Estrogen Receptor-positive Breast Research Articles

Identifying determinants of adherence to adjuvant endocrine therapy following breast cancer: A systematic review of reviews.

In oestrogen-receptor positive breast cancer, daily oral adjuvant endocrine therapy (ET) for at least 5 years significantly reduces risks of recurrence and breast cancer-specific mortality. However, many women are poorly adherent to ET. Development of effective adherence support requires comprehensive understanding of influences on adherence. We undertook an umbrella review to identify determinants of ET adherence. We searched PubMed, Embase, CINAHL, PsycINFO, Cochrane and PROSPERO (inception to 08/2022) to identify systematic reviews on factors influencing ET adherence. Abstracted determinants were mapped to the World Health Organization's dimensions of adherence. Reviews were quality appraised and overlap assessed. Of 5732 citations screened, 17 reviews were eligible (9 quantitative primary studies; 4 qualitative primary studies; 4 qualitative or quantitative studies) including 215 primary papers. All five WHO dimensions influenced ET non-adherence: The most consistently identified non-adherence determinants were patient-related factors (e.g. lower perceived ET necessity, more treatment concerns, perceptions of ET 'cons' vs. 'pros'). Healthcare system/healthcare professional-related factors (e.g. perceived lower quality health professional interaction/relationship) were also important and, to a somewhat lesser extent, socio-economic factors (e.g. lower levels of social/economic/material support). Evidence was more mixed for medication-related and condition-related factors, but several may be relevant (e.g. experiencing side-effects, cost). Potentially modifiable factors are more influential than non-modifiable/fixed factors (e.g. patient characteristics). The evidence-base on ET adherence determinants is extensive. Future empirical studies should focus on less well-researched areas and settings. The determinants themselves are numerous and complex in indicating that adherence support should be multifaceted, addressing multiple determinants.

Bexmarilimab-induced macrophage activation leads to treatment benefit in solid tumors: The phase I/II first-in-human MATINS trial

Macrophage Clever-1 contributes to impaired antigen presentation and suppression of anti-tumor immunity. This first-in-human trial investigates the safety and tolerability of Clever-1 blockade with bexmarilimab in patients with treatment-refractory solid tumors and assesses preliminary anti-tumor efficacy, pharmacodynamics, and immunologic correlates. Bexmarilimab shows no dose-limiting toxicities in part I (n= 30) and no additional safety signals in part II (n= 108). Disease control (DC) rates of 25%-40% are observed in cutaneous melanoma, gastric, hepatocellular, estrogen receptor-positive breast, and biliary tract cancers. DC associates with improved survival in a landmark analysis and correlates with high pre-treatment intratumoral Clever-1 positivity and increasing on-treatment serum interferon γ (IFNγ) levels. Spatial transcriptomics profiling of DC and non-DC tumors demonstrates bexmarilimab-induced macrophage activation and stimulation of IFNγ and Tcell receptor signaling selectively in DC patients. These data suggest that bexmarilimab therapy is well tolerated and show that macrophage targeting can promote immune activation and tumor control in late-stage cancer.

Abstract P2-17-04: Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis

Abstract Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis Benson C. Selvanesan1,2, Sheelu Varghese1,2, Justyna Andrys5, Ricardo H. Arriaza6, Rahul Prakash6, Purushottam B Tiwari7, Cara Olsen8, Daniel Hupalo2,4, Yuriy Gusev5, Megha N. Patel6, Sara Contente1, Miloslav Sanda9, Aykut Uren7, Matthew D. Wilkerson3,4, Clifton L. Dalgard3,4, Linda S. Shimizu6, Maksymilian Chruszcz6, Tomasz Borowski5, Geeta Upadhyay 1,3,7. Affiliations 1 Department of Pathology, 2 Henry M. Jackson Foundation, 3 Murtha Cancer Center, 4 Department of Anatomy, Physiology, and Genetics 8 Department of Preventive Medicine and Biostatistics Uniformed Services University of the Health Sciences, Bethesda, MD, USA. 5 Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Cracow, Poland. 6 Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, USA. 7 Department of Oncology, Georgetown University Medical Center, Washington, DC, USA. 9 Max Planck Institute for Heart and Lung Research, Ludwigstrasse, 43, 61231 Bad Nauheim, Germany. Correspond Disclaimer The opinions expressed herein are those of the authors and are not necessarily representative of the official policies of the Uniformed Services University of the Health Sciences (USUHS), the Department of Defense (DOD), the United States Army/Navy/Air Force, the U.S. Government, or any other funding agencies Conflict of Interest None Acknowledgments NIH, NCI, R01 CA227694. NIH, NCI, R21CA256424. DOD, USUHS, VPR-NFP-74-9824. Biomedical Instrumentation Center, USUHS. The American Genome Center, USUHS. Antibody Characterization Program, Clinical Proteomics Tumor Analysis Consortium (CPTAC), National Cancer Institute, National Institute of Health. The Polish Grid Infrastructure, Cracow, Poland. NIH P30CA51008 and 1S10OD019982-01 to Biacore Molecular Interaction Shared Resource (BMISR), Georgetown University. ABSTRACT Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including triple-negative and estrogen receptor-positive breast, ovarian, gastric, head and neck, neuroblastoma, bladder, and lung cancers. Inhibition of LY6K signaling is an ideal therapeutic approach for cancer, since the LY6K protein is not involved in vital organ function. Previously, we identified the small molecule NSC243928 as a binder of LY6K using surface plasmon resonance screening and showed that its activity was dependent on LY6K expression in triple-negative breast cancer cells. Here, we demonstrate the structural basis of the molecular interaction of NSC243928 with LY6K protein and the subsequent inhibition of LY6K function in mitosis and cell division via Aurora B-histone pathway. We observed that LY6K interacts with phosphorylated histones and Aurora B kinases during mitosis and that this interaction was disrupted in the presence of NSC243928. Disruption of LY6K function in mitosis/cytokinesis leads to DNA damage, senescence, and apoptosis of cancer cells. We observed that NSC243928 led to increased binding of LY6K to phosphorylated gammaH2X at S139, which was dependent on NSC243928 interaction with LY6K on phenylalanine 79. Furthermore, we observed increased levels of phosphorylated gammaH2X at S139 and increased caspase-3 activation in the tumor isografts of 4T1 and E0771 mammary tumors treated with NSC243928. These data reveal that LY6K is a novel cell cycle target for therapeutic development in triple-negative breast cancer and other solid cancers with high expression of LY6K, such as bladder cancer, head and neck, and lung cancer. Citation Format: Benson Selvanesan, Sheelu Varghese, Justyna Andrys, Ricardo Arriaza, Rahul Prakash, Purushottam Tiwari, Cara Olsen, Daniel Huplo, yuriy Gusev, Megha Patel, Sara Contente, Miloslav Sanda, Matthew Wilkerson, Clifton Dalgard, Linda S. Shimizu, Maksymilian Chruszcz, Tomasz Borowski, Geeta Upadhyay. Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-04.

Co-designed weight management intervention for women recovering from oestrogen-receptor positive breast cancer

BackgroundWeight gain is commonly observed during and after breast cancer treatment and is associated with poorer survival outcomes, particularly in women with oestrogen receptor-positive (ER +) disease. The aim of this study was to co-design (with patients) a programme of tailored, personalised support (intervention), including high-quality support materials, to help female breast cancer patients (BCPs) with ER + disease to develop the skills and confidence needed for sustainable weight loss. MethodsER + BCPs were recruited from two UK National Health Service (NHS) Trusts. The selection criteria included (i) recent experience of breast cancer treatment (within 36 months of completing primary treatment); (ii) participation in a recent focus group study investigating weight management perceptions and experiences; (iii) willingness to share experiences and contribute to discussions on the support structures needed for sustainable dietary and physical activity behaviour change. Co-design workshops included presentations and interactive activities and were facilitated by an experienced co-design researcher (HH), assisted by other members of the research team (KP, SW and JS).ResultsTwo groups of BCPs from the North of England (N = 4) and South Yorkshire (N = 5) participated in a two-stage co-design process. The stage 1 and stage 2 co-design workshops were held two weeks apart and took place between Jan–March 2019, with each workshop being approximately 2 h in duration. Guided by the Behaviour Change Wheel, a theoretically-informed weight management intervention was developed on the basis of co-designed strategies to overcome physical and emotional barriers to dietary and physical activity behaviour change. BCPs were instrumental in designing all key features of the intervention, in terms of Capability (e.g., evidence-based information, peer-support and shared experiences), Opportunity (e.g., flexible approach to weight management based on core principles) and Motivation (e.g., appropriate use of goal-setting and high-quality resources, including motivational factsheets) for behaviour change.ConclusionThis co-design approach enabled the development of a theoretically-informed intervention with a content, structure and delivery model that has the potential to address the weight management challenges faced by BCPs diagnosed with ER + disease. Future research is required to evaluate the effectiveness of the intervention for eliciting clinically-important and sustainable weight loss in this population.

GPER-mediated stabilization of HIF-1α contributes to upregulated aerobic glycolysis in tamoxifen-resistant cells.

Tamoxifen is a first-line therapeutic drug for oestrogen-receptor positive breast cancer; however, like other therapeutics, its clinical use is limited by acquired resistance. Tamoxifen-resistant cells have demonstrated enhanced aerobic glycolysis; however, the mechanisms underlying this upregulation remain unclear. Here, we demonstrated that G-protein coupled oestrogen receptor (GPER) was involved in the upregulation of aerobic glycolysis via induction of hypoxia-inducible factor-1α (HIF-1α) expression and transcriptional activity in tamoxifen-resistant cells. Additionally, GPER stabilized HIF-1α through inhibiting its hydroxylation and ubiquitin-mediated degradation, which were associated with upregulation of C-terminal hydrolase-L1 (UCH-L1), downregulation of prolyl hydroxylase 2 (PHD2) and von Hippel-Lindau tumour suppressor protein (pVHL), induction of HIF-1α/UCH-L1 interaction, and suppression of HIF-1α/PHD2-pVHL association. The GPER/HIF-1α axis was functionally responsible for regulating tamoxifen sensitivity both in vitro and in vivo. Moreover, there was a positive correlation between GPER and HIF-1α expression in clinical breast cancer tissues, and high levels of GPER combined with nuclear HIF-1α indicated poor overall survival. High levels of the GPER/HIF-1α axis were also correlated with shorter relapse-free survival in patients receiving tamoxifen. Hence, our findings support a critical role of GPER/HIF-1α axis in the regulation of aerobic glycolysis in tamoxifen-resistant cells, offering a potential therapeutic target for tamoxifen-resistant breast cancer.

Abstract 649: Targeting progesterone receptor (PR) with the antiprogestin onapristone in patient-derived xenograft (PDX) models of estrogen receptor positive (ER+), PR positive (PR+) bone metastasis of breast cancer

Abstract Background: Onapristone (ONA) is a progesterone receptor (PR) antagonist that prevents PR-mediated DNA transcription. ONA is currently being evaluated in metastatic breast cancers (MBC), as well as other hormone-dependent cancers. Recently, therapeutic options for metastatic estrogen receptor (ER) positive breast cancers have been expanded by the introduction of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), administered in combination with endocrine treatments, as first line therapy. Moreover, the selective PI3K inhibitor alpelisib (ALP) has been approved for the treatment of PIK3CA-mutated endocrine resistant MBC. The objective of this study was to evaluate the efficacy of ONA in combination with fulvestrant (FUL) and palbociclib (PAL) or ALP in different PDX models established from ER and PR positive breast cancers. Methods: PDX models were established from primary tumors or biopsies from bone metastases from endocrine therapy patients with progressing tumors. ER and PR expression were analyzed by immunohistochemistry (IHC) and western blot (WB) analysis in 17 PDX models. The anti-tumor activity of onapristone alone or combined with FUL and PAL was tested in 2 ER+/PR+ PDX studies, while the combination with ALP+FUL was tested in one PIK3CA-mutated PDX study. Phosphorylation of both PR and ER-PR interactions were analyzed in treated tumors by WB and by the in situ Proximity Ligation Assay (PLA), respectively. Results: PR expression was found in 1/9 PDX established from primary breast tumors and 4/8 PDX established from bone metastases. ONA in vivo activity was tested in 2 PDX of bone metastases: PDX BC1101 (PR low) and BC1117 (PR high). BC1101 showed amplification of FGFR1 and CCND1 genes, while BC1117 has an activating mutation of PIK3CA gene. In the low PR PDX BC1101, treatment with ONA was ineffective in both the monotherapy and combination setting. Conversely, in the PDX with high expression of PR, BC1117, ONA treatment, given as monotherapy, decreased tumor growth. The anti-tumor activity of ONA+FUL+PAL combination was significantly increased as compared to FUL+PAL, with the majority of xenografts showing tumor regression. Expression of PR and phospho-PR were inhibited in the tumors treated by FUL, ONA+FUL, and ONA+FUL+PAL. Interaction between ER and PR, analyzed by the in situ PLA assay, was inhibited in the ONA+FUL treated xenografts ±PAL. Finally, treatment with the triple combination of ONA+FUL+ALP was also highly effective and significantly greater than the combination of FUL+ALP. RNAseq analysis of treated xenografts is ongoing to identify transcriptomic changes in the different treatment arms. In conclusion, our study demonstrates that ONA improved the response to endocrine treatment using CDK4/6 or PI3K inhibitors in a MBC-derived PDX model with high PR expression. Citation Format: Martin Lehr, Ahmed Dahmani, Léa Huguet, Rania El-Botty, Charlène Thiebaut, Muriel Romancer, Paul Cottu, Elisabetta Marangoni. Targeting progesterone receptor (PR) with the antiprogestin onapristone in patient-derived xenograft (PDX) models of estrogen receptor positive (ER+), PR positive (PR+) bone metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 649.

Abstract 2741: A communication theory framework for modeling cytokine-mediated signaling in healthy and breast cancer derived peripheral blood immune cells

Abstract Previous studies have shown that approximately 40% of estrogen receptor positive (ER+) breast cancer (BC) patients harbor immune signaling defects in their blood at diagnosis that predicts overall survival. The aim of this work is to apply communication theory and signal processing methods to model cytokine-mediated signaling errors in immune cells in the peripheral blood. Our approach uses communication theory concepts to develop a communication system model consisting of a transmitter, receiver, and channel, applied to the JAK/STAT signaling pathway and tested using multi-color flow cytometry analysis of peripheral blood mononuclear cells stimulated with IFNγ, IL-2, IL-6, IL-4, IL-10, IL-12, or TGFβ in samples from 2 BC patients and 4 healthy controls. We use our model to estimate the detection error rate when one of the 7 cytokines is being transduced through the JAK/STAT signaling pathway, but its signal is being received on multiple phosphorylated signal transducers and activators of transcription (pSTAT) molecules. In addition, we extend the measured error rate value to an error rate curve by varying the signal-to-noise ratio available to the receiver in our system model. Our results show a trend towards higher detection error rates in immune cells from BC patients as compared to healthy controls, which may indicate altered immune signaling and incorrect cell decisions in these patients. Also, considering different cell subtypes, our results show two orders of magnitude increase in error rate in CD4+ memory T cells compared to CD4+ naïve T cells in BC patients. With further development, we believe that our communication theory model and quantification of optimal signal detection in cells will provide a better understanding into how immune cells can accurately “decode” information to respond properly to various signals and how the immune signaling is altered in BC patients, causing better or worse outcomes. The benefit of our novel approach is that it integrates all the transmitted (cytokines) and received signals (pSTATs) into a single communications framework that can be efficiently analyzed together. Citation Format: Adina Matache, Aleksandra Karolak, Sergio Branciamore, Andrei Rodin, Peter P. Lee, Russell C. Rockne. A communication theory framework for modeling cytokine-mediated signaling in healthy and breast cancer derived peripheral blood immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2741.

Abstract 1772: EGFR signaling as a mechanism of resistance to CDK4/6 inhibitors in Palbociclib-resistant ER+ breast cancer cells

Abstract Breast cancer driven by estrogen receptor (ER) is responsible for approximately 60-70% of cases among women. Selective inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) and ER signaling is now standard-of-care therapy for ER positive (ER+) metastatic breast cancer. CDK4/6 inhibitors, including palbociclib (PD), used in combination with endocrine therapy have shown improvement in progression-free survival compared to endocrine therapy alone in the metastatic setting. However, the inevitable development of acquired resistance significantly limits the efficacy of this targeted therapy. In ER+ breast cancer resistant cells, rewired signaling driven by different oncogenes, including the epidermal growth factor receptor (EGFR), overcomes the inhibition of CDK4/6 by PD, which, in turn, allows cell cycle progression contributing to PD-resistance. We investigated the dependency of ER+ breast cancer cell lines sensitive and resistant to PD on EGFR signaling in vitro. We evaluated the expression and activity of EGFR in two different matching pairs of ER+ palbociclib-sensitive (pS) and palbociclib-resistant (pR) breast cancer cell lines (MCF7 and T47D) and observed elevated EGFR expression in MCF7/pR cells. Increased EGFR expression in MCF7/pR cells correlated with elevated ER alpha phosphorylation at S118 and S167 sites, suggesting a direct cross-talk between EGFR and ER signaling in PD-resistant cells. Serum starvation significantly decreased EGFR activity, cdk2 and cyclin D1 expression in both MCF7 and T47D PD-resistant cells compared to the parental cell lines, confirming their dependency on the presence of growth factors. Notably, stimulation with EGF promoted ER phosphorylation and expression of cell cycle related targets: cdk2, cyclin E and wee1 in both MCF7 and T47D cell lines sensitive and resistant to PD. Furthermore, stimulation with estrogen promoted EGFR activation in MCF7/pS and MCF7/pR cells, indicating a direct cross-regulation between these molecular targets. Further, exposure to the EGFR inhibitor erlotinib dramatically inhibited the proliferation of MCF7/pR cells compared to the parental cells, suggesting that EGFR could be an important bypass signaling regulator contributing to resistance to PD. We found that dual treatment with erlotinib and PD significantly inhibited Rb phosphorylation in MCF7/pR cells improving the effect of PD on cell cycle arrest. Moreover, dual treatment of MCF7/pR cells attenuated ER expression suggesting the higher dependency of ER signaling on EGFR activity in resistant cells. Taken together, our findings suggest that EGFR signaling is a mechanism of resistance in ER+ cell lines with acquired resistance to PD in vitro and that EGFR targeting can be a promising approach to improve palbociclib efficacy in breast cancer patients. Citation Format: Nadiia Lypova, Lilibeth Lanceta, Susan M. Dougherty, Jason A. Chesney, Yoannis Imbert-Fernandez. EGFR signaling as a mechanism of resistance to CDK4/6 inhibitors in Palbociclib-resistant ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1772.

Abstract 4043: KSHN001034: An intramuscular prodrug of fulvestrant to treat estrogen-receptor (ER) positive advanced metastatic breast cancer

Abstract Purpose: Breast cancer (BC) remains a considerable health concern, with over 75% of total cases accounting for ER+ BC. Fulvestrant, a selective estrogen receptor degrader (SERD), is the primary drug of choice for treating advanced metastatic ER+, BC. Currently, Faslodex® (500 mg) is given as an intramuscular (IM) depot that achieves the steady-state plasma concentration of 24-28 ng/mL of fulvestrant from day 28 onwards. Additionally, clinical studies suggest that current Faslodex® (500 mg) achieves insufficient blockade of ERα receptor plausibly due to suboptimal physicochemical and pharmacokinetic (PK) properties. Hence, achieving higher fulvestrant plasma concentrations is a significant unmet need to improve efficacy and inhibit the development of drug resistance. To overcome these issues, we modified the physicochemical properties of the fulvestrant to enhance its solubility by developing a prodrug, KSHN001034, with the potential to deliver higher fulvestrant plasma exposures. Methods: KSHN001034 was evaluated for its physicochemical and ADME properties. Thermodynamic solubility and liver microsomal stability were performed. KSHN001034 was formulated as 100 mg/mL compared with Faslodex (50 mg/mL). Single-dose PK studies were performed using KSHN001034 in Sprague Dawley (SD) rats and Beagle Dogs post-IM administration for 7 and 14 days, respectively. The uterotrophic activity was investigated by determining changes in uterine weight in juvenile female SD rats for KSHN001034 (10 mg/kg, IP) and fulvestrant (25 mg/kg, SC). The ERα expression was also evaluated in rat uterus tissue via a simple western assay. Results: A remarkable improvement (~1100 fold) was achieved for KSHN001034 in aqueous solubility (1.112 mg/mL) vs. fulvestrant (<1 µg/mL). Significant improvement was observed in metabolic stability vs. fulvestrant. KSHN001034 showed significant improvement in PK exposure of fulvestrant upon IM administration in rats and dogs with higher AUC and Cmax compared to that of Faslodex. Relative bioavailability of fulvestrant post-KSHN001034 treatment was found to be >6-fold in rats and ~2.5 fold in dogs compared to Faslodex. Treatment of KSHN001034 antagonized estradiol (E2)-mediated uterine stimulation, exhibiting significant inhibition (85%) of uterotrophic activity vs. fulvestrant (73%). KSHN001034 reduced E2-induced uterus weight, supporting efficacy due to released fulvestrant as it is a true SERD. Additionally, KSHN001034 significantly inhibited the E2-induced ERα expression in rat uterus. Conclusions: Overall, KSHN001034 indicates the potential to deliver higher fulvestrant concentrations that would effectively block the ERα receptor resulting in improved efficacy and minimal emergence of resistance. Further, enhanced solubility and reduced injection volume would result in better patient compliance. Citation Format: Heta Pandya, Ganesh Sangle, Vishal Unadkat, Vishal Goswami, Parva Purohit, Chirag Mehta, Sonam Sinha, Ketan Variya. KSHN001034: An intramuscular prodrug of fulvestrant to treat estrogen-receptor (ER) positive advanced metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4043.

Abstract 5450: New se-nsaid hybrid molecules as potent and selective cytotoxic agents against breast cancer cells

Abstract Breast cancer is currently the most frequently diagnosed cancer and one of the leading causes of mortality among women worldwide. The introduction of a selenium atom (Se) in the structure of organic molecules has been demonstrated to be a valid approach in the design of novel chemotherapeutic agents with several biological effects, including antitumoral activity. In addition, several epidemiological, preclinical, and clinical studies support the chemopreventive and chemotherapeutic potential use of non-steroidal anti-inflammatory drugs (NSAIDs).On the basis of these findings, and taking into account the results of previous studies by our research group, we have developed a new series of twenty-five NSAID analogs bearing Se in the form of selenoesters modulated with different functional groups. The new compounds were tested in four cancer cell lines (HTB-54, DU-145, HT-29 and MCF-7) and two non-malignant cells (184B5 and BEAS-2B). The cytotoxicity of the five most potent and selective molecules was further studied in other cancer cell lines as well. Compounds 4a and 4d stood out as the most selective compounds with a selectivity index at least 5-fold higher in breast cancer cells. Compound 4d showed IC50 values below 10 µM and was found to be more potent than 4a in almost every cell line tested. Additionally, 4d effectively induced apoptosis in triple negative (MDA-MB-231) and estrogen receptor-positive (MCF-7) breast cancer cell lines. Taken together, this study supports the design of new derivatives containing Se as potential anticancer agents. Citation Format: Sandra Ramos-Inza, Ignacio Encío, Asif Raza, Arun Sharma, Carmen Sanmartín, Daniel Plano. New se-nsaid hybrid molecules as potent and selective cytotoxic agents against breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5450.

Abstract 3462: Modeling drug resistance in hormone receptor positive breast cancer using patient derived organoid cultures and cancer associated fibroblasts

Abstract Breast cancer is the most prevalent cancer among women worldwide. Estrogen receptor-positive (ER+) breast cancer constitutes roughly 70% of all breast cancers. Early-stage ER+ breast cancers usually respond well to ER-targeted therapies. However, only 20-40% of patients with advanced ER+ breast cancer respond to ER-targeted therapy. Recently, three inhibitors for the cyclin-dependent kinase 4/6 (CDK4/6) were approved as first-line and/or second-line treatment in combination with ER-targeted therapies. Unfortunately, despite initial response to the combination treatment, patients acquire resistance to both ER-targeted therapies and CDK4/6 inhibitors over time. As a result of this, the 5-year survival of women with metastatic breast cancer is 28%. A large contribution to this poor survival rate is the desmoplastic nature of the tumor microenvironment. Desmoplasia is characterized by the abnormal growth of fibroblasts and a dense extracellular matrix. The role of desmoplasia in breast cancer and its contribution to promoting a pro-tumor environment by limiting nutrients and oxygen has been extensively studied. However, the role of secreted factors such as cytokines and metabolites by cancer-associated fibroblasts (CAFs) has been poorly studied in the context of targeted drug resistance and its effects on tumor behavior. Therefore, to investigate the mechanism of stroma-mediated resistance to targeted therapies, we have propagated patient-derived organoids and CAFs from treatment-naïve and treatment-resistant ER+ breast tumors. Our data shows that conditioned media from CAFs have variable ability to drive resistance to hormone therapy and CDK4/6 inhibitors. To identify secreted factors that drive resistance, we have performed cytokine arrays and untargeted metabolomics of the CAF lines that were the strongest drivers of resistance. We have identified several cytokines, such as IL-5, IL-8 and YKL40, that were secreted by all CAF lines. Furthermore, many CAF lines secreted the TCA cycle intermediates α-ketoglutarate and succinate, which have been implicated as oncometabolites in solid tumors. Our preliminary data suggests that YKL40, and succinate induce drug resistance. Together, this suggests that these factors secreted by CAFs play a role in supporting a pro-tumor environment by promoting drug resistance. Defining this mechanism of resistance to ER-targeted therapy and CDK4/6 inhibitors will elucidate potential therapeutic targets to enhance patient response to therapy. Citation Format: JENNY HOGSTROM, Kayla Cruz, Jaymin Patel, Tejas Mehta, Angelica Warren, Taru Muranen. Modeling drug resistance in hormone receptor positive breast cancer using patient derived organoid cultures and cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3462.

The correlation analysis of 21-gene assay use in uninsured and underrepresented minorities with estrogen receptor (ER) positive HER2 negative breast cancer.

e12531 Background: 21-gene breast cancer recurrence-score assay (Oncotype DX) have been used to predict recurrence risks and guide adjuvant chemotherapy. However, access to such tests is limited, especially in a population concentrated with uninsured and underserved patient populations. Previously equations such as the Magee equation has been proposed to predict correlation of the recurrence score and the estimated recurrence score with high concordance rate. This study examines the concordance of such an assay to predicted scores in uninsured and underrepresented minority heavy public hospital. Methods: We performed a retrospective chart review of 68 women with hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer seen at University Hospital (public safety net hospital in Newark, New Jersey with greater majority of the patient population consists of underrepresented minorities such as Black/African Americans and Latino/Hispanics). 6 patients did not have pathology records, 9 patients did not have the Oncotype information available, and 4 patients were waiting on the results. Of the pre-menopausal eligible patients, two did not have any treatment or follow up information available and were included as part of the correlation assay, however, no. Results: Of the 47 eligible patients with follow-up information, 13 had a low recurrence score of up to 10 if post-menopausal and up to 15 if pre-menopausal, 22 had an intermediate recurrence score of 11 to 25 if post-menopausal and 16 to 25 if pre-menopausal, and 12 had a high recurrence score of greater than 26 according to the national comprehensive cancer network guidelines. The Pearson correlation between the Magee predicted score and Oncotype recurrence score was 0.73 (p < 0.001). All patients received hormone therapy and 5 patients total had recurred. 2 were pre-menopausal and had received adjuvant chemotherapy. 3 were post-menopausal in low and intermediate categories who had not received adjuvant chemotherapy. Conclusions: This study shows a high correlation between the predicted Magee score and Oncotype recurrence score. This reflects a possible use in the prediction scoring system as another resource for prognostication and guidance in treatment especially in the underinsured population.

NOHA: A sensitive, low-cost, and accessible blood-based biomarker to determine breast cancer estrogen receptor status in low-resource settings.

580 Background: Significant challenges to breast cancer control in low- and middle-income countries (LMICs) include late-stage disease presentation because of few/no early detection programs, inadequately staffed and equipped pathology laboratories, and constrained treatment options. Estrogen receptor (ER) expression is critical to determining candidacy for cost efficient and accessible hormonal agents in LMICs; however, access to standard immunohistochemistry (IHC)-based ER analysis is grossly limited/nonexistent due to cost and technical requirements. We have identified Nw-hydroxy-L-Arginine (NOHA) as a low cost and accessible blood-based biomarker to distinguish estrogen-receptor negative (ER–) from estrogen-receptor positive (ER+) breast cancer, differentiate ER– high grade versus low grade tumors, and correlate ER– molecular phenotype with ethnic variation. Our studies with US patients suggest the NOHA threshold of <4nM as a reliable indicator of ER– versus ER+ disease (Table 1). Here we examine the clinical utility of NOHA as an alternative to IHC in distinguishing ER– from ER+ breast tumors in Tanzanian patients. Methods: Following informed consent, 70 newly diagnosed breast cancer patients were recruited at Kilimanjaro Christian Medical Center (KCMC; Moshi, Tanzania). Prior to any treatment, a needle prick amount of blood was collected from each patient on a Noviplex plasma card (Shimadsu, U.S.) and stored at -80˚C. Plasma cards and unstained tumor pathology slides were shipped at 2-3 months intervals to US labs for NOHA and immunohistochemistry (IHC) ER testing. Statistical difference was set at p<0.01, with NOHA and IHC assay operators blinded to patient clinical status. Results: Our early data show correlation between NOHA levels and ER IHC results, providing a means to distinguish ER– from ER+ breast cancer in the low-resource setting. Plasma cards stored at -80˚C for up to 3 months retained NOHA stability in assays involving a proprietary antibody-based ELISA, and by LC-MS. Conclusions: This study suggests the clinical utility of NOHA as a cost-effective, accessible replacement for standard IHC testing in determining ER status among breast cancer patients in LMICs, promising to extend access to cost efficient and available hormonal agents and improving outcomes and quality of life. The present study provides foundational knowledge for broader studies of NOHA utility in global breast cancer control, as well as in ongoing development of NOHA rapid-testing technologies. [Table: see text]

Tumor copy number alterations were associated with primary endocrine resistance in ER+ breast cancer.

e12567 Background: Several studies has showed alterations in genes were associated with endocrine resistance in breast cancer. Nevertheless, genomic landscape in primary endocrine-resistant breast cancer has not been thoroughly reported. Whether the genomic landscape of primary endocrine-resistant breast cancer is different from that of secondary endocrine-resistant breast cancer is unknown. Methods: We analyzed the genomic landscape of primary tumor of consecutive patients with estrogen-receptor positive (ER+) breast cancer in real-world clinical practice in our center between September, 2019 to December, 2020. According to the duration of endocrine treatment, we classified them into primary endocrine resistance group and secondary endocrine resistance group. Results: A total of 41 tumor samples of which 25 cases in primary endocrine resistance and 16 cases in secondary endocrine resistance were included for analysis. TP53 (65.9%, 27/41), PIK3CA (46.3%, 19/41), MYC (36.6%, 15/41), MCL1 (31.7%, 13/41), CCND1 (31.7%, 13/41) were the most frequently altered genes in all 41 BCs. Copy number amplification of MYC was common in primary endocrine resistant group (48.0%, 12/25), while lower in secondary endocrine resistant group (18.8%, 3/16, P = NS). Copy number alteration (CNA) of CCND1, MCL1, FGF19, ZNF217, ZNF703 and FGFR1 were commonly observed in primary endocrine resistance group. Percentage of CNA in all mutation types was significant increased in primary endocrine resistance group than in secondary resistance group (33.0% vs. 21.0%, P = 0.03). Then we further analyzed and found CNA burden, which was defined as number rather the percentage of the CNA event, was associated with the prognosis in ER+ breast cancer. When defining 6 as the cut-off value of CNA-High, DFS, PFS and OS were practically poorer in patients with CNA-High than those with CNA-Low. Conclusions: Copy number alterations were more commonly seen in primary endocrine-resistant breast cancer and associated with prognosis in ER+ breast cancer. This founding urged to further investigate the prognostic value of CNA burden in predicting the effectiveness of endocrine therapy.

Phase Ib/II study of BCL-2 inhibitor lisaftoclax (APG-2575) safety and tolerability when administered alone or combined with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor in patients with estrogen receptor-positive (ER+) breast cancer or advanced solid tumors.

TPS1122 Background: Various cancers evade apoptosis by overexpressing BCL-2 proteins. Investigational agent lisaftoclax (APG-2575) is a novel, potent, selective BCL-2 inhibitor, while palbociclib inhibits cyclin-dependent kinases (CDK) 4 and 6. In preclinical studies, palbociclib decreased proliferation of ER+ breast cancer cell lines by arresting them in the G1 cycle phase. Preclinical data demonstrate favorable, complementary effects of palbociclib when combined with a BCL-2 inhibitor and support this combination in patients with ER+/ human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer. Methods: This global multicenter open-label dose escalation and dose expansion study is assessing the safety of lisaftoclax monotherapy in patients with histologically or cytologically confirmed advanced solid tumors that have progressed on standard therapy or when combined with CDK4/6 inhibitor palbociclib in physiologically postmenopausal women with ER+/ HER2- metastatic breast cancer that has progressed or relapsed after treatment with a CDK4/6 inhibitor. This trial consists of 2 parts: a phase Ib dose escalation phase using a standard 3+3 design to determine the maximum tolerated dose (MTD) of lisaftoclax as a single agent in patients with solid tumors, as well as both the MTD and recommended phase 2 (RP2D) dose of lisaftoclax when combined with palbociclib in women with ER+/ HER2- metastatic breast cancer. Phase II of this study is a signal-seeking expansion of lisaftoclax at RP2D when combined with palbociclib in women with ER+/HER2- metastatic breast cancer. This phase is being conducted using Simon’s Minimax two-stage design. The primary objective for phase II is to determine clinical benefit response, and secondary efficacy endpoints include overall response rate, duration of response, time to response, and progression-free survival. Lisaftoclax is being administered orally once daily in a 28-day cycle at the assigned dose. Clinical trial information: NCT04946864.

Adherence to EndoPredict test scores for extended endocrine therapy management in the prospective EndoPredict Extended Endocrine Trial (EXET).

537 Background: Current guidelines advise that individuals with estrogen receptor-positive (ER+) breast cancer (BC) undergo 5 years of endocrine therapy with consideration of up to 10 years of extended endocrine therapy (EET). The relative benefit of EET beyond 5 years is minimal for many patients, yet a significant group of patients at higher risk of late recurrence may benefit. EndoPredict is a gene expression assay to stratify those with high versus low BC recurrence risk and is validated to predict early and late distant metastasis up to 15 years using an EPclin score. In the prospective EXET study we evaluated adherence to EndoPredict test results when making decisions regarding EET. Methods: Patients with prior ER+, human epidermal growth factor 2 negative (HER2-) BC diagnosis and 4-6.5 years of endocrine therapy underwent EndoPredict testing. To evaluate the impact on decision making for EET, chart review was performed at or after test results were clinically disclosed. Univariable analysis was used for EPclin risk classification (high, low), multivariable analysis was used to evaluate the EPclin score with clinical variables and Firth logistic regression was used to assess the association of a given variable with the therapy decision. Results: The cohort consisted of 411 patients with an average age of 62 years (range 27, 90), 107 received adjuvant chemotherapy (26%), and 71 were lymph node positive (17.3%). The rate of EET was significantly different for those with low-risk scores (EPclin Score ≤ 3.3) compared to high-risk scores (EPclin Score > 3.3; OR 8.5×10-4 [95% CI 6.8×10-6, 6.0×10-3], p-value 5.7×10-68). All patients with high-risk scores received EET. Within the low-risk group, those treated with adjuvant chemotherapy and those who were lymph node positive chose to extend endocrine therapy at higher rates. The EPclin score provided significant information in predicting EET status, even when accounting for clinical variables such as node status, tumor grade, age at diagnosis, adjuvant chemotherapy status and tumor stage (EPclin Score OR 20.2 [10.1, 44.0]; p-value=3.1 × 10-25). Conclusions: Even after adjusting for other clinical factors, EndoPredict is a significant predictor of clinical decision on EET. High adherence to EndoPredict test results in EET decision making was noted in our study. Clinical trial information: NCT04016935. [Table: see text]

High expression of SLC20A1 is less effective for endocrine therapy and predicts late recurrence in ER-positive breast cancer.

Estrogen receptor-positive (ER+) breast cancer intrinsically confers satisfactory clinical outcomes in response to endocrine therapy. However, a significant proportion of patients with ER+ breast cancer do not respond well to this treatment. Therefore, to evaluate the effects of endocrine therapy, there is a need for identification of novel markers that can be used at the time of diagnosis for predicting clinical outcomes, especially for early-stage and late recurrence. Solute carrier family 20 member 1 (SLC20A1) is a sodium/inorganic phosphate symporter that has been proposed to be a viable prognostic marker for the luminal A and luminal B types of ER+ breast cancer. In the present study, we examined the possible association of SLC20A1 expression with tumor staging, endocrine therapy and chemotherapy in the luminal A and luminal B subtypes of breast cancer. In addition, we analyzed the relationship between SLC20A1 expression and late recurrence in patients with luminal A and luminal B breast cancer following endocrine therapy. We showed that patients with higher levels of SLC20A1 expression (SLC20A1high) exhibited poorer clinical outcomes in those with tumor stage I luminal A breast cancer. In addition, this SLC20A1high subgroup of patients exhibited less responses to endocrine therapy, specifically in those with the luminal A and luminal B subtypes of breast cancer. However, patients with SLC20A1high showed good clinical outcomes following chemotherapy. Patients tested to be in the SLC20A1high group at the time of diagnosis also showed a higher incidence of recurrence compared with those with lower expression levels of SLC20A1, at >15 years for luminal A breast cancer and at 10–15 years for luminal B breast cancer. Therefore, we conclude that SLC20A1high can be used as a prognostic biomarker for predicting the efficacy of endocrine therapy and late recurrence for ER+ breast cancer.

Limiting systemic endocrine overtreatment in postmenopausal breast cancer patients with an ultralow classification of the 70-gene signature

PurposeGuidelines recommend endocrine treatment for estrogen receptor-positive (ER+) breast cancers for up to 10 years. Earlier data suggest that the 70-gene signature (MammaPrint) has potential to select patients that have an excellent survival without chemotherapy and limited or no tamoxifen treatment. The aim was to validate the 70-gene signature ultralow-risk classification for endocrine therapy decision making.MethodsIn the IKA trial, postmenopausal patients with non-metastatic breast cancer had been randomized between no or limited adjuvant tamoxifen treatment without receiving chemotherapy. For this secondary analysis, FFPE tumor material was obtained of ER+HER2− patients with 0–3 positive lymph nodes and tested for the 70-gene signature. Distant recurrence-free interval (DRFI) long-term follow-up data were collected. Kaplan–Meier curves were used to estimate DRFI, stratified by lymph node status, for the three predefined 70-gene signature risk groups.ResultsA reliable 70-gene signature could be obtained for 135 patients. Of the node-negative and node-positive patients, respectively, 20% and 13% had an ultralow-risk classification. No DRFI events were observed for node-negative patients with an ultralow-risk score in the first 10 years. The 10-year DRFI was 90% and 66% in the low-risk (but not ultralow) and high-risk classified node-negative patients, respectively.ConclusionThese survival analyses indicate that the postmenopausal node-negative ER+HER2− patients with an ultralow-risk 70-gene signature score have an excellent 10-year DRFI after surgery with a median of 1 year of endocrine treatment. This is in line with published results of the STO-3-randomized clinical trial and supports the concept that it is possible to reduce the duration of endocrine treatment in selected patients.

Conventional and digital Ki67 evaluation and their correlation with molecular prognosis and morphological parameters in luminal breast cancer

Digital counting methods were developed to decrease the high intra- and inter-observer variability of immunohistochemical markers such as Ki67, with most presenting a good correlation coefficient (CC). Since Ki67 is one of the major contributors to Oncotype DX, it is conceivable that Ki67 expression and the recurrence score (RS) obtained by the multigene panel are positively correlated. We decided first to test to what extent conventional and digital Ki67 quantification methods correlate in daily practice and, second, to determine which of these methods correlates better with the prognostic capacity of the Oncotype DX test. Both Ki67 evaluations were performed in 89 core biopsies with a diagnosis of estrogen receptor (ER) positive HER2-negative breast cancer (BC). Cases were, thus, classified twice for surrogate subtype: first by conventional analysis and then by digital evaluation. The Oncotype RS was obtained in 55 cases that were subsequently correlated to Ki67 evaluation by both methods. Conventional and digital Ki67 evaluation showed good concordance and correlation (CC = 0.81 (95% CI 0.73–0.89)). The correlation of Oncotype DX risk groups and surrogate derived subtypes was slightly higher for the digital technique (rs = 0.46, p < 0.01) compared to the conventional method (rs = 0.39, p < 0.01), even though both were statistically significant. In conclusion, we show that digital evaluation could be an alternative to conventional counting, and also has advantages for predicting the risk established by the Oncotype DX test in ER-positive BC. This study also supports the importance of an accurate Ki67 analysis which can influence the decision to submit ER-positive HER2-negative BC to prognostic molecular platforms.

ECM1 is associated with endocrine resistance in ER+ breast cancers

ABSTRACT Extracellular matrix protein 1 (ECM1) is associated with a poor prognosis of breast cancers. However, the role of ECM1 with endocrine resistance in estrogen receptor-positive (ER+) breast cancers has not been elucidated yet. We show that ECM1 promotes endocrine resistance in ER+ breast cancers. ECM1 is overexpressed in luminal breast cancer patients compared to the basal type of breast cancer. Significantly, higher expression of ECM1 is associated with poor response to endocrine therapies in luminal B breast cancer patients. We found that ECM1 is upregulated in CAMA1 and MDA-MB-361 cells grown in long-term estrogen-deprived (LTED) conditions. Moreover, the ablation of ECM1 significantly inhibited the proliferation of CAMA1 LTED and MDA-MB-361 LTED cells. Finally, an interrogation of a dataset containing transcriptome and proteome of breast cancer cell lines revealed that the level of ECM1 mRNA is positively correlated with that of phosphorylated Src. Based on these findings, we strongly suggest that ECM1 significantly contributes to the acquisition of endocrine resistance in ER+ breast cancers by the activation of Src.