Modelling anti-estrogen resistance in estrogen receptor-positive breast cancer: molecular mechanisms and emerging therapeutic strategies

Objective: Endocrine therapy (ET) now is the standard of care (SOC) for ER+ breast cancer treatment. The representative drug of ET is tamoxifen, a selective estrogen receptor modulator (SERM) that inhibits ER+ breast cancer growth by antagonizing ER function. Tamoxifen displays promising efficacy but also brings problems such as increased endometrial cancer risk and acquired resistance. SERD fulvestrant controls ER+ breast cancer by degrading in-cell ER. It shows improved efficacy in advanced or tamoxifen-resistant (TamR) breast cancer patients and is recently approved as first-line treatment for ER+ breast cancer. However, due to its poor PK profile, this drug could only be administrated intramuscularly, which restricts its applicable patient population. Thus, developing an oral SERD is still an unmet medical need. Method & Results: We have discovered an oral SERD, LX-039. LX-039 is a novel indole series compound. It robustly degraded ER in MCF-7 breast cancer cells (IC50 = 1.53 nM) and inhibited MCF-7 cell proliferation (IC50 = 2.56 nM). Besides, LX-039 exhibited good PK profile across species, F for mice, rat and dog were 32.2%, 44.5% and 48.1% respectively. Dose to p.o. AUC showed linear relationship, and very high p.o. AUCs could be obtained at high doses. For rat @500 mpk, p.o. AUC reached to 4.52M nM.h, and dog @300 mpk, p.o. AUC reached to 1.5M nM.h. LX-039 displayed promising in vivo antitumor efficacy. In naive MCF-7 breast cancer model, TGI was 87% @ 20 mpk; in TamR MCF-7 model, TGI was 70% @100 mpk. What makes LX-039 more advantageous is its safety profile. LX-039 showed purer ER antagonism. In rat uterine growth inhibition assay, the inhibition rate reached to 94% while inhibition rate of GDC-0810 and AZD-9496 were 65% and 89%, respectively. It had a clean CYP inhibition profile by various CYP assays, which indicates a low DDI risk. LX-039 selectively antagonizes ER without affecting other nuclear receptors such as AR, GR and PR. It showed a clean inhibition profile for a selected kinase/GPCR panel (IC50 >3 μM) and was negative in the GABA gated ion channel assay (IC50 > 10 μM) and the mini Ames test. In the 14 day rat non-GLP tox study, NOAEL of LX-039 was determined to be 300 mpk. Conclusion: The antitumor efficacy of LX-039 as demonstrated in the preclinical tumor models is highly encouraging and warrants testing the compound in the clinic as an oral SERD for treating ER+ breast cancer. The IND enabling studies of LX-039 are ongoing, and IND filing scheduled in the mid of 2018. Citation Format: Jianyu Lu, Lihong Hu, Ping Wang, Yusong Zhu, Peng Zou, Dongdong Wu, Charles Z. Ding, Shuhui Chen. Novel indole compound LX-039 as an oral selective estrogen receptor degrader (SERD) for treating estrogen receptor positive (ER+) breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1804.

Approximately 75% of breast cancer incidences are Estrogen Receptor positive (ER+). Treatment of ER+ breast cancer with antiestrogen endocrine therapy targets the proliferative mechanisms of ER using selective ER modulators (SERMs), such as tamoxifen, and selective ER downregulators (SERDs), such as fulvestrant. The resistance to endocrine therapy, either innate or acquired, is clinically diagnosed in up to 50% of patients within 5 years of treatment. Antiestrogen therapy of ER+ mammary cancer cells in vitro and in xenografts does not cause complete cell death or regression. Through the use of combination therapy to target the remaining sub-population of cells, complete regression can be predicted. Our lab has developed endocrine tamoxifen-resistant models through prolonged exposure to tamoxifen, estrogen deprivation, and overexpression of PKCα, a pro-survival pathway component, in addition to a fulvestrant-resistant model through prolonged exposure to fulvestrant. Investigation of the resistance mechanisms will provide insight into potential, combinatorial targets for overcoming endocrine resistance in ER+ breast cancer. Use of spheroidal 3D cell culture provides a physiologically relevant model with sufficient throughput for drug discovery. Establishing spheroids and subsequent treatment predicts the efficacy of combination treatment on tumor regression. Targeting pathways associated with increased prevalence of resistance, such as CDK 4/6, can be employed for clinical therapy. Using both novel in-house and clinical SERMs and SERDs, we have used these multiple cell lines to discover combinatorial targets using both mechanistic and unbiased screening approaches. The use of combination endocrine therapy in tamoxifen-resistant models has shown antiproliferative synergy, and is able to enhance tumor regression in xenografts. By optimizing combinatorial endocrine treatment against both fulvestrant- and tamoxifen-resistance, novel therapeutic approaches are being developed for ER+ breast cancer. Citation Format: Lauren M. Gutgesell, Rui Xiong, Jiong Zhao, Debra A. Tonetti, Gregory R. Thatcher. Optimizing combination therapy against antiestrogen-resistance in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3603. doi:10.1158/1538-7445.AM2017-3603

Introduction: Fulvestrant is a selective estrogen receptor down-regulator (SERD). Recent studies have shown that the efficacy of fulvestrant is dose-related. However, at the higher dose (500 mg/month) most cancers develop resistance and progress. We previously reported expression of several markers, including estrogen receptor (ER) and BCL-2, on breast cancer circulating tumor cells (CTC) using CellSearch®. We now report pilot data showing inter-patient heterogeneity of these markers on CTC in patients with known ER positive breast cancer whose disease is progressing on fulvestrant. Methods: We conducted a pilot trial to determine the analytical validity of measuring expression of markers of endocrine sensitivity (ER, BCL-2) or resistance (HER-2, Ki-67) with fluorescent-labeled antibodies using the CellSearch® system. Patients with ER positive metastatic breast cancer (MBC) whose disease was progressing on any type of therapy were eligible after signed informed consent. This report is limited to the subjects who were progressing on fulvestrant. Whole blood (WB) was characterized for CTC counts and each of the four molecular markers using the CXC CellSearch® kit. Results: Of 50 enrolled patients, seven were progressing on fulvestrant. Two patients had no detectable CTC, while five patients had an average of ≥5 CTC/7.5 mL WB. Results are shown in a table below: CTC-ERCTC-BCL-2Patient #Fulvestrant dose (mg/month)Days since last doseN CTC/7.5 mL of WB% of CTC-ER+N CTC/7.5 mL of WB% of CTC-BCL-2+295002880%110%4550028170%170%2250341010%714%850031812%1735%172507728%367% These exploratory data suggest widely different mechanisms of resistance to fulvestrant in different patients with ER positive MBC. In two of the patients (29, 45) treated with 500 mg/month, both CTC-ER and CTC-BCL-2 expression were absent, suggesting no signaling through the ER pathway. We hypothesize either that fulvestrant was actively down-regulating ER, but the cancers had adopted other growth and survival pathways, or that ER negative, hormone-independent clones had evolved. In the other three cases, ER was clearly present with evidence of signaling, based on BCL-2 expression. Two of these patients (2, 17) were on the lower dose of fulvestrant, now considered to be less effective. However, the third (8) was on the higher dose and yet still had evidence of ER signaling. This observation suggests that some patients may benefit from even higher doses of SERD therapy. Conclusions: These pilot results suggest heterogeneous biological or pharmacological mechanisms of resistance to SERD therapy. These data suggest that CTC-ER and CTC-BCL-2 expression could serve as pharmacodynamic monitoring tools for dose escalation of fulvestrant or other SERDs. Further molecular analysis might provide biological bases for resistance to fulvestrant. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-4.

Taking a Second Look at Neoadjuvant Endocrine Therapy for the Treatment of Early Stage Estrogen Receptor Positive Breast Cancer During the COVID-19 Outbreak.

Background: Despite treatment, many estrogen receptor positive (ER+) breast cancer (BC) patients, relapse with de novo or acquired endocrine resistant disease. Using a kinome siRNA library screen, we identified MPS1, which is required for recruitment of the spindle assembly checkpoint complex, as strongly associated with resistance to both endocrine therapy and CDK4/6 inhibitor palbociclib, a contemporary first-line combination for advanced ER+ BC. Until now, the target population for MPS1 inhibitors has been triple negative BC. Here we show for the first time, potential efficacy of MPS1 inhibitors in endocrine resistant BC models. Methods: A panel of ER+ BC cell lines adapted to estrogen independent growth (LTED) or after additional resistance to palbociclib (palboR), was subjected to a siRNA kinome screen targeting 709 genes. Kinases causative of resistance were identified using Z-scores. Cell viability upon MPS1 inhibition (MPS1i) with BOS172722 was assessed 2D and 3D, and the class effect confirmed with other compounds targeting MPS1. Impact of MPS1i on ER-mediated transcription was assessed using luciferase reporter assays. Effect of MPS1i on chromosomal alignment and time spent in mitosis was established by time lapse and confocal microscopy. Cell cycle and DNA content were analysed by FACS. Apoptosis was assessed using PARP cleavage. Efficacy of BOS172722 in a xenograft model of LTED was evaluated. Results: Kinome knockdown identified MPS1 as the top common hit in LTED and palboR cell lines. Increased MPS1 was evident in MCF7-LTED at the transcript and protein level. Notably, BOS172722 significantly reduced viability of the majority of LTED and palboR cell lines tested (IC50 between 25-100nM), an effect shown to be class specific using a second MPS1i. Upon MPS1i, cells demonstrated shorter time in mitosis, aberration of cell cycle, increased mitotic errors, culminating in apoptosis. Of note, as little as 48hr exposure to BOS172722 reduced colony formation over 3 weeks suggesting early accumulation of errors is sufficient to provide a long term anti-proliferative effect. To evaluate the clinical relevance of MPS1 in ER+ BC treated with endocrine therapy, we interrogated publicly available datasets from patients treated with neoajuvant AI therapy. In the anastrozole cohort, on-treatment MPS1 expression was significantly (p<0.0001) associated with poor response to anastrozole based on a 2-week residual Ki67 score <10%. In the letrozole cohort, increased on-treatment expression of MPS1 (p=0.0118) was associated with poor response based on tumor shrinkage <50%. Finally, in a xenograft model of AI relapse, BOS172722 caused significant tumor shrinkage, compared to vehicle. Conclusion: For the first time, we show that MPS1 inhibitors are capable of inducing mitotic aberrations and apoptosis in ER+ BC resistant to endocrine therapy and palbociclib providing a new therapeutic strategy. Citation Format: Joanna Nikitorowicz-Buniak, Sunil Pancholi, Nikiana Simigdala, Ricardo Ribas, Spiros Linardopoulos, Mitch Dowsett, Stephen Johnston, Lesley-Ann Martin. MPS1 as a novel target in endocrine- and palbociclib-resistant estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4950.

<div>Abstract<p><b>Purpose:</b> Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line therapy for the management of estrogen receptor (ESR1)–positive breast cancer. However, ESR1 mutations or other ligand-independent ESR1 activation mechanisms limit the duration of response. The clinical efficacy of fulvestrant, a selective estrogen receptor downregulator (SERD) that competitively inhibits agonist binding to ESR1 and triggers receptor downregulation, has confirmed that ESR1 frequently remains engaged in endocrine therapy–resistant cancers. We evaluated the activity of a new class of selective estrogen receptor modulators (SERM)/SERD hybrids (SSH) that downregulate ESR1 in relevant models of endocrine-resistant breast cancer. Building on the observation that concurrent inhibition of ESR1 and the cyclin-dependent kinases 4 and 6 (CDK4/6) significantly increased progression-free survival in advanced patients, we explored the activity of different SERD– or SSH–CDK4/6 inhibitor combinations in models of endocrine therapy–resistant ESR1<sup>+</sup> breast cancer.</p><p><b>Experimental Design:</b> SERDs, SSHs, and the CDK4/6 inhibitor palbociclib were evaluated as single agents or in combination in established cellular and animal models of endocrine therapy–resistant ESR1<sup>+</sup> breast cancer.</p><p><b>Results:</b> The combination of palbociclib with a SERD or an SSH was shown to effectively inhibit the growth of MCF7 cell or ESR1-mutant patient-derived tumor xenografts. In tamoxifen-resistant MCF7 xenografts, the palbociclib/SERD or SSH combination resulted in an increased duration of response as compared with either drug alone.</p><p><b>Conclusions:</b> A SERD– or SSH–palbociclib combination has therapeutic potential in breast tumors resistant to endocrine therapies or those expressing ESR1 mutations. <i>Clin Cancer Res; 21(22); 5121–30. ©2015 AACR</i>.</p><p><i>See related commentary by DeMichele and Chodosh, p. 4999</i></p></div>

<div>Abstract<p><b>Purpose:</b> Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line therapy for the management of estrogen receptor (ESR1)–positive breast cancer. However, ESR1 mutations or other ligand-independent ESR1 activation mechanisms limit the duration of response. The clinical efficacy of fulvestrant, a selective estrogen receptor downregulator (SERD) that competitively inhibits agonist binding to ESR1 and triggers receptor downregulation, has confirmed that ESR1 frequently remains engaged in endocrine therapy–resistant cancers. We evaluated the activity of a new class of selective estrogen receptor modulators (SERM)/SERD hybrids (SSH) that downregulate ESR1 in relevant models of endocrine-resistant breast cancer. Building on the observation that concurrent inhibition of ESR1 and the cyclin-dependent kinases 4 and 6 (CDK4/6) significantly increased progression-free survival in advanced patients, we explored the activity of different SERD– or SSH–CDK4/6 inhibitor combinations in models of endocrine therapy–resistant ESR1<sup>+</sup> breast cancer.</p><p><b>Experimental Design:</b> SERDs, SSHs, and the CDK4/6 inhibitor palbociclib were evaluated as single agents or in combination in established cellular and animal models of endocrine therapy–resistant ESR1<sup>+</sup> breast cancer.</p><p><b>Results:</b> The combination of palbociclib with a SERD or an SSH was shown to effectively inhibit the growth of MCF7 cell or ESR1-mutant patient-derived tumor xenografts. In tamoxifen-resistant MCF7 xenografts, the palbociclib/SERD or SSH combination resulted in an increased duration of response as compared with either drug alone.</p><p><b>Conclusions:</b> A SERD– or SSH–palbociclib combination has therapeutic potential in breast tumors resistant to endocrine therapies or those expressing ESR1 mutations. <i>Clin Cancer Res; 21(22); 5121–30. ©2015 AACR</i>.</p><p><i>See related commentary by DeMichele and Chodosh, p. 4999</i></p></div>

Endocrine therapy is the mainstay of treatment for metastatic estrogen receptor (ER) positive breast cancer. The selective ER degrader (SERD) fulvestrant is increasingly relevant due to its high therapeutic efficacy and often used in combination with other targeted treatments in the metastatic setting. However, most metastatic patients inevitably progress during therapy and develop resistance, constituting a major clinical problem. Our aim is to identify mechanisms conferring resistance to fulvestrant and evaluate potential therapeutic interventions. We have generated several fulvestrant-resistant (FR) in vitro models through chronic exposure of established breast cancer cell lines (i.e. MCF7, Cama1, T47D and HCC1428) to increasing concentrations of fulvestrant. The models have been characterized by assessing gene expression alterations using microarrays, protein expression by western blots, cell cycle profiling by flow cytometry and drug sensitivity by proliferation assays. These established FR models demonstrate a marked decrease in sensitivity to fulvestrant and ability to progress through the cell cycle in the presence of fulvestrant despite downregulation of ERα and decrease in ER signaling. Gene Ontology analysis revealed involvement of mitotic cell cycle, cell proliferation and DNA replication and repair genes in development of resistance. Investigation of cell cycle regulation showed cell line specific alterations in the FR models. Striking molecular alterations included upregulation of cyclin D3, as well as cyclin E2 and its binding partner CDK2 in Cama1 FR cells, and upregulation of CDK6 in MCF7 FR cells. These changes indicate importance of cyclin D-CDK4/6 and cyclin E-CDK2 nodes in supporting cell cycle progression of cells that have developed resistance to fulvestrant. Subsequently, we evaluated response of the FR cells to pharmacological inhibition of CDK4/6 by palbociclib and CDK2 by dinaciclib. MCF7 parental and FR cells were equally responsive to palbociclib treatment, while Cama1 FR cells displayed less sensitivity to palbociclib than their parental counterpart. In contrast, dinaciclib induced comparable growth inhibition in Cama1 parental and FR cells, while demonstrating lower efficacy in MCF7 FR model comparing to parental cells. Over all, our findings show heterogeneous mechanisms of endocrine resistance and demonstrate that our in vitro models can represent diverse mechanisms of fulvestrant resistance. This heterogeneity likely occurs also in breast cancer patients whose tumors develop resistance to endocrine therapy. The FR models display ER-independent growth, for which the underlying mechanisms include alterations in cell cycle regulation. Importantly, some of the changes could predispose FR cells to respond better to specific therapeutic interventions, such as treatment with CDK4/6 or CDK2 inhibitors. Citation Format: Kamila K. Kaminska, Nina Akrap, Johan Staaf, Åke Borg, Ana Bosch, Gabriella Honeth. Fulvestrant resistance in estrogen receptor positive breast cancer models is driven by heterogeneous ER independent transcriptional programs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5907.

Background: Modulation of estrogen activity and/or synthesis is the mainstay therapeutic strategy in the treatment of ER+ BC. However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand binding domain that drive ER-dependent transcription and proliferation in the absence of estrogen. ER antagonists that are efficacious against ligand-dependent and ligand-independent, constitutively active ESR1 mutant tumors may be of substantial therapeutic benefit. GDC-0927 (formerly known as SRN-927) is a novel, potent, non-steroidal, orally bioavailable, selective ER antagonist/ER degrader (SERD) that induces tumor regression in ER+ BC patient-derived xenograft models. Methods: A phase I dose escalation study with 3+3 design was conductedin postmenopausal women with ER+ (HER2-) metastatic BC (progressing ≥ 6 months on endocrine therapy and with ≤ 2 prior chemotherapies in the advanced or metastatic setting) to determine the safety, pharmacokinetics (PK) and the recommended Phase 2 dose (RP2D) of GDC-0927. Pharmacodynamic (PD) activity was assessed with [18F]-fluoroestradiol (FES)-PET scans. Plasma PK samples (after single dose and at steady state), CT scans, and when feasible, pre and on-study tumor biopsies were obtained Results: From March 16, 2015 to March 17, 2017 patients (pts) with a median age of 53 years (range 44-69) and a median number of prior therapies for MBC 4 (range 1-7) were enrolled at 3 total daily dose levels (600, 1000, 1400 mg) once daily (QD) given orally with fasting (n = 12). Increases in GDC-0927 exposure were approximately dose proportional. Treatment related adverse events (AEs) were all grade 1 or 2. The most common treatment-related AEs were nausea (54%, n = 7), diarrhea (46%, n = 6), elevated aspartate aminotransferase (39%, n = 5) and anemia, constipation, (each 31%, n = 4). Treatment interruption was required for 2 pts due to nausea and vomiting. Of those pts with FES-PET avid disease at baseline (9 of 12), all post-therapy scans showed complete or near complete (> 90%) suppression of FES uptake to background levels, including pts with ESR1 mutations. Evidence of reduced ER levels and Ki67 staining was observed in on-treatment biopsies. Five of 12 pts (1 at 600 mg and 4 at 1400 mg) were on study ≥ 24 weeks (CBR = 41.6 %) with the best overall response of stable disease with 1 patient (ESR1 mt+ D538G) on study for over 490 days. There were no dose limiting toxicities and no SAEs related to study drug. R2PD was 1400 mg and was selected for single arm dose-expansion which is now complete with last patient enrolled on March 17, 2017. Updated results from dose-escalation and dose-expansion will be presented at the meeting (N = 43). Conclusions: GDC-0927 appears well-tolerated to date with PK exposure supporting QD dosing, evidence of robust PD target engagement, and encouraging anti-tumor activity in heavily pretreated pts with advanced or metastatic ER+ BC, including pts with ESR1 mutations. Citation Format: Dickler MN, Villanueva R, Perez Fidalgo JA, Mayer IA, Boni V, Winer EP, Hamilton EP, Bellet M, Urruticoechea A, Gonzalez-Martin A, Cortes J, Martin M, Giltnane J, Gates M, Cheeti S, Fredrickson J, Wang X, Friedman LS, Spoerke JM, Metcalfe C, Liu L, Li R, Morley R, McCurry U, Chan IT, Mueller L, Milan S, Lauchle J, Humke EW, Bardia A. A first-in-human phase I study to evaluate the oral selective estrogen receptor degrader (SERD), GDC-0927, in postmenopausal women with estrogen receptor positive (ER+) HER2-negative metastatic breast cancer (BC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD5-10.

Background:Based on randomized controlled trials demonstrating no survival benefit of axillary dissection in elderly breast cancer patients, the SSO/Choosing Wisely campaign recommended against the routine use of sentinel lymph node biopsy (SLNB) in clinically node negative patients aged ≥70 with estrogen receptor (ER) positive breast cancer in 2016. SLNB is still performed in >80% of such patients and we have previously shown that at our institution, SLNB positivity influences adjuvant therapy decisions in this population. In this study, we sought to validate the association of SLNB positivity and adjuvant treatment in a larger population-based cohort, and to evaluate the impact of this finding on oncologic outcomes. Methods:The Breast Cancer Outcome Unit (BCOU) prospectively collects demographic, pathologic, treatment and outcomes data on all patients referred to BC Cancer with breast cancer in British Columbia, Canada. Female patients aged ≥70 with newly diagnosed estrogen receptor-positive invasive breast cancer who underwent SLNB from 2010-2016 were included. Patients with HER2-positive disease or those treated with neoadjuvant therapy were excluded. Multivariable analysis was used to assess the effect of SLNB positivity on adjuvant treatment. Overall survival (OS) and breast cancer specific survival (BCSS) were assessed using Kaplan-Meier analysis and Cox regression was used to assess contribution of SLNB positivity and adjuvant treatment. A nomogram was created to model the effect of nodal positivity and adjuvant treatment on BCSS. Results:We identified 2580 patients who met study criteria with a median age of 75 and a median tumor size of 15 mm. SLNB was positive in 23%. Sixty-seven percent of patients had breast conserving surgery (BCS) and 62% of patients had RT (BCS 79%, mastectomy 25%). As systemic therapy 5% of patients had chemotherapy (CT) and 78% of patients had hormone therapy (HT). Use of adjuvant therapies was associated with SLNB positivity: Systemic therapy (HR = 2.4, 95% CI: 1.84-3.14, p <0.0001), RT (HR = 4.94, 95% CI: 3.91-6.25, p <0.0001) and nodal RT (HR = 61.4, 95% CI: 26.6-141.7, p <0.0001). The 5-year OS was 86% and BCSS was 96% with a median follow-up of 4.33 years (95% CI 4.21-4.47 years). There was improved BCSS with receipt of HT (HR 0.51 95% CI 0.301-0.875, p=0.0142) and worse BCSS with grade 3 vs grade 1 disease (HR 4.09, 95% CI 2.06-8.10, p<0.0001). Age, tumor size, status of SLNB and use of RT were not significant prognostic variables. Patients with a positive SLNB who did not receive any adjuvant therapy had lower BCSS (HR 3.22 95% CI 1.235-8.418, p=0.0168) than those with a negative SLNB. However, amongst those who received any combination of CT, HT and RT, there was no significant difference in BCSS regardless of nodal status. A nomogram was developed incorporating tumor size, grade, SLNB status and adjuvant treatment. Using the nomogram, patients aged 75-79 with T1, grade 1-2 tumors, with or without positive SLNB and treated with or without adjuvant therapy had 5-year BCSS ≥95%. The nomogram also indicated that 5-year BCSS was similar for patients with positive and negative SLNB for all combinations of tumor features when patients received HT. Conclusions:In this modern, population-based cohort of patients over 70 with ER-positive breast cancer, 5-year BCSS was excellent at 96%. Although the use of adjuvant treatment was associated with a positive SLNB, BCSS was not changed based on nodal status when patients received HT. Our results support the Choosing Wisely recommendations; SLNB can be safely omitted in elderly patients willing to take HT, and we advocate that SLNB can be omitted in low-risk patients aged ≥75 even in the absence of planned HT. Citation Format: Elaine McKevitt, Rona Cheifetz, Kimberly DeVries, Alison Laws, Rebecca Warburton, Lovedeep Gondara, Caroline Lohrisch, Alan Nichol. Sentinel node biopsy should not be routine in older patients with ER positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD4-02.

Breast cancer is the most diagnosed type of cancer amongst women in economically developing countries and globally. Most breast cancers express estrogen receptor alpha (ERα) and are categorized as positive (ER+) breast cancer. Endocrine therapies such as, selective estrogen receptor modulators (SERMs), aromatase inhibitors (AIs), and selective estrogen receptor downregulators (SERDs) are used to treat ER+ breast cancer. However, despite their effectiveness, severe side-effects and resistance are associated with these endocrine therapies. Thus, it would be highly beneficial to develop breast cancer drugs that are as effective as current therapies, but less toxic with fewer side effects, and less likely to induce resistance. Extracts of Cyclopia species, an indigenous South African fynbos plant, have been shown to possess phenolic compounds that exhibit phytoestrogenic and chemopreventive activities against breast cancer development and progression. In the current study, three well characterized Cyclopia extracts, SM6Met, cup of tea (CoT) and P104, were examined for their abilities to modulate the levels of the estrogen receptor subtypes, estrogen receptor alpha and estrogen receptor beta (ERβ), which have been recognized as crucial to breast cancer prognosis and treatment. We showed that the Cyclopia subternata Vogel (C. subternata Vogel) extracts, SM6Met and cup of tea, but not the C. genistoides extract, P104, reduced estrogen receptor alpha protein levels while elevating estrogen receptor beta protein levels, thereby reducing the ERα:ERβ ratio in a similar manner as standard of care breast cancer endocrine therapies such as fulvestrant (selective estrogen receptor downregulator) and 4-hydroxytamoxifen (elective estrogen receptor modulator). Estrogen receptor alpha expression enhances the proliferation of breast cancer cells while estrogen receptor beta inhibits the proliferative activities of estrogen receptor alpha. We also showed that in terms of the molecular mechanisms involved all the Cyclopia extracts regulated estrogen receptor alpha and estrogen receptor beta protein levels through both transcriptional and translational, and proteasomal degradation mechanisms. Therefore, from our findings, we proffer that the C. subternata Vogel extracts, SM6Met and cup of tea, but not the C. genistoides extract, P104, selectively modulate estrogen receptor subtypes levels in a manner that generally supports inhibition of breast cancer proliferation, thereby demonstrating attributes that could be explored as potential therapeutic agents for breast cancer.

Three inhibitors of CDK4/6 kinases have been recently approved for use in combination with endocrine therapy. These inhibitors considerably increase the progression-free survival of patients with advanced Estrogen receptor (ER)-positive breast cancer in first-line treatment. As the resistance to CDK4/6 inhibitors is currently a major problem faced in pre-clinical and clinical drug development, the identification of novel treatment combinations and predictive markers is of high importance. BET proteins, specifically BRD2/4, have important roles in cell cycle regulation and proliferation. Thus, BET inhibitors (BETi)have the potential to overcome some pathways of resistance, especially for those mediated by upregulation of specific transcription factors. We have investigated the response to BET inhibition in patient-derived xenographs (PDXs) from patients with advanced ER+ breast cancer ex vivo and in vivo. Specifically, 14 breast cancer PDXs (10 ER+/HER2- and 4 ER+/HER2+) that were resistant to CDK4/6i in vivo were treated with ZEN-3694, a selective BET inhibitor currently in clinical development. Nine of the PDXs were sensitive to BET inhibition in monotherapy (65%). The non-responder PDXs showed an improved response when they were treated with ZEN-3694 in combination with fulvestrant (a selective ER degrader) or with a CDK4/6 inhibitor (ribociclib or abemaciclib). RNA-seq and computational analyses comparing responder versus non-responder PDXs revealed 493 differential expressed genes for responders, and 252 for non-responders. From those, 142 genes were differentially expressed in both conditions (untreated and treated). Furthermore, phosphorylation of BRD4 was observed in the ZEN-3694 non-responder PDX group, a mechanism of resistance previously observed in triple-negative breast cancer cell lines. This phosphorylation was abrogated by adding a CDK4/6 inhibitor. In the PDXs, resistance to single agent CDK4/6 and BET inhibitors might be overcome using a combination of both inhibitors. Finally, the molecular mechanism involved in phosphorylation of BRD4 was characterized in a MCF-7 cell line resistant to CDK4/6 inhibitors. Our results show that CDK2 could be one of the kinases involved in the phosphorylation of BRD4. Conclusion: BET inhibition has an impact in advanced ER+ breast cancer cells, either used alone or in combination with endocrine therapy and CDK4/6 inhibitors. Detection of phosphorylated BRD4 (as a mechanism of resistance to BETi) has high potential for use as a biomarker for selecting patients who could benefit from a combined treatment of BET and CDK4/6 inhibitors. Citation Format: Marc Cosín, Tian Tian, Winona Oliveros, Olesya A. Kharenko, Edward H. van der Horst, Reena G. Patel, Cyrus Calosing, Eric Campeau, Ravi Jahagirdar, Sanjay Lakhotia, Marta Melé, Marta Palafox, Joaquín Arribas, Mafalda Oliveira, Cristina Saura, Violeta Serra, Sandra Peiro. Overcoming resistance to inhibitors of CDK4/6 and BET in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1282.

Purpose In breast cancer, response to endocrine therapy depends on estrogen receptor and progesterone receptor status. However, poor prognosis is conferred on patients with hormone receptor (HR)-positive breast cancer. We aimed to examine weakly positive HR breast cancer by comparing weakly positive HR to strongly positive HR and negative HR breast cancer. Methods We examined the clinical and biological features of 1,496 women with breast cancer, and these patients were categorized according to HR status as weakly positive, strongly positive, and negative HR breast cancer. Results In this study, among 1,496 patients with breast cancer, negative HR breast cancer was found in 374, weakly positive HR breast cancer in 90 and strongly positive HR breast cancer in 1,032 patients. Our multivariate analysis showed that there were differences in T stage, tumor–node–metastasis stage, vascular invasion, histologic grade and type, and Ki-67 index. Patients with weakly positive HR breast cancer had an increased risk of death and recurrence compared with those with strongly positive HR breast cancer and had similar prognosis as patients with negative HR breast cancer. Conclusion Patients with weakly positive HR breast cancer received endocrine therapy because they were regarded as having positive HR breast cancer. However, their prognosis of overall survival and relapse-free survival was similar to that in patients with negative HR breast cancer. Therefore, we need to closely observe and consider active treatment for patients with weakly positive breast cancer. Keywords: Breast neoplasms; Estrogen receptors; Progesterone receptors; Triple-negative breast neoplasms

Background: The majority of breast cancers are defined as estrogen receptor positive (ER+) breast cancer. Despite availability of standard therapies, such as aromatase inhibitors, many women eventually relapse with aggressive disease due to acquisition of endocrine resistance, including ESR1 mutations. To help address some of the challenges associated with current therapies including exposure limitations and intramuscular administration, we have developed RAD1901, a novel, non-steroidal, oral selective estrogen receptor degrader (SERD). Preclinical studies with RAD1901 have demonstrated a dose dependent degradation of ER consistent with a SERD mechanism of action. In multiple in vivo models of breast cancer, including patient-derived xenograft models that are sensitive or resistant to standard endocrine therapies, RAD1901 has anti-tumor efficacy both as a single agent and in combination with palbociclib and everolimus. Importantly, RAD1901 has shown superior efficacy compared to fulvestrant in a number these models including those harboring and ESR1 mutation. Methods: RAD1901-005 is a Phase 1 study currently enrolling ER+ advanced metastatic breast cancer patients (ClinicalTrials.gov identifier: NCT02338349) with a dose escalation cohort based on a standard 3+3 design followed by a safety expansion cohort at a tolerated dose. Key inclusion criteria include postmenopausal women aged 18 years or older, with advanced ER+, HER2-negative breast cancer, who have received ≤ 2 prior chemotherapy regimens in the metastatic setting and > 6 months of prior endocrine therapy. In addition, circulating tumor DNA (ctDNA) was evaluated to determine ESR1 mutation status and to correlate it with clinical response. Results: As of the cut-off date in March, 13 patients were enrolled in the dose escalation part of the study (3+3 design) at doses of 200 mg qd, 400 mg qd and 600 mg qd. RAD1901 exposure was dose dependent and the PK profile was comparable to PK data from a previous study in healthy volunteers. RAD1901 was well tolerated with the most common adverse events being low-grade nausea and dyspepsia. No DLTs were observed. A safety expansion cohort (Part B, n=20) was opened. At the cut-off date 4 of the 13 patients had been on study 4 or more months. Updated outcomes and biomarker data, including ctDNA, will be presented at the meeting. Conclusion: RAD1901, a novel, non-steroidal, oral SERD, is well-tolerated with manageable adverse effects, and is associated with preliminary evidence of clinical activity in patients with advanced ER+ advanced postmenopausal breast cancer, including patients with ESR1 mutant tumors. Citation Format: Kaklamani VG, Kabos P, Elledge R, Harb W, Purandare D, O'Neill A, Garner F, Bardia A. A phase 1 study of RAD1901, a novel, oral selective estrogen receptor degrader, for the treatment of ER-positive advanced breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-08-06.

Breast cancer is subdivided into three types: hormone (estrogen and progesterone) receptor (ER and PR) positive, Her2-neu positive and triple negative breast cancers. In general, surgical and radiation treatments are similar, but drug treatment for different subtypes of breast cancers is different. Endocrine therapy (ET) is specifically used for the treatment of ER and PR positive breast cancers. This review discusses every aspect of endocrine therapy: ovarian suppression agents, selective estrogen receptor modulators (SERMs) and downregulators, and aromatase inhibitors (AIs). The most famous agents for the treatment of HER2 positive breast cancers are trastuzumab and its derivative Kadcyla (ado-trastuzumabemtansine). Other agents for the treatment of this subtype of breast cancers are also discussed. For the treatment of triple-negative breast cancers (TNBC) and other breast cancers, the following agents are discussed: anthracyclines and related regimens, taxanes, combination therapy of platinum with taxanes, combination therapy to counter drug resistance, ixabepilone and other epothilones, angiogenesis inhibitors. The lack of known specific molecular targets has promoted abundant research in order to find possible "vulnerabilities" in TNBC. For the first time, we propose thetranslocator protein (TSPO) 18 kDa as a potential target for TNBC. Furthermore, currently Photodynamic Therapy (PDT) is way under-explored for the treatment of breast cancers. In this review, PDT for the treatment of breast cancers is discussed. We also discuss imaging-guided therapy for breast cancers. Finally, from a perspective point of view, we call on the development of more potent agents for differentiation therapy.