had a similar outcome to patients treated with chemotherapy. A cut-off value of 20% appeared to be most appropriate for distinguishing between the high and low Ki-67 groups. To summarize, a simple visual whole slide Ki-67 assessment turned out to be a reliable method for clinical decision-making in early breast cancer patients. We confirmed Ki-67 as an important prognostic and predictive biomarker.

ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer.

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

Abstract 6576: Discovery of potent and selective novel small molecule drugs to target estrogen receptor mutants in endocrine-resistant breast cancer

Abstract Introduction: The acquisition of estrogen receptor alpha (ERα) gene (ESR1) mutations is a key driver for the development of resistance to current endocrine therapy in breast cancer. Clinical studies have shown that ESR1 mutations are frequently observed in metastatic ER-positive breast cancer patients and are associated with poor survival. It is well appreciated that activating ESR1 somatic mutations, especially Y537S and D538G, can drive estrogen-independent activities. In addition, these ESR1 mutations diminish the potency of the current standard-of-care agents (tamoxifen and fulvestrant) that bind ERα directly. Therefore, it is an unmet need to develop next-generation antiestrogens that inhibit ERα mutant signaling in breast cancer to improve patient survival. Here, we search for small molecule inhibitors against ERα mutants Y537S and D538G using DNA-encoded chemical library (DEL). Methods: ERα ligand binding domain (LBD) proteins corresponding to wild type (WT), Y537S and D538G mutants were expressed in E. coli and purified by Ni-NTA, anion exchange, and size exclusion chromatography. The ability of these purified proteins to bind ligands was tested in biochemical assays to validate their use in DEL selections. We conducted a DEL affinity selection using our in-house collection of 6 billion small molecule compounds against WT, Y537S and D538G mutants ER LBD proteins in the presence and absence of estradiol. Selection hits that enriched with these targets were resynthesized off-DNA and tested in biochemical and functional studies using CRISPR-Cas9 knock-in Y537S or D538G mutant breast cancer cells. Results: We have successfully purified microgram amounts of ERα LBD for WT, Y537S, and D538G proteins. In our biochemical assay, the SRC3 peptide binds to the WT ERα LBD in the presence of estradiol, whereas Y537S and D538G LBDs bind the SRC3 peptide in the absence of estradiol, consistent with these mutants constitutively binding to SRC3. Our multibillion small molecule collections of DELs can screen and identify several hits in WT and mutant ERα LBDs. To confirm the selection output, we synthesized off-DNA compounds and validated these in biochemical and cell-based studies. Compounds CDD-1274 and CDD-1802 dramatically decrease the WT and mutant ERα protein levels in multiple ER+ breast cancer cells. These compounds greatly reduce protein levels for GREB1, TFF1, c-MYC, E2F1, and survivin in wildtype and mutant breast cancer cells, and they increase levels of cleaved PARP, a marker for apoptosis in wildtype and mutant ER+ breast cancer cells but not in ER-negative breast cancer cells. Conclusion: Our study rapidly identified potent and novel small molecule drugs to target estrogen receptor mutants using our DNA-encoded chemical library platform. Acknowledgments: Funding sources (NIH/NCI R03 CA259664 and CPRIT RP220524 to MP). Citation Format: Anil Kumar Devakrishnan, Chandrashekhar Madasu, Suzanne A.W Fuqua, Martin M. Matzuk, Murugesan Palaniappan. Discovery of potent and selective novel small molecule drugs to target estrogen receptor mutants in endocrine-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6576.

Abstract 6279: Estrogen signaling in low-grade serous ovarian carcinoma

Abstract High level of estrogen in the blood of women has been linked to the progression and possibly development of ovarian cancer. Almost all low-grade serous ovarian cancer (LGSOC) expresses estrogen receptor alpha (ERα protein encoded by the gene ESR1) and develops during reproductive age of women. However, recent studies suggested that a higher expression of ERα correlates with better survival. In a phase II clinical trial, hormonal therapies have been shown to be associated with a clinical benefit rate of 61% for recurrent ER-positive LGSOC patients. The study also indicated that women with recurrent LGSOC who received hormonal therapies had a significantly longer progression-free survival but not overall survival. Endocrine therapies have proven to be effective in treating breast cancer. Unfortunately, in ovarian cancer clinical trials (GOG0281 and PARAGON), these treatments have not achieved comparable results, with an objective response rate of only approximately 14%. To understand the ESR1 regulation in LGSOC, we have performed RNAseq and ChIPseq analyses of LGSOC tumor tissues. We also forced over-expression of ESR1 in LGSOC cell lines. Using RNAseq data, we aligned the sequencing reads to all the known ESR1 transcript variants. We found that ESR1 transcript variant 1, 2, 3 and X11 were the major transcripts expressed. Variants 1, 2 and 3 encode the regular 66 kDa ESR1 protein, while the X11 variant encodes a 52 kDa ESR1 protein. Both 66 kDa and 52 kDa ESR1 proteins were also detected in LGSOC tumor tissues by western blots. To further explore the function of ESR1 66kDa in LGSOC, we over-expressed ESR1 variant 1 in two LGSOC cell lines (HOC-7 and HCC5075) by transfecting with an expression plasmid. Total proteins were extracted from the mock control and transfected cells for functional proteomic analysis by reverse phase protein arrays (RPPA). From the RPPA data, overexpressing ESR1 in LGSOC cell lines suppressed DNA repair pathways but activated cell cycle and mitosis pathways. To further delineate the ESR1 DNA binding sites in LGSOC, we performed ChIPseq analysis of these tumor samples as well as two ovarian cancer cell lines that express ESR1 protein. Our results demonstrated that ESR1 regulated “cistromes” in ER positive ovarian carcinoma is very different from that in ER positive breast cancer as much less ESR1 binding sites were found in LGSOC. There are ESR1 binding sites common to both types of cancers, but we found quite a few unique ESR1 binding sites. We are trying to integrate the ChIPseq and RNAseq data and hopefully identify, for the first time, an ovarian cancer specific ERα cistrome. ER expression may not always correlate with activation of ESR1 signaling pathways, which may be one of the reasons underlying endocrine therapy resistance in ovarian cancer. The results of an integrated analyses will also give us insight on the management of ER positive LGSOC by identifying new genetic targets that may be used in combination with endocrine therapy. Citation Format: Yvonne TM Tsang, Chun Wai Oscar Ng, David Gershenson, K-Kwok wong. Estrogen signaling in low-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6279.

Abstract 2994: MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling

Abstract Background: Estrogen receptor (ER) positive breast cancer is the most common subtype of breast cancer. Elucidation of the regulatory network in estrogen receptor pathway is critical to develop and deliver potent targeted therapy for ER-positive breast cancer. The Mir-99a-let-7c cluster host gene MIR99AHG is a long non-coding RNA involving in a variety of tumor formation and disease progression processes. However, the biological functions of MIR99AHG in ER-positive breast cancer have not been reported. Methods: The Western-blot, qRT-PCR, Immunofluorescence and fluorescence in-situ hybridization were used to identify potential target genes and regulatory pathway of estrogen receptor α (ERa), which is a key proliferative driver of ER-positive breast cancer cells. The cell proliferation assay, clone formation assay, xenograft tumor model and rescue assay were used to investigate the effect of differential expression of MIR99AHG on the proliferative capacity of ER-positive breast cancer cells MCF7 and T47D. In addition, correlation analysis was performed to verify the relation of the expression of MIR99AHG, LATS1, LATS2, CYR61, CTGF and ESR1 from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. And survival analysis was used to predict the prognostic impact of MIR99AHG in breast cancer patients. Results: MIR99AHG expression was significantly higher in ER-positive breast cancer cells MCF7 and T47D than in ER-negative breast cancer cells SKBR3, MDA-MB-231 and BT549. The high expression of MIR99AHG predicted poor prognosis in ER-positive breast cancer patients. In vitro and in vivo assays demonstrated that MIR99AHG positively regulated the proliferation of MCF7 and T47D cells. Importantly, the Hippo signaling was identified to be down-stream mediators of MIR99AHG in modulating ERα transcription. Furthermore, XMU-MP-1, which targets the Hippo signaling upstream kinase MST1/2, was able to reverse the effect of MIR99AHG on MCF7 cell proliferation. Conclusions: MIR99AHG up-regulates ERα expression and promotes proliferation in ER-positive breast cancer cells through activating Hippo signaling pathway. Targeting MIR99AHG/Hippo pathway is a potential strategy for the treatment of ER-positive breast cancer. Citation Format: Wende Wang, Danping Lin, Ziyang Lin, Yangyang Yan, Yusen Qin, Yuanke Liang, Haoyu Lin, De Zeng. MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2994.