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Background:Aromatase inhibitors (AIs) are used for the adjuvant treatment of early breast cancer. Although generally well-tolerated, they have some adverse effects. Because of AIs are used long term in adjuvant treatment, side effects are also very important. Preclinical and a little clinic data indicate that estrogens exert neurotrophic and neuroprotective actions in the brain. So estrogen appears to play an important role in cognitive function and memory. We know that AIs reduce circulating estrogen to very low levels. For this reason, there has been concern that these agents may have a negative effect on cognitive functions. However, this situation is still unclear. The purpose of our study is to evaluate the relationship between duration of treatment and cognitive functions in patients with breast cancer who use AIs in adjuvant therapy. Methods:Patients diagnosed with breast cancer who use AIs as adjuvant treatment and followed up at the Akdeniz University Medical Oncology Clinic were included. The patients were surveyed for demographic characteristics. Montreal Cognitive Assessment (MoCA) and Standardized Mini-Mental State Examination (SMMT) tests were applied to patients by the same investigator to evaluate their cognitive functions. The total scores of the tests and the orientation, naming, short-time memory, visuospatial functions-executive functions, attention, abstract thinking, language which are the MoCA subscales were evaluated separately. In the Turkish population, the normal range of the MoCA and SMMT tests are 21-30 points and 24-30 points, respectively. Patients were grouped as 0-6, 6-12, 12-24, 24-36, 36 and more months according to the duration of AIs using time. 200 patient’s data were analyzed with SPSS package program. This study was approved by the Akdeniz University Faculty of Medicine Clinical Research Ethnics Committee and was conducted in accordance with declaration of Helsinki. Results:The median follow-up time was 55.5 months. The median duration of AIs treatment was 36.5 months. The mean age of patients was 61.3 years-old. There was no relationship between duration of treatment and MoCA and SMMT scores which indicates cognitive functions (p>0.05). In addition, no statistically relationship was found in the evaluation of MoCA subscales (p>0.05). As expected, the total MoCA and SMMT scores were affected by factors such as age, education level, and employment status. Interestingly, although it was not our main purpose, a causal relationship between the presence of hypertension and cognitive decline was shown in our study (p: 0.004). A statistically strong correlation was found between the MoCA and SMMT scores. So, our data are reliable. The duration of treatment with AIs and MoCA and SMMT scores of the patients were given in Table 1. Conclusions:As a result, despite conflicting results in the literature, we showed with 200 patients that adjuvant treatment with AIs does not affect cognitive functions in post-menopausal hormone receptor-positive breast cancer patients. Table 1: Relationship between the duration of treatment with AIs and MoCA and SMMT scoresDuration of AInMoCA*Mean±SDMoCAMed (min-max)SMMT**Mean±SDSMMTMed (min-max)0-6 m2319,7±5,221 (10-26)25±425 (15-30)6-12 m2420,7±5,421,5 (8-28)24,7±3,624 (16-30)12-24 m1418,2±4,118,5 (13-27)24,3±425 (13-29)24-36 m2920,3±4,920 (11-29)24,1±3,225 (17-30)36 m and more11020,3±4,521 (8-29)25±3,225 (14-30)Total20020.1±4,721 (8-29)24,8±3,425 (13-30)AI: Aromatase inhibitors, m: Mounths, n: Number, MoCA: Montreal Cognitive Assessment, SMMT: Standardized Mini-Mental State Examination, SD: Standart Deviation, Med: Median*p: 0.550 **p:0.533 Citation Format: Rukiye Guler Ilhan, Yusuf Ilhan, Sema Sezgin Goksu, Ali Murat Tatli, Hasan Senol Coskun. The effect of using adjuvant aromatase inhibitors on cognitive functions in postmenopausal women with hormone receptor-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 PS9-71.

INTRODUCTION: Estrogen receptor-positive (ER+) breast cancer accounts for nearly 70% of all cases. Common targeted anti-estrogen therapies such tamoxifen, fulvestrant and aromatase inhibitors have shown success in the clinic, but unfortunately, often lead to resistance. The fatty acid synthase enzyme (FASN) is responsible for endogenously synthesizing long-chain fatty acids, such as palmitate, which can contribute to protein modification, phospholipid biosynthesis for membranes, and lipid raft signaling that favors tumorigenesis. The depletion of intracellular palmitate leads to an alteration of lipid composition within lipid rafts of the plasma membrane as well as lipids within the endoplasmic reticulum membrane. Endoplasmic reticulum stress elicits an inhibition of protein translation through the phosphorylation of eukaryotic initiating factor 2-α (p-eIF2α) and is activated by various stimuli including altered phospholipid composition within the membrane. Our preliminary findings illustrated a degradation of the ERα upon treatment with the FASN inhibitor, TVB-3166, in tamoxifen-resistant breast cancer both in vivo and in vitro. Moreover, previous studies have illustrated FASN inhibition to induce endoplasmic reticulum stress concomitant with a loss of the androgen receptor (AR) in castration-resistant prostate cancer. Additionally, palmitate treatment rescued AR expression that was accompanied by an attenuation in endoplasmic reticulum stress. HYPOTHESIS: We hypothesize FASN inhibition leads to a degradation of ERα in tamoxifen-resistant breast cancer through the induction of endoplasmic reticulum stress. METHODS: Patient tumor explants were incubated for 72h on gelatin sponges in culture medium in the absence or presence of 200nM TVB-3166. Tissue were fixed in 10% formalin and processed into paraffin blocks and stained for ERα and Ki67. To investigate TVB induced endoplasmic reticulum stress, tamoxifen-Resistant (TamR) MCF-7 and MCF-7 cells were treated with TVB-3166 or vehicle control followed by treatment with either palmitate or ER stress inhibitor, 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Expression of ERα, p-eIF2α, eIF2α were measured by western blot. RESULTS: TVB-3166 treatment of primary tumor explants decreased their proliferation (Ki67) compared to untreated controls (14% vs 36%, p Citation Format: Bryan Mcclellan, Aleksandra Gruslova, Christopher Jolly, Linda deGraffenried, Andrew Brenner. Fatty acid synthase inhibition targets ERα in tamoxifen-resistant 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 PS17-26.

Background Despite endocrine therapy is the mainstay of treatment for HR+/HER2- metastatic breast cancer (MBC), patients with high disease burden or those at risk of visceral crisis are still offered first-line chemotherapy (CT). Chemotherapy is generally followed by maintenance hormonal therapy. The MAIN-A study is an investigator-driven, randomized phase III trial designed to compare maintenance everolimus (EVE) combined with aromatase inhibitors (AI) versus AI alone in pts with disease control after first-line CT.Methods Postmenopausal pts achieving disease control (stable disease, partial response or complete response) after first-line CT were randomly assigned to EVE 10 mg po daily plus AI or to AI alone. Primary aim was PFS in the ITT. We present here overall survival (OS) results and the impact of tumor characteristics on PFS.Results 110 pts were randomized to EVE+AI (n=52) or to AI (n= 58). Primary aim results have been already presented (Guarneri V, ESMO Breast 2019), showing a non-significant prolongation of median PFS in the ITT population for EVE+AI (9.9 mos vs 7.2 mos, HR 0.764, 95% CI 0.501-1.164. Patients with visceral metastases tended to experience shorter PFS as compared with patients with patients with bone/soft tissues metastases (median 11.1 mos vs 6.4 mos, p=0.0746). The levels of estrogen receptor expression (>or< 50%) did not impact PFS, overall and by treatment arm. At the time of this writing, a total of 61 death events have been recorded. No difference in OS was observed between the two arms (median 33.9 mos for EVE+AI vs 33.5 mos for AI, HR 0.97, 95% CI 0.59-1.61).Conclusions Maintenance EVE+AI did not significantly impact on the outcome of metastatic breast cancer patients deemed suitable for first line chemotherapy. Citation Format: Valentina Guarneri, Saverio Cinieri, Maria Vittoria Dieci, Carmelo Bengala, Gabriella Mariani, Giancarlo Bisagni, Antonio Frassoldati, Claudio Zamagni, Laura Orlando, Carlo Alberto Giorgi, Gian Luca De Salvo, PierFranco Conte. Maintenance therapy with everolimus plus aromatase inhibitors vs aromatase inhibitors as after first-line chemotherapy in HR+/HER2- metastatic breast cancer: Updated analyses of the phase III randomized MAIN-A trial [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 PS12-22.

Background: Everolimus in combination with exemestane (EVE+EXE) was initially FDA approved to treat metastatic hormone receptor-positive breast cancer (mHRBC) in patients previously treated with nonsteroidal aromatase inhibitors based on the BOLERO-2 trial. However, none of the patients in the BOLERO-2 trial received prior CDK4/6 inhibitors, which have become standard of care for mHRBC. There is limited data to support the use and clinical benefit of EVE+EXE in mHRBC patients previously treated with CDK4/6 inhibitors. Methods: We reviewed patients ≥18 years old with mHRBC treated with EVE+EXE at our institution from January 1, 2012, to April 1, 2020. Patients were excluded if they received EVE+EXE ≤30 days. Data collected included patient and tumor characteristics, prior therapies in the metastatic setting, adverse drug events, and clinical outcomes. The primary objective was to compare progression free survival (PFS) for EVE+EXE between patients with and without prior exposure to CDK4/6 inhibitors. Secondary outcomes included overall survival (OS) and safety. Group comparisons were performed by two-sample t test or Wilcoxon rank sum test for continuous variables and Pearson’s chi-square test or Fisher’s exact test for categorical variables. Log-rank test was used to compare the PFS and OS between groups. Results: One-hundred ninety two patients were included in the study; 79 patients had received prior CDK4/6 inhibitor based therapies, while 113 patients did not. Baseline patient characteristics such as histology, menopause status, de novo metastatic disease, presence of lung or liver metastases, and bone only disease were similar between groups. There was a median of 2 lines of treatment in the metastatic setting prior to receiving EVE+EXE in both groups. Fewer patients received prior chemotherapy in the metastatic setting in those who received prior CDK4/6 inhibitors (32.9% vs 49.6%, p=0.032). In patients who received a prior CDK4/6 inhibitor, median PFS was 3.9 months (95% CI: 3.5 to 4.8) compared to 5.5 months (95% CI: 4.0 to 6.3) in those who did not receive a prior CDK4/6 inhibitor (HR for progression, 1.46; 95% CI: 1.08 to 1.97, p=0.013). Overall survival between groups was not significantly different. A total of 32 (16.7%) patients discontinued EVE+EXE due to intolerance or adverse drug events. Conclusion: Patients who received a prior CDK4/6 inhibitor had a lower median PFS benefit from EVE+EXE compared to those who did not. Citation Format: Hanjie Mo, Catherine E Renna, Halle CF Moore, Jame Abraham, Megan L Kruse, Alberto J Montero, Susan B LeGrand, G Thomas Budd. Efficacy of everolimus and exemestane for the treatment of metastatic hormone receptor-positive breast cancer in patients previously treated with CDK4/6 inhibitors [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 PS12-18.

Background: Estradiol (E2) is known to promote breast cancer. However, several small-scale prospective clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)- resistant cases of hormone receptor-positive postmenopausal breast cancer. Considering that the practicality and safety of E2 treatments are still undefined, the objective of this study is to uncover the mechanisms of E2-induced tumor regression, which will lead to a better design of E2 therapies. Methods: While most of our ER+ breast cancer patient-derived xenograft (PDX) models are positively regulated by E2, an estrogen-suppressive model (named GS3) was established from an AI resistant ER+/PR−/HER2− brain metastatic breast cancer. E2 pellets (1mg each) or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC), RNA sequencing, and reverse phase protein array (RPPA) analyses of PDX specimens were conducted to decipher molecular changes after E2 treatments. Importantly, since the cancer tissue has a very heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist (MPP), not by ERβ antagonist (PHTPP). IHC showed that PR expression appeared, Ki-67 and CEACAM5 (CEA) expressions decreased, and the number of apoptotic cells significantly increased after E2 treatment. RNA sequencing indicated that estrogen-regulated genes, e.g., PGR, EGR3, PDZK1, and GREB1, were up-regulated, while tumor growth was repressed by E2. Single cell RNAseq analysis demonstrated that cells from E2-treated and Placebo-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes (HVGs). Importantly, after one-week, many cells of E2-treated PDXs were arrested within the G1 phase of the cell cycle (G1, 60%; S, 19%; G2M, 21%), whereas, cells of placebo-treated samples advanced to the S and G2M phases (G1, 47%; S, 27%; G2M, 26%). Gene Set Variation Analysis of cells from E2-treated PDXs revealed enrichment of genes associated with reactive oxygen species (ROS) mechanism. E2 increased cells expressing pro-apoptotic genes, such as TP53, BOK,IL24, and PGLYRP2. Gene expressions including mitogen-activated protein kinase (MAPK) signaling pathway, such as GADD45A, MAP3K5 (ASK1), MAPK9 (JNK2), and especially MAPKAPK2 were also increased in E2 treated PDXs. Conclusions: E2/ERα-mediated suppression of GS3 tumor growth was accompanying the inhibition of the cell cycle progression and increased expression of pro-apoptotic genes. It is known that classical MAPK pathway induces cell proliferation, meanwhile, JNK MAPK pathway induces p53 signaling pathway and apoptosis. We hypothesize that E2 induces ROS and promotes apoptosis of GS3 due to JNK MAPK pathway activation. This could be an unexpected outcome of AI resistance. Using this valuable estrogen-down-regulated ER+ breast cancer PDX and informative integrative omics analyses, critical molecular mechanisms on E2-mediated apoptosis are being revealed in our laboratory. The ongoing research using E2-sensitive PDXs will identify key markers to select AI resistant patients who are expected to respond effectively to estrogen-induced apoptosis treatments as well as to evaluate the efficacy of drug combination with estrogen, including CDK 4/6 inhibitors. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest and apoptosis in aromatase inhibitor-resistant breast cancer: Insights from single cells analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-08-01.

Background: Abemaciclib plus nonsteroidal aromatase inhibitors (AI) demonstrated efficacy with a tolerable safety profile as initial treatment for postmenopausal women with HR+, HER2− advanced breast cancer in the MONARCH 3 trial. In the intent-to-treat (ITT) population, the updated progression-free survival (PFS) with 12 months of additional follow-up was 28.2 versus 14.8 months (HR [95% CI]: 0.525 [0.415, 0.665]; p Methods: In MONARCH 3, a randomized, double-blind, phase III trial, 493 postmenopausal women with HR+, HER2− advanced breast cancer and no prior systemic therapy in the advanced setting, received an AI (anastrozole or letrozole) plus abemaciclib or placebo. From the October 31, 2018 cutoff, secondary endpoints including objective response rate ([ORR], complete response [CR] + partial response [PR]), time to response (TTR), and duration of response ([DoR], time from a confirmed CR or PR until disease progression or death) were assessed in the ITT population and exploratory subgroups previously reported as significantly prognostic. Waterfall plots will be used to illustrate the magnitude of change in tumor size for each subgroup (to be presented). Results: In patients with measurable disease, the ORR was 62.5% (95% CI: 56.7, 68.2) in the abemaciclib plus AI arm and 44.7% (95% CI: 36.2, 53.2) in the placebo plus AI arm (p≤.001). Additional follow-up confirmed that all prognostic subgroups received benefit from the addition of abemaciclib to AI, consistent with the ITT population, as evidenced by change in ORR, with the largest effects observed in subgroups of patients with liver metastases, progesterone receptor-negative tumors, high grade tumors, or treatment-free interval of Conclusions: Extended follow-up in the MONARCH 3 trial further confirmed that the addition of abemaciclib to AI is associated with durable tumor responses, including those in patients with clinically poor prognostic characteristics. References: Di Leo A et al. NPJ Breast Cancer. 2018;4:41. Clinical trial information: NCT02246621 This study was funded by Eli Lilly and Company Table: Objective Response RateAbemaciclib + AIPlacebo + AIORR ChangeNn (%)Nn (%)%Measurable disease population269168 (62.5)13259 (44.7)17.8Liver metastasesYes4727 (57.45)306 (20.00)37.45No222141 (63.51)10253 (51.96)11.55Progesterone receptor statusNegative6137 (60.66)298 (27.59)33.07Positive206130 (63.11)10351 (49.51)13.60Treatment-free interval Citation Format: Joyce O’Shaughnessy, Stephen Johnston, Miguel Martin, Jens Huober, Masakazu Toi, Valerie AnneMarie Andre, Holly Rene Martin, Molly Catherine Hardebeck, Angelo Di Leo, Matthew Philip Goetz. Updated subgroup tumor response of abemaciclib plus aromatase inhibitor for hormone receptor positive (HR+), HER2 negative advanced breast cancer (MONARCH 3) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-09.

Background: There are some cases in which estrogen addition therapy with ethinyl estradiol (EE2) is effective in cases in which aromatase inhibotor (AI) acquired resistance has been achieved after prolonged administration. In such case, ESR1 mutation was reported to be frequently seen. In this study, we examined the relation between histopathological factors, or ESR1 mutations detected by multiplex droplet digital PCR from cell free DNA (cfDNA) in patients’ and the effect of EE2 treatment. Patients and methods: From 2011 to 2016, we conducted a prospective observational study of EE2 therapy (0.5 to 3 mg / day PO) in our department (registration No. UMIN000002831). ER/PgR expression by IHC in a part cases, and serum E2 and FSH levels and blood cell counts in all cases were evaluated before/after treatment. Droplet digital PCR (ddPCR) system by the QX200™ Droplet Digital™ PCR System (Bio-Rad laboratories, Hercules, CA, USA) which makes thousands of droplets and each of them contained one or no copy of the sequence of ESR1 gene reacted with a pair of primers and two TaqMan probes. The PCR data were quantified using QuantaSoft™ software (Bio-Rad laboratories) and data are expressed as a percentage of mutant to total (mutant plus wild type) for each sample. Results: In this series, partial response (PR) was seen in 30% (12/40), long stable disease 12.5% (5/40), clinical benefit rate (CBR) 42.5% (17/40). The median EE2 dosing period in cases with CB was 8.4 months. AI was re-administered to 17 cases for which efficacy was obtained by EE2, and 8 cases had CB. After that, EE2 was re-challenge to 4 cases obtained effective cases. After EE2 treatment, serum E2 level increased and FSH decreased. In addition, the neutrophil-lymphocyte ratio increased, but its biological significance was unknown. Point mutations (Y537S, Y537N, D538G) of the ESR1 gene in cell free DNA in plasma were searched in 21 cases before EE2 administration, and High frequent ESR1 mutations were seen in 10 cases (47%) in any mutative site. In the patients with wild type of ESR1 before treatment, there were tended to be more effective for EE2 treatment, but they were not statistically significant. Additionally, no relationship between PIK3CA mutations (E542x, E545x, H1047x, G1049x) and EE2 effect was seen. Conclusion: The action mechanisms of EE2 treatment were reported as estrogen-induced apoptosis, which may not be related to ESR1 nor PIK3CA mutations. Further research related to EE2 treatment with biologic factors, such as gene alterations of the related factors, must be required. Citation Format: Hirotaka Iwase, Takashi Takeshita, Mutsuko Ibusuki, Aiko Sueta, Mai Tomiguchi, Yoshitaka Fujiki, Yutaka Yamamoto. Clinical value of ESR1 mutations from cell free DNA in ethynil estradiol treatment for metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-11-12.

Introduction: Mutations in the ESR1 gene (ESR1m) are important mechanisms of resistance to endocrine therapy (ET) in estrogen receptor-positive (ER+) advanced breast cancer and have been recognized as a prognostic and predictive biomarker as well as a potential therapeutic target. ESR1m prevalence has been described as 9-45% in cohorts of ET-resistant metastatic tumors in a variety of publications. We recently reported a prevalence of 25% in a cohort of ER+ breast cancer patients with visceral metastasis. However, the role of ESR1m as a mechanism of resistance to ET used in early-stage disease is not well studied. Neoadjuvant endocrine therapy (NET) is being increasingly explored, not only to allow less extensive surgery but also as a scientific tool, exploring biomarkers to predict outcomes. The preoperative endocrine prognostic index (PEPI) combining Ki67 score, ER Allred score, tumor size, and nodal status after NET is a surrogate of endocrine sensitivity and can identify a subgroup of patients with primary resistance to ET. In this study, we evaluated the prevalence of ESR1m in tumor samples from patients with ER+ breast cancer that were primarily resistant to neoadjuvant aromatase inhibitor (AI) therapy. Methods: We followed a prospective cohort of postmenopausal patients with ER+ HER2- stages II-III breast cancer treated with neoadjuvant endocrine therapy (NET). Patients were treated with anastrozole for a recommended period of at least three months. Tumor samples from patients with a pattern of primary endocrine-resistant tumors (defined as a PEPI Score higher than 3) were selected and analyzed for the presence of ESR1m. ESR1m were evaluated in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue using real-time quantitative polymerase chain reaction (RT-qPCR). Mutations in codons 380, 537, and 538 of the ESR1 gene were analyzed. Results: 127 patients were included in the cohort, of which 100 (79%) had completed NET and had surgery. Among these patients, the PEPI Score ranged from 0 to 3 in 70% (70/100), and 30% (30/100) had a PEPI Score of 4 or more and were selected. 23 patients were included in the analysis (6 did not consent or were lost to follow-up, and one was HER2-positive). Patients characteristics are summarized in Table 1. The median duration of NET was 22 weeks. All samples of tumor tissue from the surgical specimens after NET were evaluable. Quantification of DNA extraction and reference gene cycle threshold values confirmed that the material was adequate for the analysis. We compared these findings with a study from our group using the same methodology in patients with advanced disease where ESR1 mutations were detected in 25% (n=32) of patients with visceral metastasis of ER+ breast cancer resistant to endocrine therapy. ESR1 mutations were not identified in any of the 23 patients with early-stage ER+ breast cancer resistant to NET (p 0.01, Fisher´s exact test) Discussion: Growing evidence supports the notion that there are different mechanisms of primary and secondary endocrine resistance. Our study suggests that ESR1 mutations do not evolve rapidly and do not represent a common mechanism of primary endocrine resistance in the neoadjuvant setting. Therefore, ESR1m should be considered a mechanism of acquired endocrine resistance in the context of advanced disease. Further research should be conducted to identify factors associated with intrinsic resistance to ET. Citation Format: Tomas Reinert, Susana Ramalho, Vivian CA Vasconcelos, Leonardo R Silva, Ana Elisa R Silva, Camila A Andrade, Maria Beatriz PL Kraft, Guilherme P Coelho, Jovana Mandelli, Monique Binotto, Cesar Cabello, Geisilene RP Silva, Jose Bines, Carlos H Barrios, Matthew J Ellis, Marcia S Graudenz. ESR1 mutations are not a mechanism of primary resistance to aromatase inhibitors in ER-positive breast cancer treated with neoadjuvant endocrine therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-08.

Background: Non-adherence to aromatase inhibitors (AIs) for breast cancer is common and increases risk of recurrence. Few prospective studies have systematically evaluated reasons for adherence failure. S1105 randomized women to receive text message reminders or usual care, and no effect of the intervention was observed on the rates of adherence failure. We analyzed baseline characteristics to evaluate factors associated with non-adherence prospectively over 3-years.Methods: Patients enrolled in SWOG S1105 were required to have been on an AI for ≥30 days at enrollment. Patients were assessed for non-adherence of AIs every 3 months for 36 months, with non-adherence defined as urine AI metabolite assay results satisfying any of the following: median) vs low ( Citation Format: Dawn L Hershman, Joeseph M Unger, Anna Moseley, Grace C Hillyer, Kathryn B. Arnold, Julie R Gralow, N. Lynn Henry, Scott D Ramsey, Alfred I Neugut. Baseline patient reported outcomes predict aromatase inhibitor adherence failure among women in enrolled in a randomized trial of text-messaging [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD10-08.

Aromatase inhibitors (AIs) are currently the standard treatment for postmenopausal women with estrogen-dependent metastatic breast cancer. While effective, patients develop resistance leading to tumor relapse, metastasis, and more aggressive phenotypes. Previous research suggested that AI resistance is due to upregulation of growth factor pathways (i.e., HER2 and EGFR) and increased cellular motility. Our lab previously demonstrated that a novel phytoalexin, glyceollins, inhibits proliferation, survival, and migration of hormone independent letrozole-resistant breast cancer cells (LTLT-Ca). However, many postmenopausal women with AI-resistance tumors remain hormone dependent. Therefore, there is a need to understand distinctions between estrogen receptor (ER+) positive and ER negative (ER-) AI resistant tumors and their response to therapy. We hypothesize that treating ER+ letrozole-resistant breast cancer using a combination approach of glyceollin and lapatinib (a dual EGFR/HER2 inhibitor) will reduce growth factor signaling, inhibit proliferation and motility. To test this hypothesis, T47Darom cells (T47D cells which stably overexpress aromatase) and T47DaromLR cells (T47Darom cells that are letrozole resistant) were treated with lapatinib, glyceollin, or the combination. To evaluate viability, resazurin assays were performed and results demonstrated that glyceollin and/or lapatinib had no effect on proliferation in the T47Darom cells. However, glyceollin alone and glyceollin + lapatinib inhibited proliferation of T47DaromLR cells by 46% and 59%, respectively. Cell survival was assessed by colony formation assays and glyceollin caused a 33% reduction in cell survival and glyceollin + lapatinib caused a 60% reduction in the colony formation of T47Darom cells. Interestingly, in T47DaromLR cells glyceollin and the combination significantly inhibited cell survival by 94% alone and 96%. To further evaluate changes that occur as AI sensitive cells transition to AI resistance, a quantitative proteomic analysis of the whole cell lysates of T47DaromLR (AI-resistant) versus T47Darom cells (AI-sensitive) was performed to identify significant protein expression changes. Results demonstrated a 3.195-fold-increase (p Citation Format: Rashidra R Walker, Jankiben Patel, A. Michael Davidson, Karen Gallegos, Syreeta L Tilghman. A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-15.

Background. Preclinical and observational evidence supports cancer prevention activity of non-steroidal anti-inflammatory drugs (NSAIDs) in the breast via suppression of prostaglandin E2 (PGE2) synthesis by cyclooxygenase-2 (COX2). Evidence includes linking of PGE2 to aromatase activity and estrogen synthesis in breast adipose tissue, as well as effects on collagen and breast density (BD). Methods. In an open-label trial, we evaluated the effect of the non-selective NSAID sulindac at 150 mg bid for 12 months on BD in 52 postmenopausal women taking adjuvant aromatase inhibitors (AIs) for breast cancer. BD was measured using a fat-water decomposition MRI based BD measure (MRD) previously shown to be more quantitative than mammographic density. A non-randomized observation cohort of 46 postmenopausal women on AI without NSAID use was conducted in parallel to assess the effect of AI on MRD over 12 months. Eligible participants were recruited at two study sites and included women with an intact, unaffected contralateral breast and BI-RADS ≥ 2. Each subject’s MRI_BD measures at baseline and at 6 and 12 months were included in linear mixed models for longitudinal data. Log-transformation was applied to the outcome of BD. Covariates included log-transformed baseline BD, time on AI, and baseline body mass index (BMI) and change in BMI. Breast tissue collagen fiber alignment for 30 women with paired breast biopsies, before and after 6 months on sulindac, was examined using Second-Harmonic Generation (SHG) microscopy and analysis of the distribution (histogram) of ‘straight’ fibers in three randomly selected areas of breast tissue. Straightness of individual fibers was calculated as the linear length of a fiber divided by the distance along the fiber. Results. Participants on sulindac intervention had a significant change in BD relative to baseline BD at 6 (p=0.05) and 12 months (p Citation Format: Patricia Thompson, Chuan Huang, Betsy Wertheim, Christina Preece, Jie Yang, Jessica Martinez, Denise Roe, Pavani Chalasani, Alison Stopeck. Sulindac reduces breast density and alters collagen alignment in patients on aromatase inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD3-09.

Introduction: Breast cancer is the most common cancer in women. In postmenopausal women with early stage estrogen receptor positive breast cancer, aromatase inhibitors (AI) are a common treatment option. AI's are reported to lead to a high early discontinuation rate in younger post-menopausal women due to poor tolerance. The most common side effects reported to lead to early discontinuation are arthralgia, hot flashes, fatigue, and night sweats. The reported tolerance to AI therapy in women age 75 or older is not well documented. Our study looks at women, ages 75 and older, diagnosed with early stage breast cancer who were placed on adjuvant AI therapy and focuses on tolerability, incidence of common side effects, rate of treatment changes, and on discontinuation rates. Objective: This study evaluates the tolerability, treatment side effects and the discontinuation rate of AI in women over the age of 75 with early stage breast cancer. Methods: Our study is a retrospective chart review of 58 patients' ages 75 to 95 with early stage breast cancer treated with adjuvant AI. Charts of patients were reviewed and duration of treatment, patient reported side effects, treatment changes, and discontinuation rate were recorded. Results: Data analysis showed that 36/55 (65.5%) of patients did not report significant side effects to AI. 6/55 (10.9%) patients required therapy changes due to side effects. 5/6 required one treatment change and 1/6 required multiple treatment changes. In 5/6 therapy was changed to another AI. Only 2/55 (3.6%) of patients discontinued therapy. In both patients who discontinued AI, therapy was discontinued due to medical complications unrelated to AI therapy. Average time to discontinuation was 11 months. The most common reported side effects were arthralgia 9/55 (16.4%), fatigue 3/55 (5.5%), hot flashes 4/55 (7.3%), rash 3/55 (5.5%) and hair thinning 3/55 (5.5%). The most common reported side effect which led to treatment change was arthralgia 4/6 (66.7%). With a median follow up time of 24 months, breast cancer specific mortality was 1/55 (1.8%). Reported Side Effects on AIn=55 patientsPatients who noted symptomsPatients who changed therapy due to symptomsFatigue30Arthralgia94Hot flashes40Rash31Vaginal dryness10Hair thinning31 Conclusion: Our study evaluated the tolerance of AI in older women diagnosed with early stage breast cancer. 36/55 (65.5%) of elderly patients reported no significant side effects suggesting that AI's are well tolerated in this population and the known side effects are significantly less common than previously reported in a younger cohort in whom arthralgia as well as vasomotor symptoms affect as many as 30% of women. This improved tolerance led to a significantly lower early discontinuation rate than previously reported in the younger cohort: discontinuation rate of 3.6% by 24 months in the 75 or older population versus 20% by 24 months in the younger cohort of post-menopausal women treated with AI. Reference: 1 Wagner, L.I., Zhao, F., Goss, P.E. et al. Breast Cancer Res Treat (2018) 169: 537. https://doi-org.proxy.library.stonybrook.edu/10.1007/s10549-018-4713-2 Citation Format: Chaudhry B, Baer L, Kudelka A, Cohen J, Stopeck AT. Aromatase inhibitors are significantly better tolerated by early stage breast cancer patients 75 or older and with significantly lower early discontinuation rate [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-11-22.

Background: There is a debate concerning risk of cardiovascular disease (CVD) from adjuvant therapy with aromatase inhibitors (AI). Randomized trials have indicated higher CV risk and one observational study higher risk of myocardial infarction, whereas other studies have pointed out no elevated risk. The first morphological sign of developing atherosclerosis is a thickening of the arterial intima layer, whereas in the longer perspective the media layer gets thinner - a process not captured when using conventional Carotid intima-media-thickness (CIMT). We therefore used high resolution ultrasound (HRU), to separately estimated the intima and media layers and calculate the I/M thickness ratio. A thicker intima and higher I/M thickness ratio are signs of adverse vascular effects. This principle of estimating vascular aging can strongly distinguish 70-yr old subjects with and w/o prevalent CVD, yielding C-values up to 0.90 in ROC-analysis. Patients and Methods: We included 150 postmenopausal breast cancer women of whom 50 were treated with AI during at least 3 years, 50 with at least 3 years of tamoxifen and 50 were without endocrine therapy. Patients were investigated with non-invasive HRU (22MHz) of the common carotid artery (CCA). Non-parametric statistical methods were used, including Bootstrap quantile regression to obtain 95% confidence intervals (CI) and adjustment for potential confounders: age, smoking, use of statins, HbA1c and previous HRT. Results: Of 150 included women, 132 remained having a technically acceptable ultra sound scans and no AI therapy before tamoxifen therapy; AI (#48); tamoxifen (#40) and no anti-hormone therapy (#44). Corresponding median ages were 66, 65 and 65 yrs and median treatment durations were 54 (AI) and 58 (tamoxifen) months. AI therapy, compared to tamoxifen, showed a significantly thicker CCA-intima layer, p = 0.031. However, mean values of both CCA-intima thickness and I/M thickness ratio were very similar in the tamoxifen and the no anti-estrogen group; Thus, AI compared to the other two groups combined, had a significantly thicker intima layer (p = 0.009) and higher I/M ratio (p = 0.047), both indicating adverse vascular effects from AI therapy. After adjustment for potential confounders, AI therapy still showed a thicker CCA intima layer of 0.015 mm (95% CI .003, .027); p = 0.016. In contrast, corresponding analysis using conventional Carotid intima-media thickness (CIMT), revealed no significant group differences or association to AI treatment duration. Conclusion: More than three years of adjuvant therapy with aromatase inhibitors, compared to women having no anti-estrogen therapy or tamoxifen, was associated with signs of adverse vascular effects; thicker intima layer and higher intima/media ratio of the common carotid artery. After adjustment, the thicker intima layer remained significant. These findings are in accordance with reports of increased risk of cardiovascular events following adjuvant therapy with aromatase inhibitors. Citation Format: Lindman H, Dahlberg L, Larsson M, Naessén T. A study of adverse vascular effects of adjuvant therapy with aromatase inhibitors in women with breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-14-06.

Approximately 30-40% of breast cancer cases are estrogen receptor negative (ENBC), including the most aggressive triple-negative subtype (TNBC) which spread early and recur frequently with a poor prognosis. ENBCs do not respond to selective ER modulators such as tamoxifen or aromatase inhibitors, and therefore, there is an urgent need of non-ER-based therapies for ENBC and TNBC. We recently developed a novel small molecule JMX0293 based on our lead drug candidate HJC0152. Both HJC0152 and JMX0293 were able to suppress cell proliferation of both ER+ (MCF-7, T-47D) and ER- (MDA-MB-231 and MDA-MB-468) breast cancer cell lines in a dose-dependent manner, with significantly lower toxicity against normal mammary epithelial cells (MCF-10A) in vitro. JMX0293 exhibited moderately improved anti-proliferative effects against highly invasive TNBC cell line MDA-MB-231 and ER+ breast cancer cell line MCF-7 with IC50 values of 0.92 and 2.24 μM, respectively. JMX0293 was also demonstrated to induce apoptosis of MDA-MB-231 cells dose-dependently. Mechanistically, JMX0293 inhibited STAT3 phosphorylation at Tyr705 residue, leading to the down-regulation of Bcl-2 and upregulation of apoptotic proteins including Bax, cleaved-caspase 3 and cleaved-PARP. In addition, JMX0293 reduced the ratio of Bcl-2 to Bax. In vivo, JMX0293 treatment for 2 weeks at a dose of 10 mg/kg significantly suppressed the growth of TNBC xenograft tumors in mice with minimal impact on general health and body weight. Our results suggest that JMX0293 induces TNBC cell apoptosis and contributes to its anti-proliferative effect through inhibiting transcription factor STAT3, and JMX0293 could be further characterized and optimized for developing a promising therapeutic for treating ENBC and TNBC. This work was supported by grant (R01 CA231150) from the NIH/NCI. Citation Format: Dong J, Xu J, Kim H, Zhou J, Shen Q. A novel small molecule JMX0293 inhibits the growth of triple-negative breast cancer via suppressing STAT3 and inducing apoptosis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-13.

Background: Aromatase inhibitors (AI), which block the conversion of androgen to estrogen, are the mainstay therapy for estrogen receptor–positive (ER+) breast cancer (BC). However, many patients relapse. Purpose: To identify molecular alterations associated with long-term neoadjuvant AI therapy of ER+ BC. Methods: We retrospectively identified 137 patients diagnosed with ER+ BC and treated with neoadjuvant AI for at least 1 month at the Royal Marsden Hospital (2003–2016). Paired pre- (diagnosis) and post-AI (surgery) biopsies with >40% invasive cell areas were available from 87 ER+ BC patients. In all samples, we evaluated ER, PR and Ki67 immunostaining, ESR1 hot-spot mutations by droplet digital PCR and expression of 801 genes associated with BC and response to endocrine therapy by NanoString. Results: Mean time on neoadjuvant treatment was 26 weeks (range 5.6–92.3). Cell proliferation remained suppressed in most tumours indicating little evidence for acquired resistance: 56/87 (64%) showed residual Ki67 (Ki67r) 10%. This was paralleled by reduced expression of proliferation genes, ER (immunostaining and gene expression) and estrogen-regulated genes (ERG) at surgery compared with the diagnostic samples (all p6 months, giving a prevalence in this cohort of 5/34 (15%). Tumours with ESR1 mutations showed less suppression of ERGs (p=0.002) and proliferation (p=0.039) and increased ESR1 (p=0.016) expression at surgery compared with tumours without mutation. Pathway analysis confirmed lack of inhibition of estrogen response (FDR>5%) and less inhibition of cell cycle [enrichment score: -0.49 vs -0.78) and E2F targets (-0.47 vs -0.8) in tumours with mutation. Tumours without ESR1 mutation but with Ki67r>10% also showed reduced ERGs response (p=0.006) compared to tumours with Ki67r Conclusion: Overall most tumours showed no evidence for the emergence of resistant disease after neoadjuvant AI therapy even after many months of treatment. However, we detected an enrichment of ESR1 mutations (15% of cases) after long-term treatment as a putative driver of ERG expression and proliferation and thus reduced AI response. Therefore, mutant ER appears to be associated with ligand-independent ERG activity supporting the clinical validity of dual blockade with a selective ER down-regulator combined with a CDK4/6 inhibitor targeting the RB/E2F axis in this scenario. Citation Format: Leal MF, Haynes BP, Schuster E, Yeo B, Afentakis M, Dodson A, Buus R, Cheang MC, Martin L-A, Dowsett M. Acquired ESR1 mutation and persistent expression of estrogen regulated genes in ER+ breast cancers on long-term neoadjuvant treatment with aromatase inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-15-01.

Background. The NEO-LET-EXE trial examines the neoadjuvant use of sequential administration of the aromatase inhibitor letrozole (Femar / Femara) and the aromatase inactivator exemestane (Aromasin). Although both drugs nearly completely inhibit aromatase, resistance to both is developed with time. However, when used sequentially, in some patients after switching to the alternative drug and progressing on the first choice, new responses may appear. The mechanism behind this clinical observation is currently not known. The solution may lead to a novel strategy to re-sensitize tumors to hormonal treatment. Prior studies have examined genomics at the four month time point, but not at both two months and four months. Material. Postmenopausal patients with estrogen receptor (ER) positive (>50%), HER-2 negative locally advanced breast cancer may be enrolled. Age: 18+ (no upper limit). Present accrural and target accrural: 49 out of planned 100 patients have been enrolled so far. The last patient is expected to enter the trial in Q4 2019. Study design. In the neoadjuvant, randomized, open-label, intra-patient cross-over trial NEO-LET-EXE biopsies are taken before treatment, after two months on one aromatase inhibitor and swap to the other aromatase inhibitor, and at surgery at four months. Results. In order to explain the phenomenon of a lack of cross-resistance between steroidal and non-steroidal aromatase inhibitors we profiled biopsies at three time points per patient by whole exome and whole transcriptome sequencing from FFPE from 25 patients. A total of 56 DNA whole exomes and 41 RNA seq transcriptomes were generated from FFPE samples available. When grouping both arms together, mutational burden decreased at two months, while clonality of mutations increased, providing evidence of selection. At four months, mutational burden increased from the two month timepoint. In particular, PIK3CA somatic variants present at the first time point were not detected at two months. However, these were detected again at significant variant allele fractions at four months after switch of treatment. The majority of gene expression changes happen in the initial two months, with fewer changes between two and four months. Instead, significant changes in alternative splicing at two months and four months were observed, for example for FGFR1, which does not experience a large fold change in expression between these two points. Our preliminary results show significant DNA and RNA changes in the first two months of aromatase inhibition leading to fewer, more clonal variants. Comparison of the four month to two month time point shows fewer RNA changes than the prior two months and an increase in the number of somatic variants compared to the two month timepoint. Citation Format: Vaske CJ, Parulkar R, Bahrami N, Sauer T, Loeng M, Gravdehaug B, Aljabri B, Bemanian V, Lindstrøm J, Lüders T, Kristensen V, Geisler J. Time-course DNA and RNA profiling of tumors from intra-patient cross-over trial of sequential use of aromatase inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-11.

Breast Cancer is the main cause of cancer-associated mortality in women worldwide. The estrogen receptors (ER's) play an important role in normal mammary gland development, as well as in breast cancer. Estrogen Receptor α is expressed in 70% of breast cancers, where it contributes to increased cell proliferation and decreased cell death. Endocrine therapies such as anti-estrogens and aromatase inhibitors target ERα signaling and improve outcomes of these patients. Syngeneic, immunocompetent mouse models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of breast cancer. In contrast to the tumor-inducing role of ERα, ERβ has been shown to have tumor suppressive activities in various cancer, including the breast cancer. Compounds that selectively activate ERβ hold promise because they could potentially avoid the unwanted effects of ERα activation, while exploiting the tumor-suppressive function of ERβ. In the present study, we assessed the antitumor effects of ERβ agonists using three different syngeneic mouse models; D2A1 (BALB/c) and MM51 (FVB) syngeneic models and ex-vivo culture of highly metastatic cell line E0771 (C57/B6). Effect on in vitro cancer cell growth was evaluated by cell proliferation and clonogenic assays. Cell cycle distribution was analysed by flow cytometry. Our results demonstrate that ERβ agonists LY500307 and S-Equol not only inhibited the growth of all three mouse mammary tumor cell lines, but also reduced the colony formation ability. ERβ agonists also induced the cell-cycle arrest in time and dose-dependent manner. In mechanistic studies, ERβ agonists LY500307 and S-Equol, modulated the protein levels of cyclin-dependent kinases (CDKs) (4, 6, and 2), cyclins (D1 and E), in a differential manner in these three cell lines. Our in vivo studies of D2A1 and MM51 cells demonstrates that ERβ agonist LY500307 inhibited the tumor growth and the effect was more pronounce in combination with aromatase inhibitor letrozole. Ex-vivo model of E0771 cells showed that LY500307 has potential to dramatically reduce the proliferation of mouse mammary tumor growth.Together, these results identify potential molecular targets and anticancer effects of ERβ agonists in mouse mammary tumors. Citation Format: Ramasamy K, Samayoa C, Krishnegowda NK, Thurlapati A, Vadlamudi RK, Tekmal RR. Estrogen receptor β agonists inhibits syngeneic mammary tumor growth through cell-cycle arrest by modulating cell-cycle regulators [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-04-01.

Endothelial dysfunction in breast cancer survivors on aromatase inhibitors (AIs) over time Background: AIs reduce breast cancer-related mortality however they may increase cardiovascular (CV) risk. Our previously published cross-sectional study suggested women on AIs were more likely to have endothelial dysfunction when measured by EndoPAT ratio as compared to healthy postmenopausal women. Reductions in EndoPAT ratio ( Methods: Fourteen women with locally advanced breast cancer prescribed an AI underwent vascular testing at baseline (pre-AI) and at 6 months. Subjects with tobacco use, hypertension or hyperlipidemia were excluded. Consented subjects underwent biomarker analysis and radial artery pulse wave analysis using the HDI/Pulse Wave CR-2000 CV Profiling System and pulse contour analysis using the Endo-PAT2000 system. Biomarkers were obtained using a fasting blood draw to evaluate the following lipids and inflammatory markers: serum ultrasensitive estradiol, serum glucose, total cholesterol (TC), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and triglycerides (TG), CRP, plasminogen-activator 1 (PA1), and tissue-type plasminogen activator (tPA). Changes between baseline and follow-up using Wilcoxon signed-rank tests were analyzed. Results: Mean baseline age was 59 years and median body mass index was 26.5 kg/m2. Median systolic blood pressure and total cholesterol were 120/70 mm/Hg and 228 mg/dL, respectively. Baseline ultrasensitive estradiol levels were 7 pg/mL and hsCRP was 2.45 mg/dL. Prior to AI therapy, endoPAT ratio was 2.18 (1.19, 2.43). After six months, EndoPAT ratio declined to a median 1.12 (0.85, 1.86) (p=0.045). There were no statistically significant changes in serum glucose, TC, LDL, HDL, hsCRP, PA1 and tPA. HsCRP remained elevatedat median 2.98 mg/L. At six months, estradiol levels decreased to a median of 2 pg/mL (p=0.052), however, there appeared to be no linear association between changes in EndoPAT and estradiol (p=0.91). Conclusion: Breast cancer survivors on AIs have endothelial dysfunction, a predictor of adverse CV disease. These changes develop while on AIs. Underlying pathophysiology requires further evaluation. Cardiovascular markersMeasuresBaselineFollow-Up at 6 MonthsChangeP-valueBMI (kg/m2)26.5 (24.4, 31.6)27.1 (23.9, 32.9)0.5 (0.0, 1.3)0.056SBP (mmHg)120 (115, 124)123 (114, 127)-0.8 (-7.4, 3.6)0.91DBP (mmHg)70 (61, 73)69 (62, 71)0.0 (-3.0, 2.6)0.88Total Cholesterol (mg/dL)228 (202, 244)213 (210, 229)-1 (-18, 27)0.70HDL (mg/dL)64 (58, 69)73 (61, 77)2 (-3, 14)0.44LDL (mg/dL)143 (121, 159)129 (120, 142)6 (-11, 14)0.65Estradiol (pg/mL)7 (4, 15)2 (2, 3)-8 (-12, -3)0.05hsCRP (mg/dL)2.45 (1.14, 6.07)2.98 (0.90, 4.81)-8 (-12, -3)0.85EndoPAT Ratio2.18 (1.19, 2.43)1.12 (0.85, 1.86)-0.16 (-1.45, -0.02)0.0451.Summaries shown are median (1st quartile, 3rd quartile). Citation Format: Blaes AH, Petersen A, Beckwith H, Potter D, Florea N, Yee D, Vogel R, Duprez D. Endothelial dysfunction in breast cancer survivors on aromatase inhibitors (AIs) over time [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-12-06.

Introduction: Breast cancer is the most common cancer in women. In estrogen receptor positive breast cancers aromatase inhibitors (AI) are a common treatment option. AIs are associated with a reduction in bone mineral density, and patients with osteopenia at baseline have a higher risk of developing subsequent osteoporosis while on AI therapy. Women age 75 and older are a fast growing subset of breast cancer patients and commonly have osteopenia or osteoporosis at time of breast cancer diagnosis. Studies of long-term effects of AI on bone density in these older women who are at higher risk of osteoporosis and musculoskeletal events are lacking at this time. Objective: To evaluate the objective change in bone density in women over the age of 75 diagnosed with early stage breast cancer and treated with AI. Methods: A retrospective chart review of 49 patients ages 75 to 95 diagnosed with early stage breast cancer and treated with AI. Pretreatment DEXA scan results were recorded as well as prevalence of bone targeted therapy at the time of breast cancer diagnosis. Incidence of bone targeted therapy initiated subsequent to cancer diagnosis and changes in T score on follow up DEXA scans were collected as well. Incidence of musculoskeletal events and osteonecrosis of the jaw were recorded. Results: 40/49 (81.6%) of study women were found to have osteopenia (23/49 [46.9%]) or osteoporosis (17/49 [34.7%]) on pre-treatment DEXA scans. Only 16/49 (32.7%) of patients were on bone-targeted treatment prior to breast cancer diagnosis. Of the patient with baseline osteoporosis, only 4/17 (23.5%) were on bone targeted treatment prior to breast cancer diagnosis. 25/49 (51%) of women were initiated on bone targeted therapy subsequent to breast cancer diagnosis and following review of pretreatment DEXA scan results. 5/49 (10.2%) of women were started on bisphosphonates and 7/49 (14.3%) were started on Denosumab. On the first subsequent DEXA scan at a median follow up of 2 years, 14/21(66.7%) of women were noted to have stable DEXA findings (defined as change in T score less than 0.5). 7/21 (33.3%) had a worsening T score on repeat DEXA. Of those patients with worsening T score, 3/7 (42.9%) changed categories (either from normal density to osteopenia or from osteopenia to osteoporosis. 3/49 (6%) of patients sustained a fracture while on AI therapy. There were no reported events of osteonecrosis of the jaw. Subsequent DEXA showing stabilitySubsequent DEXA showing worsening T scoreFractureBaseline Normal bone density1 (n=3)2 (n=3)1 (n=9)Baseline Osteopenia9 (n=12)3 (n=12)0 (n=12)Baseline Osteoporosis4 (n=6)2 (n=6)2 (n=17) Conclusion: Many elderly women are found to have osteopenia or osteoporosis at the time of breast cancer diagnosis and AI initiation. Most elderly patients had stable findings on subsequent bone density testing. Women with known osteoporosis initiated on bone-targeted therapy and AI did not have significant worsening in bone health. With appropriate treatment and monitoring elderly women with baseline decreased bone density can be treated safely with aromatase inhibitors. Citation Format: Baer L, Chaudhry B, Kudelka A, Cohen J, Stopeck AT. Aromatase inhibitors and bone health in women 75 and older treated for early stage breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-11-24.

Background: Approximately 75% of all breast cancers diagnosed are estrogen receptor-positive (ER+) and currently approved endocrine therapies rely heavily on blocking of the ER signaling pathway. In recent years, the combination of an endocrine agent with other targeted agents have been evaluated to address endocrine resistance and improve progression-free survival (PFS). Recently, it was demonstrated that the addition of a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor to an endocrine agent roughly doubles PFS, leading to the approval and use of certain CDK4/6 inhibitors in combination with either aromatase inhibitors in the first-line metastatic setting or in combination with the selective estrogen receptor degrader (SERD), fulvestrant, in the second-line metastatic setting. While combining CDK4/6 inhibitors and endocrine therapy can result in significantly increased PFS, patients eventually progressed on these combinations, and to date, there is no cure for patients with advanced metastatic ER+ breast cancer. Given the increased use of CDK4/6 inhibitors in the ER+ breast cancer treatment paradigm, it will be important to understand how treatment resistance to CDK4/6 inhibitors manifests in order to optimize therapeutic strategies to target this patient population. We have previously described elacestrant (RAD1901), a novel and orally bioavailable SERD, as an inhibitor of ER+ breast cancer growth in in vitro models and in in vivo patient-derived xenograft (PDX) models. Importantly, elacestrant inhibited the growth of PDX models that were derived from heavily pretreated patients, models harboring mutations in ESR1, and models insensitive to standard of care endocrine therapies. Given these results, we hypothesized that elacestrant would have anti-tumor activity in a CDK4/6 inhibitor-resistant setting. Herein, we describe elacestrant activity in multiple in vitro and in vivo models of CDK4/6 inhibitor resistance in both wild-type and mutant ESR1 backgrounds. Methods: In vitro models of estrogen-independent ER+ breast cancer, harboring either wild-type or mutant ER, were exposed to increasing concentrations of approved CDK4/6 inhibitors: palbociclib, ribociclib, or abemaciclib. ER expression/signaling, changes in cell cycle mediators, and the effects of elacestrant and other SERDs were examined in these representative models. Results: Despite prolonged exposure to CDK4/6 inhibitors, the resistant cell lines retained ER, ER signaling, and importantly, ER-driven proliferation. Elacestrant induced dose-dependent growth inhibition in CDK4/6 inhibitor-resistant cells, and this effect was independent of the CDK4/6 inhibitor used to generate resistance. Elacestrant also demonstrated in vivo tumor growth inhibition of CDK4/6 inhibitor-resistant ER+ PDX models. Conclusions: Our preclinical data demonstrate that elacestrant is a SERD that can inhibit tumor growth in a CDK4/6 inhibitor-resistant setting and provides rationale for examining elacestrant in patients that have progressed on a combination of endocrine therapy with a CDK4/6 inhibitor. Citation Format: Patel H, Tao N, Arlt H, Bihani T. Elacestrant (RAD1901) demonstrates anti-tumor activity in models resistant to CDK4/6 inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-13-03.

Supported by a PHRC grant (#09-18-005) Background: Recent literature has suggested that germline genetic variants of drug-metabolizing enzymes or CYP19A1 (coding for aromatase) may be involved in the systemic aromatase inhibitors (AI) concentrations or the occurrence of side effects (Hertz et al. Pharmacogenomics 2017). A prospective multicentre 3-year follow-up study was carried out to investigate the relationships between pharmacogenetics (PG), pharmacokinetics (PK) and toxicity in breast cancer patients treated with adjuvant AI (n=1098) or tamoxifen (n=879). The clinical results and the tamoxifen PG/PK analyses are described elsewhere (abstracts #851544 and #850248). Methods: SNP genotyping of 95 SNPs was performed on the Biomark (Fluidigm) with Taqman assays and was available for 373, 515 and 151 patients treated with anastrozole (ANA), letrozole (LETRO) and exemestane (EXE) respectively. CYP2A6 metaboliser status (MS) (poor, intermediate or normal) was determined based on alleles function (*1, *9, *2) and number of CYP2A6 copies. Trough plasma concentrations of each drug were determined 6 months after the start of the study by UPLC-MS/MS and were available for 342, 463 and 130 patients of the ANA, LETRO and EXE arms. Patients with AI concentrations below the limit of quantification were excluded for non-compliance (9 patients for ANA, 8 patients for LETRO and 7 patients for EXE). Toxicity was measured as a binary outcome (occurrence or worsening of hot flushes, fatigue, pain, arthralgia, vaginal dryness). All genetic associations were adjusted for multiple testing. Results: ANA concentration was significantly higher in patients experiencing pain (p=0.025) and was associated with rs28365063 (UGT2B7 g.372A>G). LETRO concentrations were strongly associated with CYP2A6 metabolizer status (p=0.0001) but did not differ in patients with or without toxicity. In the EXE arm, patients with hot flushes or arthralgia had a significantly lower level of exemestane (p= 0.0002 and p=0.023 respectively) but since the metabolism of EXE leads to active 17-hydroexemestane, we can hypothesize that the lower EXE concentration is an indirect reflection of the metabolite formation. A SNP (rs2307424) in NR1I3 gene (coding for the constitutive androstane receptor CAR) was associated with EXE concentrations. CAR has been shown to regulate CYP2B6, which is involved in the formation of 6-hydroxy-methyl-exemestane (inactive metabolite). Regarding the relationships between PG and toxicity, in the ANA arm, 3 SNPs of CYP19A1 gene tended to be associated with hot flushes worsening (rs934635) and arthralgia (rs10046 and rs2304463) but did not remain significant after multiple tests correction. In the EXE arm, several SNPs in NR1I3 gene were associated with fatigue. In the LETRO arm, patients with a poor CYP2A6 MS had a higher risk of experiencing depression. Conclusions: Our study confirms the predominant role of CYP2A6 in LETRO PK. To our knowledge, this is the first study to report on the role of UGT2B7 rs28365063 in ANA and NR1I3 in EXE PK and side effects. These relationships need to be re-evaluated with the drug concentrations obtained during the 3-year follow-up. Citation Format: Thomas F, Marquet P, Pinguet F, White-Koning M, Robert J, Tafzi N, Solassol I, Despax R, Levasseur N, Ellis S, Massoubre A, Mbatchi L, Le Morvan V, Roché H, Chatelut E, Evrard A. Pharmacogenetic determinants of aromatase inhibitors pharmacokinetics and side effects: 6-month results of the adjuvant breast cancer longitudinal PHACS study (NCT01127295) [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 P3-12-07.

Background: Aromatase inhibitors (AIs) reduce the risk of breast cancer in women at increased risk and reduce recurrence in those with ductal carcinoma in situ (DCIS) (IBIS-2, MAP.3, NSABP B-33). The effectiveness of AIs depends on full adherence. We have previously reported adherence figures for the International Breast Cancer Intervention Studies 2 (IBIS-2) when 5 years of active treatment was not completed. Here, we assess reports of early symptoms on 5-year adherence with anastrozole in the prevention (versus placebo) and DCIS (versus tamoxifen) IBIS-2 after active treatment has been completed by all women. Methods: In IBIS-2, 3864 postmenopausal women in the prevention study were randomised to placebo vs. anastrozole (1mg/day) and 2980 postmenopausal women with DCIS were randomised to tamoxifen (20mg/day) vs. anastrozole (1mg/day). Women were excluded from the analyses (n=491 [262 prevention; 229 DCIS]) due to breast cancer, death, major adverse events, or failure to initiate preventive therapy. Adherence ( Results: In the IBIS-2 prevention study (N=3615), overall adherence to treatment was 67.7% and was statistically not significantly different between anastrozole (66.5%) and placebo (69.0%) (OR=0.89 (0.78-1.03), P=0.11). Adherence was significantly lower regardless of treatment allocation for those who developed arthralgia (68.3% vs. 72.8%, P=0.008) or gynaecological symptoms (vaginal changes, irregular bleeding) (65.1% vs. 72.2%, P=0.007), but not for those who reported hot flushes (71.1% vs. 71.8%, P=0.92), compared with those who did not report these symptoms at 6 months. In the IBIS-2 DCIS study (N=2759), adherence to treatment was 70.1% overall (anastrozole (70.2%) or tamoxifen (70.0%) (OR=1.01 (0.86-1.19), P=0.92)). Women treated with anastrozole reported significantly more arthralgia (30.6% vs. 20.5%, P Conclusions: In the IBIS-2 trials, we observed no significant differences in adherence between either anastrozole vs. placebo (prevention), or anastrozole vs. tamoxifen (DCIS). Significant associations between early symptoms and adherence were observed only in the prevention study, regardless of treatment allocation. Reporting symptoms in the first 6 months of preventive and adjuvant therapy is unlikely to explain non-adherence to medication. Further research is required to identify modifiable factors which may be altered by behavioural interventions to improve adherence. Citation Format: Sestak I, Smith S, Sleeth M, Howell A, Cuzick J. Participant-reported symptoms as predictors of long-term adherence of endocrine therapy in the International breast cancer intervention studies 2 (IBIS-2) [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 P5-15-01.

Background Polymorphisms of genes involved in estrogen production have been linked to breast cancer risk, prognosis and treatment response. Polymorphisms of the aromatase gene CYP19A1 influence its activity. The UGT2B17 catalyzes glucuronic acid transfer to a variety of substrates, including steroids and drugs like the aromatase inhibitor exemestane. We investigated the impact of two variants of CYP19A1 (rs10046, rs4646) and the UGT2B17 gene deletion on disease outcome in 125 postmenopausal women operated for ER-positive primary breast cancer enrolled in a randomized pre-surgical trial. Patients Briefly, upon informed consent, postmenopausal patients with ER-positive breast cancer (stage T1–2, N0–1, M0) eligible for surgery were randomized to receive either exemestane (25 mg/day), or celecoxib (800 mg/day), or placebo for 6 weeks prior to surgery at the European Institute of Oncology (2004-2008). Exemestane showed a significant 10% absolute reduction in Ki67 labeling index compared to the other two arms. Serum and whole blood was taken at baseline and the day before surgery and stored at -80°C until assayed. Methods DNA was extracted from blood by QIAamp DNA Blood Kits. The CYP19A1 rs1004/rs46466 were analyzed by Taqman genotyping assays in real-time PCR. The UGT2B17 deletion was estimated by copy number assay (Lifetechnologies). Serum estradiol (E2) and estrone (E1) levels were measured by gas chromatography tandem mass spectrometry detection (GS-MS/MS) after liquid-liquid extraction. The lower limit of quantitation were 0.625 pg/mL for estradiol and 1.56 pg/mL for estrone. The association of genetic polymorphisms with “any event” was assessed by the Cox proportional hazards models adjusted for confounders. Results The genetic polymorphisms did not deviate from Hardy-Weinberg equilibrium (P-value >0.41) and minor allele frequency of rs10046 (A/G), rs4646 (C/A), and UGT2B17Del were 0.45, 0.22, and 0.31, respectively. The rs10046 A and rs4646 C alleles were associated with higher estrogen levels. Carriers of rs10046 AA had median levels of 7.57 pg/ml E2 and 35.9 pg/mL E1 versus 3,9 pg/mL E2 and 27.4 E1 pg/mL in CA/AA genotypes (P Conclusions Our analysis confirms previous findings of an association of CYP19A1 rs10046/rs4646 with estrogen levels in postmenopausal women. Interestingly, the carriers of the variants associated with lower estrogen levels at diagnosis had better prognosis. Further genomic profiling in larger trials aimed to enhance tailored treatment efficacy in endocrine-responsive postmenopausal breast cancer are warranted. Citation Format: Johansson H, Gandini S, Aristarco V, Macis D, Guerrieri-Gonzaga A, Serrano D, Pruneri G, Lazzeroni M, Viale G, Toesca A, Rajasekaran A, Bonanni B, DeCensi A. Impact of common polymorphisms of CYP19A1 and UGT2B17 gene deletion on early endocrine-responsive postmenopausal breast cancer [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 P4-08-05.

Background: Randomized trials (RCTs) have reported improvements in breast cancer outcomes from extending treatment with aromatase inhibitors (AIs) beyond the initial 5 years after diagnosis. It is uncertain whether this effect is consistent in different subgroups. Methods: We identified RCTs that compared extended AIs to placebo or no treatment using a systematic search of MEDLINE. The search was supplemented by a review of abstracts from the American Society of Clinical Oncology and San Antonio Breast Cancer Symposium meetings between 2013 and 2016. Hazard ratios (HRs) and 95% confidence intervals (CI) for disease-free survival (DFS) were extracted or estimated from forest plots and included in a meta-analysis using generic inverse variance and random effects modelling. Pre-specified subgroups included age (2 cm), nodal status (positive vs. negative), hormone receptor status (estrogen [ER] and progesterone receptor [PR] positive vs. ER or PR positive) and administration of adjuvant chemotherapy (yes vs. no). Results: Seven trials comprising 16,349 patients were analyzed. Studies designs and prior endocrine therapy are shown in Table 1 Table 1: Characteristics of included studies.TrialTreatment ArmsSample sizePrior endocrine treatmentABCSG 6aAnastrozole 3 years vs. none387/ 469Tamoxifen± aminoglutethimide: 100%, 5 yearsMA 17Letrozole 5 years vs. placebo2572/ 2577Tamoxifen: 100%, ∼5 yearsNSABP B-33Exemestane 5 years vs. placebo783/ 779Tamoxifen: 100%, ∼5 yearsDutch DATAAnastrozole 6 years vs,. anastrozole 3 years827/ 833Tamoxifen: 100%, 2-3 yearsIDEALLetrozole 5 years vs. letrozole 2.5 years903/ 898Any endocrine treatment (tamoxifen/AIs/sequence of tamoxifen+ AIs): 100%, 5 yearsMA.17RLetrozole 5 years vs. placebo959/ 959AIs: 100%, ∼5 years Prior tamoxifen: 79.3%NSABP B-42Letrozole 5 years vs. placebo1959/ 1964Any endocrine treatment (AIs/sequence of tamoxifen+ AIs): 100%, 5 years . The pooled effect of prolonged treatment with AIs in different subgroups is shown in the Table 2. Table 2: Intra-subgroup comparison of longer AIs treatment effect by subgroupsSubgroup ASubgroup BHR (95% CI) Subgroup AHR (95% CI) Subgroup BP for differenceAge 2 cmT ≤2 cm0.77 (0.55-1.06)0.88 (0.68-1.13)0.44N positiveN negative0.72 (0.63-0.83)0.83 (0.64-1.08)0.22ER and PR positiveER or PR positive0.68 (0.44-1.04)1.03 (0.53-2.02)0.27Adjuvant chemotherapyNone0.71 (0.59-0.86)0.80 (0.65-0.98)0.51 Overall, the effect of prolonged AIs was similar in all subgroups. However, non-significantly greater effect sizes were seen in patient with larger tumors, nodal involvement, presence of both ER and PR expression and those treated with adjuvant chemotherapy. Conclusions: Extended treatment with adjuvant AIs is associated with similar relative improvements in DFS in all subgroups analyzed. The greater effect size seen in node positive and large tumor subgroups and the higher baseline risk of recurrence will likely translate to a higher absolute benefit from extended AIs in these groups. Citation Format: Goldvaser H, Algorashi I, Ribnikar D, Majeed H, Ocana A, Seruga B, Templeton AJ, Amir E. Efficacy of extended adjuvant aromatase inhibitors in subgroups of women with early breast cancer [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 P3-12-04.

Background: Elevated body mass index (BMI) is associated with increased risk of estrogen receptor-positive postmenopausal breast cancer. Mechanistically, most individuals with elevated BMI have breast white adipose tissue inflammation (WATi) which confers increased breast cancer risk, particularly in those with existing benign breast disease. Individuals with WATi have elevated in-breast expression of aromatase and several systemic changes that increase breast cancer risk, including hyperinsulinemia and higher levels of C-reactive protein. However, women with normal BMI but high levels of body fat are also likely to harbor WATi and are at increased risk of postmenopausal breast cancer. The accuracy of BMI for assessing adiposity and predicting obesity-related disorders, including cancer, varies across race and ethnicity. Whether the association between BMI and WATi varies by race is unknown. Here we aimed to characterize relationships among breast WATi and clinicopathologic features in a racially diverse cohort undergoing mastectomy for breast cancer treatment. Methods: Non-tumorous breast tissue and fasting blood were collected from women undergoing mastectomy for breast cancer treatment or prevention at a single center serving a racially diverse patient population. Breast WATi was detected by the presence of crown-like structures in the breast (CLS-B), which are composed of a dead/dying adipocyte surrounded by CD68+ macrophages. Clinicopathologic data were abstracted from electronic medical records. Associations among categorical variables were examined using Fisher's exact test. Relationships between continuous variables were examined using the Spearman correlation. Results: As of May 18, 2018 62 patients have been accrued; median age 55 (range 32 to 84). Self-reported race distribution was: 36 (58%) Asian, 5 African American (8%), 20 (32%) Caucasian, and 1 (2%) unknown. Breast tissue has been analyzed for WATi in 60 cases thus far. Clinicopathologic features stratified by the presence or absence of breast WATi are presented in. Breast WAT inflammation was associated with obesity (P=0.02) and a trend to association was observed with dyslipidemia (P VariableBreast WATi Absent (n=25)Breast WATi Present (n=35)Age, years Median (range)51 (32 to 71)59 (36 to 80)BMI, kg/m2 Median (range)22.5 (18.1 to 35.3)28.0 (19.2 to 38.9)BMI Category Underweight1 (4%)0 (0%)Normal16 (64%)10 (29%)Overweight5 (20%)18 (51%)Obese3 (12%)7 (20%)Race, n (%) Asian15 (60%)19 (54%)African American2 (8%)3 (9%)Caucasian8 (32%)12 (34%)Unknown0 (0%)1 (3%)Menopausal Status, n (%) Pre10 (40%)12 (34%)Post15 (60%)23 (66%)Hypertension, n (%)7 (28%)16 (46%)Diabetes mellitus, n (%)0 (0%)5 (14%)Dyslipidemia, n (%)1 (4%)11 (31%) Conclusions: Breast adipose inflammation is associated with elevated BMI and possibly metabolic syndrome disorders in a racially diverse population. These findings are consistent with observations from predominantly Caucasian cohorts. Race-specific characteristics will also be examined. Study accrual is ongoing and updated results will be presented. Citation Format: Iyengar NM, Siegel B, Malik M, Giri DD, Tsai J, Hughes M, Adam A, Williams S, Zhou XK, Rodgers W, Ginter P, Patel A, Yong F, Cherian A, August P, Dannenberg AJ. Obesity, adipose inflammation, and race in patients with early stage breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-05.

Background. ESR1 mutations are acquired frequently in hormone receptor positive breast cancer after treatment with aromatase inhibitors (AI) in the metastatic setting. In prior analysis of the SoFEA phase III randomised trial, we demonstrated the detection of ESR1 mutations in circulating tumor DNA (ctDNA) predicted for greater benefit of fulvestrant compared to exemestane (Fribbens et al JCO 2016). Methods. The phase III EFECT study randomised 693 patients with ER+ metastatic breast cancer who had progressed on a prior non-steroidal AI, between fulvestrant loading dose and then 250mg q28 days and exemestane (Chia S, et al. J Clin Oncol 2008). Baseline serum samples were available from 227 patients in EFECT, and were analysed for the 8 most common ESR1 mutations by multiplex digital PCR. The association between baseline ESR1 status and time to progression (TTP) was analysed using Kaplan-Meier methods. Results. ESR1 mutations were successfully analysed in 98% (222/227) of patients with baseline serum samples, with ESR1 mutations detected in 23.4% (52/222) samples. Overall, detection of ESR1 mutations at baseline was associated with shorter TTP (hazard ratio [HR] 2.03, 95% CI 1.26-3.29, p=0.004). In patients with ESR1 mutation detected, TTP was 2.0 months (95%CI, 1.7-2.4) on exemestane and 3.5 months (95%CI, 1.9-5.0) on fulvestrant (HR 0.67, 95%CI 0.37-1.19, p=0.17). In patients without ESR1 mutations detected, TTP was 4.5 months (95%CI, 3.7-5.6) on exemestane and 3.7 months (95%CI, 3.3-5.2) on fulvestrant (HR 1.05, 95%CI 0.75-1.45, p=0.78). A meta-analysis of SoFEA and EFECT will be presented at the conference. Conclusions. Historical serum samples may be used for ctDNA analysis, illustrating the potential for future research of sample archives. Patients with ESR1 mutation detected in ctDNA have poor outcome when treated with exemestane, replicating prior results from SoFEA and demonstrating clinical utility for ESR1 mutation ctDNA analysis. Citation Format: Turner N, Swift C, Kilburn L, Garcia-Murillas I, Johnston S, Budzar A, Robertson J, Gradishar W, Piccart M, Schiavon G, Chia S. Baseline circulating ESR1 mutation analysis in the randomised phase III EFECT study of fulvestrant versus exemestane in advanced hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-04.

The ESR1 ligand–binding mutations were unveiled a number of years ago and are the most common genetic mechanism of acquired resistance to endocrine treatment, particularly, to aromatase inhibitors. The discovery of these mutations was enabled after advancements in sequencing technologies and when metastatic tissue samples were interrogated. The ESR1 ligand–binding domain mutations are activating mutations that lead to constitutive ligand-independent activity, which explains the emergence of these mutations under the selective pressure of aromatase inhibitors. Arnesen and colleagues have generated new models of the ESR1 mutations using CRISPR technology to generate single-cell–derived clones in which the ESR1 ligand–binding mutations were “knocked-in” and expressed under the endogenous promoter of estrogen receptor. The authors have extensively characterized these models and have shed new light on the functional consequences ESR1 mutations. See related article by Arnesen et al., p. 539

Introduction: Obesity is associated with poor breast cancer outcomes in postmenopausal women. Our prior retrospective studies have shown that use of nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with reduced recurrence in obese breast cancer patients and a doubling of time to recurrence. Because it was recently determined that CD163+ M2 macrophages were clinically associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological duct type in human primary breast tumors, the mechanism proposed was a decrease in prostaglandin E2 (PGE2) and aromatase locally in the breast with a concomitant decrease in circulating M2-activated tumor associated macrophages (TAMs). Methods: Postmenopausal women of varying body habitus were recruited at the CTRC in San Antonio and underwent randomized assignment to 1 of 3 arms: Aspirin (ASA) at 81mg daily, 1500mg of docosahexaenoic acid (DHA) and 2500mg eicosapentaenoic acid (EPA) given daily, or combined ASA and DHA/EPA. Sera were collected prior to and following 28 days of exposure, and cytokines including prostaglandin E2 were assessed via enzyme ­linked immunosorbent assay (ELISA). 28 circulating cytokines/chemokines were assessed by Luminex array using Millipore Milliplex MAP to look for associations between cytokine array profiles, PGE2 production and macrophage activation. Circulating class M-1 activated and M-2 activated macrophages were enumerated by flow cytometry to assess how PGE2 modulation influences macrophage phenotype and function. Investigators were blinded to randomization until analysis was complete. Results: A total of 122 patients were randomized with 2 drop outs and 115 completing the 28 days of intervention as planned. The median BMI was 31.4, with 12.8% normal (BMI 29.9). Patients had a median age of 63 (47-76), 91% white, and 46.0 % Hispanic. A positive correlation was observed between BMI and baseline PGE2 levels. The most consistent impact on PGE2 was observed with ASA with 81% obtaining a decrease from baseline (median change -28%); by comparison 55.1% (-1%) and 65.6% (-22%) of subjects showed decrease in the DHA/EPA and combined groups respectively. As of today, full cytokine profiling was performed on a subset of 38 patients and revealed a positive correlation with change in PGE2 and cytokines: EGF, Eotaxin, GM-CSF, IL1Ra, IL5, IL8, MIP1b, and TNFa. Conclusion: Aspirin alone most consistently impacted patient circulating PGE2 levels, and will be used in planned studies as an adjunct to adjuvant endocrine therapy in obese hormone receptor positive post-menopausal patients. Full cytokine and macrophage activation status will be reported. Citation Format: Brenner AJ, Lengfelder L, Quach DK, Cavazos DA, Ramirez RJ, Gruslova A, Kist K, Lathrup K, Kaklamani V, Beeram M, deGraffenried LA. Randomized study of COX2 inhibition on systemic inflammation in obese and non-obese subjects [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 P1-09-16.

Male breast cancer (MBC) is a rare disease dominated by a sea of pink. Issues faced by men with this disease include delayed diagnosis, lack of male-specific information, stigma about having a "female disease" and often under-treatment. MBC is often treated as a female breast cancer but differences between the two are starting to emerge. Men are often older at diagnosis and sometimes considered "too old" for more aggressive treatments such as chemotherapy. Drugs such as aromatase inhibitors may not be as effective in men as in women but are often prescribed. Drugs such as tamoxifen can cause side-effects such as weight gain, hot flushes, loss of libido and impotence. Support for patients with MBC is less advanced than that for female breast cancer. A Man's Pink, a MBC advocacy organization, mission is to promote MBC awareness, increase early detection, optimize and increase the survival rates for men diagnosed with breast cancer. Male Breast Cancer: Taking Control (BC Publishing, Boyages, 2015) empowers patients to understand their diagnosis and treatment. Apart from incidence data, prognosis and treatment options, this talk will focus on how a website (www.malebreastcancer.ca) dedicated to MBC can help dismiss some of the myths and help overcome stigmata for men facing difficulties at diagnosis, treatment and recovery. Our goal is to promote awareness to assist with earlier diagnosis and treatment to improve survival rates and the journey for men in their battle with breast cancer. Citation Format: Wagner H, Boyages J. Male breast cancer— infusing a little blue into the sea of pink [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 P4-18-05.

Estrogen receptor positive (ER+) breast cancer accounts for the majority of breast cancers diagnosed worldwide but fortunately, outcomes are markedly improved by pharmacological interventions that interrupt ER function. Unfortunately, suppression of relapse risk from early stage disease with endocrine therapy (anti-estrogens or aromatase inhibitors) is only ∼50%, and for advanced disease, pan endocrine therapy resistance is almost inevitable. While many mechanisms for intrinsic and acquired endocrine resistance have been explored, links between defects in DNA repair, the fundamental drivers of cancer pathogenesis, and endocrine therapy resistance have been understudied. Here we link mismatch repair (MMR) deficiency to poor clinical outcomes in ER+ breast cancer using whole exome DNA sequencing data and mRNA expression analysis. We subsequently demonstrate that MMR deficiency bypasses ER dependent cell cycle regulation through disruption of Chk2/p21-mediated feedback inhibition of CDK4 in breast cancer cell lines and tumor samples, as well as through correlations with human clinical data. We also show that pharmacological targeting of CDK4 significantly inhibits growth of MMR-deficient ER+ breast cancer cells in vitro and in vivo. This mechanism provides a new explanation for why endocrine therapy resistant ER+ breast cancers can respond to CDK inhibition and suggests that primary tumors exhibiting MMR deficiency are good candidates for adjuvant CDK4 inhibitor treatment. Citation Format: Haricharan S, Schmelz J, Schmidt C, Singh P, Holloway K, Anurag M, Suman V, Olson JA, Hunt K, Bainbridge MN, Ellis MJ. Mismatch repair deficiency induces endocrine therapy resistance in 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 PD2-06.

Despite the success of adjuvant endocrine therapy in early breast cancer, approximately 50% of all recurrences still occur after the initial 5 years of follow-up. Earlier studies confirmed that endocrine therapy extension after 5 years of adjuvant tamoxifen with either tamoxifen or aromatase inhibitors up to 10 years leads to an improvement in survival. However, aromatase inhibitors are currently standard of care in the initial 5 years of adjuvant therapy, and the benefit of extended use beyond 5 years of AI based therapy is still debated. The randomized phase III IDEAL trial was designed to study the optimal duration of extended adjuvant endocrine therapy after the initial 5 years of any endocrine therapy. Between April 2007 and November 2011, 1824 postmenopausal, HR-positive early breast cancer patients were included by 74 hospitals in the Netherlands. Enrolled patients earlier received 5 years of any endocrine therapy (87.9% AI based),completed this treatment no longer than 2 years before randomization, and did not have any recurrence at the moment of inclusion. The included patients were randomly allocated to either 2.5 or 5 years of extended letrozole (daily 2.5mg). Primary outcome was disease free survival (DFS), secondary endpoints were overall survival (OS), distant disease free survival (DDFS), contralateral breast cancer and safety. Results & Discussion: Results will become available soon. Citation Format: Blok EJ, van de Velde CJH, Meershoek-Klein Kranenbarg EM, Putter H, van den Bosch J, Maartense E, van Leeuwen-Stok AE, Liefers G-J, Nortier JWR, Rutgers EJT, Kroep JR. Optimal duration of extended letrozole treatment after 5 years of adjuvant endocrine therapy; results of the randomized phase III IDEAL trial (BOOG 2006-05) [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 S1-04.

Over 70% of all breast cancers are estrogen receptor positive (ER+) and are treated with endocrine therapies such as aromatase inhibitors or ER-disruptors as part of the standard of care. However, endocrine resistance remains a major clinical challenge for the successful treatment of this disease. ER+ breast cancer cells can escape endocrine therapy-mediated cell death through adaptations in the unfolded protein response (UPR), an evolutionarily conserved cellular stress response pathway that is linked to many other pathways (autophagy, metabolism, anti-oxidant response). To better understand how the UPR pathway promotes endocrine resistance, we built a mathematical model from quantitative measurements of key UPR signaling molecules following treatment of ER+ MCF7 cells (endocrine sensitive, estrogen dependent) with dithiothreitol (DTT). DTT disrupts or prevents protein disulfide bonding and is a potent inducer of the UPR. The same measurements were also made in two MCF7-derived resistant cell lines: LCC1 (sensitive to ER-disruptors, estrogen independent) and LCC9 (resistant to ER-disruptors, estrogen independent). Determining the (minimal) changes to the MCF7 mathematical model needed to adequately capture the data from the resistant cell lines allows us to create specific hypotheses for how the UPR adapts to help produce resistance to endocrine therapies. These hypotheses, in turn, can lead to drug targets for reversing resistance. Citation Format: Shajahan-Haq AN, Demas DM, Clarke R, Baumann WT. Mathematical modeling of the unfolded protein response pathway in breast cancer cells [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 P4-08-03.

Treatment of hormone sensitive breast tumors with endocrine therapy such as antiestrogens or aromatase inhibitors has improved their clinical outcomes. However, not all tumors respond and the ones that do respond may eventually acquire resistance. One of the proposed mechanisms of resistance to endocrine therapy is overexpression of ERα and cross-talk of ERα with growth factor receptors. Studies including our own have shown that downregulation of ER with pure antiestrogen fulvestrant in combination with AIs may prolong responsiveness of the tumors to endocrine agents. Fulvestrant has been employed as either first or second line treatment for ER positive breast cancers alone or in combination with AIs. Studies have suggested that further escalation of dose may provide further benefit. However, dose escalation of fulvestrant which is administered via intramuscular injection is difficult due to its poor solubility. To overcome this shortcoming of an injectable drug, a novel orally active SERD (selective estrogen receptor downregulator), AZD9496 was developed. In addition to being orally active, AZD9496 is selective for mammary ERα. In the current study, we compared the effect of AZD9496 and fulvestrant on the growth of MCF-7Ca (human estrogen receptor positive MCF-7 cells stably transfected with human placental aromatase gene) xenografts grown in ovariectomized athymic nude mice. Tumors were allowed to form with androstenedione (aromatizable source of estrogen) supplement. When the tumors reached ~250 mm3, mice were grouped such that the mean tumor volumes were not significantly different (p>0.99). Mice bearing xenografts of MCF-7Ca were then treated with fulvestrant (1 mg/d-sc) or AZD9496 (5 mg/kg/d-po), alone or in combination with anastrozole (200μg/d-sc) for 23 weeks. Tumors were measured weekly and growth rate was calculated. AZD9496 was significantly better at inhibiting the growth of tumors compared to control (p0.99 and tumor volume on week 23, p=0.99). In the second study, efficacy of AZD9496 was evaluated on against anastrozole resistant MCF-7Ca xenografts. Tumors were treated with anastrozole (200μg/d) for 13 weeks. During this time, the tumors initially regressed but eventually began to grow and had doubled in volume. At this time-point, they were regrouped to receive second line treatment. Single agent AZD9496 was marginally significant compared to continued anastrozole treatment (p=0.07). Nevertheless, second line treatment with AZD9496 was equally effective as fulvestrant (p=0.36). The combination of anastrozole with AZD9496/fulvestrant was more effective in reducing tumor growth compared to continued anastrozole treatment. Next, we measured the effect of AZD9496 on the mouse uterus. Uterine weight of mice treated with AZD9496 was not significantly different from mice that were treated with androstenedione (p=0.99). These results suggest that AZD9496 was selective for tumor ERα and had no effect on the uterine ERα. These results suggest that AZD9496 may be a better alternative to fulvestrant due to its selectivity for mammary ER and same efficacy as fulvestrant obtained upon oral administration. Citation Format: Sabnis GJ, Kazi A, Schech A, Yu S, Golubeva O, Weir H, Brodie A. A new oral SERD AZD9496 for treatment of hormone dependent postmenopausal 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 P2-09-10.

Advances in screening and treatment of breast cancer have led to reductions in breast cancer recurrence and mortality. However, systemic therapies can cause long-term and late effects that negatively impact the quality of life of patients with breast cancer. It is important for patients to be aware of the potential risks, and for providers to be knowledgeable about symptom management options. For example, taxane chemotherapies reduce the risk of disease recurrence but can cause long-lasting neuropathy that interferes with activities of daily living. Unfortunately, few effective interventions for preventing or treating neuropathy have thus far been identified. Similarly, endocrine therapies significantly reduce the risk of recurrence of hormone receptor positive breast cancer. However, tamoxifen and the aromatase inhibitors can cause menopausal symptoms and arthralgias that affect survivors of breast cancer of all ages. Interventions that can effectively treat these symptoms in some, but not all, patients have been identified. In this educational session, prevention and treatment approaches for key long-term and late effects in patients with breast cancer will be discussed. Citation Format: N Henry. Toxicities of breast cancer treatment [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 SP059.

Background: CDK4/6 inhibitors are being used in combination with aromatase inhibitors as therapy for advanced ER+ breast cancer (BC) and are being explored for use in primary BC. Few mechanisms driving resistance to added CDK4/6 inhibitors have been defined. The PALLET phase II randomized neoadjuvant trial of letrozole (LET) ± palbociclib (PALBO) in postmenopausal ER+HER2- primary BC reported that clinical response rate over 14wks was not significantly increased by adding PALBO to LET but suppression of Ki67 was significantly increased (Johnston et al, JCO 2018, 37, 178): after 14wks complete cell cycle arrest (CCCA, Ki67 Methods: 307 patients were randomized to LET (n=103) or LET+PALBO (n=204) for 14 wks. The first 2wks of LET+PALBO patients were randomised to LET, PALBO, or LET+PALBO. Biopsies were taken at baseline, 2wks and 14wks. Biomarker data are presented here for baseline only, other than Ki67 at both baseline and 14wks. IHC analyses were conducted on FFPE biopsies for ER, PgR, RB1, cyclin-E1, and cyclin-D1 (also FISH). RNA-seq was performed on fresh frozen biopsies. Association of each biomarker with CCCA was determined by logistic regression. Differentially expressed genes (DEGs) were identified between patients sensitive (CCCA) (n=94) and resistant (non-CCCA) (n=10) to treatments with or without PALBO at 14wks by DESeq2. Fifty hallmark gene sets were tested for significant enrichment with DEGs and differential gene sets were identified by using Gene Set Enrichment Analysis (GSEA). Results: The association of IHC biomarkers with CCCA is shown in the table. Lower levels of ER, higher levels of cyclin-E1, and amplification of cyclin-D1 were each significantly associated with a greater chance of non-CCCA with LET+PALBO. High cyclin-E1 levels were also associated with reduced chance of CCCA with LET only. Patients with high baseline Ki67 also exhibited higher non-CCCA with LET+PALBO at 14wks (p=0.0002). In the RNAseq data we identified 1973 DEGs between the 14wk CCCA and non-CCCA patients for LET+PALBO. E2F and MYC targets, PI3K/AKT/MTOR signalling and interferon response gene sets were among the hallmark gene sets enriched for genes with higher expression in non-CCCA patients at 14wks for LET+PALBO (FDR Conclusion: Biomarkers associated with response/resistance to added PALBO were different from LET only. PALBO resistance was associated with higher baseline expression of cyclin-E1 (both IHC and RNA), CDK2, and genes related to E2F, MYC, interferon and MTOR signalling. These results suggest that multiple identifiable mechanisms of de novo resistance to PALBO are likely to exist in primary ER+ BC. On-going WES analyses will allow the significance of alterations at the DNA level to be presented. Table 1.Continuous measurement in a logistic regression for CCCA at 14 weeks; Oddsratio calculated separately for group A and groups B,C,D and were adjusted forregion (UK vs NA); * amplified vsnon-amplifiedContinuous measurement in a logistic regression for CCCA at 14 weeksBiomarkerLET LET+PALBOOdds ratio95% CIpOdds ratio95% CIpER1.120.36, 3.480.844.471.62, 12.380.004PgR4.381.03, 18.580.053.050.50, 18.560.23RB13.010.24, 38.560.400.420.05, 38.490.83Cyclin-E10.100.01, 0.840.030.020.00, 0.200.001Cyclin-D1 IHC3.090.51, 18.490.222.560.28, 23.330.40CyclinD1 FISH*1.470.43, 4.990.530.280.06, 0.860.03 Citation Format: Eugene F Schuster, Hui Xiao, Elena Lopez-Knowles, Lucy Kilburn, Mothaffar Rimawi, David A Wheeler, Katherine Pogue-Geile, Yuan Lui, Samuel A Jacobs, Chet Cornman, Shannon Puhalla, Maggie Cheang, Judith Bliss, Stephen Johnston, Mitch Dowsett, On behalf of the PALLET Trialists. Biomarkers of resistance to palbociclib in ER+ primary breast cancer in the PALLET trial [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 PS5-01.

It is now possible to prevent many breast cancers inhigh-risk women using anti-estrogen hormonal drugs. Several Phase III breast cancer preventiontrials demonstrated that hormonal drugs such as the “selective estrogenreceptor modulators” tamoxifen and raloxifene, and aromatase inhibitors preventthe development of 50% of ER-positive breast cancers in women. However, mostwomen at high risk of breast cancer do not use these cancer preventive drugsdue to concerns about their side effects. To overcome this problem, recent studies havefocused on testing novel approaches for the prevention of breast cancer. Results of preclinical and clinical studiestesting targeted drugs and vaccines for breast cancer prevention show that itis possible to prevent breast cancer with reduced toxicity. Studies seeking to prevent all forms ofbreast cancer, including ER-positive, HER2-positive, and “triple-negative”breast cancer, will be presented and discussed. Novel approaches testing targeted therapies including signalinginhibitors, combinations of preventive agents, and vaccines, will be presented. Many of these interventions prevent breastcancer in animal models, and are now being tested in human clinicaltrials. Results from studies testing novel drugdelivery approaches will also be discussed. Topical gels containing anti-estrogen drugs are being used to reducebreast cell growth and mammographic density in Phase II clinical trials. This topical approach has the potential toprevent ER-positive breast cancers without the toxicity of oral hormonaldrugs. All of these novel preventive measuresoffer great promise to effectively prevent all forms of breast cancer withminimal toxicity. Citation Format: P Brown. Beyond Hormones: Newer Pharmacologic Approaches [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 SP020.

Background: Endocrine therapies have been associated with an overall survival benefit in MBC. However, a majority of patients eventually develop endocrine resistance and the factors associated with short versus long term response to endocrine therapies have not been well defined. These factors include key genomic alterations as well as alterations in steroid metabolism pathways. Gut bacteria play an important role in estrogen metabolism and may thus impact response to endocrine-based therapies. Our study aims to investigate both genomic and gut bacterial profiles in patients treated with endocrine therapies in MBC to further elucidate potential mechanisms of acquired endocrine resistance. Specific Aims: (1) To examine mutational profiles in cell free DNA of ER+/HER2- MBC patients treated with Aromatase Inhibitors (AI) + CDK 4/6 inhibitors to identify common oncogenic alterations associated clinical outcomes. (2) To identify gut bacterial profiles predictive of short versus long term response to endocrine therapy with AI + CDK 4/6 inhibitors. (3) To identify dietary factors associated with clinical outcomes on AI + CDK 4/6 inhibitor treatment. Patients and Methods: This is a prospective pilot study with the aim of collecting blood and fecal samples for biomarker analysis. Patients with advanced ER+/HER2- breast cancer initiating on standard of care first line palliative systemic therapy with an AI in combination with a CDK 4/6 inhibitor will be screened for eligibility. Patients with relapse on prior endocrine therapy or within 6 months of discontinuation of prior adjuvant endocrine therapy as well as patients with a history of inflammatory bowel disease, chronic diarrhea, malabsorption syndromes and prior bowel resection will be excluded. Taxonomic composition of fecal samples will be assessed by 16S rRNA gene microbiota sequencing. A subset of samples will undergo shotgun metagenomic sequencing. Targeted qPCR will be used to quantitate total bacterial density (by 16S rRNA gene qPCR), as well as genus/ species identified from published studies. Molecular alterations in cell free DNA extracted from peripheral blood samples will be analyzed using the Oncomine (ThermoFisher) Breast cfDNA assay. Droplet digital PCR assays will be designed to investigate common hotspot mutations in additional genes of interest. Dietary information will be assessed through standardized food frequency questionnaire administered at the start of treatment. Study outcomes will be mainly exploratory and hypothesis-generating. The sample size of 20 patients was determined in order to formulate an acceptable confidence interval (>95%) and to account for drop-outs and patients with insufficient number or quality of samples. The primary outcome is to compare genomic and gut bacterial profiles in patients with short ( Citation Format: Rossanna C. Pezo, Andrea Eisen, Sonal Gandhi, Ellen Warner, Katarzyna Jerzak, Maureen Trudeau, Arun Seth. Analysis of genomic alterations in cell free DNA and gut bacterial diversity in metastatic breast cancer (MBC) patients on endocrine therapy: A pilot study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-04.

Aromatase inhibitors (AI) induce painful musculoskeletal symptoms (AIMSS), which are dependent upon the pain transducing receptor TRPA1. However, as the AI concentrations required to engage TRPA1 in mice are higher than those found in the plasma of patients, we hypothesized that additional factors may cooperate to induce AIMSS. Here we report that the aromatase substrate androstenedione, unique among several steroid hormones, targeted TRPA1 in peptidergic primary sensory neurons in rodent and human cells expressing the native or recombinant channel. Androstenedione dramatically lowered the concentration of letrozole required to engage TRPA1. Notably, addition of a minimal dose of androstenedione to physiologically ineffective doses of letrozole and oxidative stress byproducts produces AIMSS-like behaviors and neurogenic inflammatory responses in mice. Elevated androstenedione levels cooperated with low letrozole concentrations and inflammatory mediators were sufficient to provoke AIMSS-like behaviors. The generation of such painful conditions by small quantities of simultaneously administered TRPA1 agonists justifies previous failure to identify a precise link between AIs and AIMSS, underscoring the potential of channel antagonists to treat AIMSS. Cancer Res; 76(23); 7024–35. ©2016 AACR.

Background: CR1447 (4-OH-testosterone, 4-OHT), a steroidal small molecule, strongly binds to the androgen receptor (AR) and has aromatase inhibiting activity. Pre-clinical studies show that CR1447 given as an ointment is efficiently absorbed and has antiproliferative activity in both ER-positive and ER-negative/AR-positive breast cancer cells. Methods: CR1447 was administered topically on a daily basis to patients with ER-positive/HER2-negative or ER-negative/PR-positive/HER2-negative advanced breast cancer pretreated with several lines of therapy. One cycle was defined as 21 days of treatment. Disease evaluation was performed at 3 and 6 months in order to determine tumor response (i.e. complete/partial remission and stable disease) in 3 cohorts of 3 evaluable patients each plus 3 confirmatory patients (dose escalation 100, 200, 400 mg). Results: 14 patients have been treated for a total of 38 cycles. Two patients are still on treatment at the time of analysis. Two patients, one in cohort 1 and one in cohort 2, showed early tumor progression and were replaced. Related adverse events were all ≤ grade 2 and included fatigue, bone and joint pain, stiffness, dry skin and mouth, nausea, sweating, urinary tract infection, rash, headache and distress. No drug-related dose limiting toxicities (DLTs) were seen. Two patients (17%) achieved stable disease at 3 months. Pharmacokinetic analysis confirmed dose-dependent transdermal uptake of CR1447, resulting in sufficient plasma concentrations of 4-OHT. 4-OH-androstenedione, a key metabolite of 4-OHT, was undetectable in most of the plasma samples. Conclusions: CR1447 administered transdermally as an ointment is well tolerated and appears to have single-agent activity in heavily pretreated ER-positive/HER2-negative and ER-negative/PR-positive/HER2-negative breast cancer patients. The recommended phase II dose is 400 mg/day. Citation Format: Schoenfeld W, Zweifel M, Thuerlimann B, Riniker S, Weder P, von Moos R, Pagani O, Bigler M, Rothgiesser KM, Pilop C, Brauchli P, Tapia C, Sessa C. Phase 1 evaluation of the androgen receptor modulator CR1447 in patients with advanced breast cancer (SAKK 21/12). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-14-05.

Background: Anastrozole (A) blocks estrogen production by inhibiting the activity of CYP19 aromatase. Fulvestrant (F) blocks estrogen receptor (ER) signaling by competitive binding, leading to ER degradation by ubiquitination. SWOG S0226 ("Phase III Randomized Trial of Anastrozole versus Anastrozole and Fulvestrant (250mg LD) as First Line Therapy for Post Menopausal Women with Metastatic Breast Cancer," ClinicalTrials.gov Identifier:NCT00075764) demonstrated that combination of A+F is superior to A alone as first-line therapy for patients with ER positive metastatic breast cancer (Mehta et al, NEJM, 2012). Our functional preclinical studies have shown that single nucleotide polymorphisms (SNPs) in SULT1A1 and UGT1A4, drug conjugation enzymes that inactivate A and F, result in decreased enzyme activity toward these drugs (Edavana et al, DMD, 2013; Edavana et al Pharmgenomics Pers Med 2013). We therefore hypothesized that these SNPs will be associated with disease outcomes in S0226 patients due to altered drug levels. Methods: Germline DNA was available for 295 (43.5%) patients enrolled in S0226 overall (157 on A and 138 on A+F). SNPs in SULT1A1 and UGT1A4 were determined either by direct sequencing or allele-specific PCR (TaqMan) assays. Results: There was no difference in progression-free survival (PFS) or overall survival (OS) comparing patients with or without available germline DNA (p = 0.86 and 0.36, respectively). The SULT1A1 G902A allele (rs6839), which confers decreased mRNA and enzymatic activity, was associated with improved PFS (GG/GA vs. AA; HR 0.74, 95% CI 0.56-0.98, p=0.033) and OS (HR 0.70, 95% 0.50-0.98, p=0.039). In exploratory subset analyses of PFS, the SULT1A1 G902A association was similar across both treatment arms (A HR=0.75; 95% CI 0.51-1.10; A+F HR=0.73; 95% CI 0.48-1.11). For OS there was some evidence of a difference by treatment (A HR=0.60; 95% CI 0.38-0.96; A+F HR=0.82; 95% CI 0.50-1.32), though no significant interaction was evident (p=0.30). The UGT1A4 G-163A promoter variant, which leads to decreased protein expression, was not associated with PFS (AA/AG vs. GG HR 0.88, 95% CI 0.68-1.14, p=0.33); however, this variant was associated with OS (HR 0.71, 95% CI 0.52-0.96, p=0.027). In subset analyses with OS, the difference was marginally stronger in the A arm (HR 0.63, 95% CI 0.42-0.97, p=0.035) compared to the A+F arm (HR 0.77, 95% CI 0.49-1.21, p=0.25), though the interaction was not significant (p=0.40). Conclusion: SULT1A1 and UGT1A4 gene variants resulting in decreased enzyme activity were associated with better PFS, OS or both in patients enrolled in SWOG S0226. Planned validation studies correlating these SNPs with drug levels and disease outcomes in additional patient cohorts will establish their clinical utility in identifying patients who benefit from A and F alone or in combination. Funding: Supported by NIH/NCI CA118981; NIH/NCI/NCTN grants CA180888, CA180819, and CA180863; and in part by AstraZeneca. Citation Format: Kadlubar SA, Barlow WE, Mehta RS, Daniels JR, Albain KS, Vandengerg TA, Dakhil SR, Tirumali NR, Lew DL, Gralow JR, Livingston RB, Hortobagiyi GN, Hayes DF, Rae JM. Association between gene variants in SULT1A1 and UGT1A4 and disease outcomes in patients enrolled in SWOG S0226 and treated with anastrozole alone or in combination with fulvestrant for metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-64.

Mammographic breast density as a predictor of breast cancer recurrence: a single centre longitudinal analysis of women with hormone receptor positive breast cancer Background Mammographic breast density has been associated with risk of development of breast cancer. To date clinical studies examining the use of tamoxifen and fall in mammographic breast density during this treatment have shown reduction in mammographic breast density examining change in mammographic breast density between baseline and a single follow-up mammogram to be predictive for disease recurrence. Aim To examine serial change in mammographic breast density over years to describe the changes which occur with use of aromatase inhibitors and tamoxifen, as well as changes following cessation of this treatment and to determine whether changes observed correlate with outcome. Method Eligible patients were identified from the Royal Perth Hospital breast unit database between January 1994-December 2011. Patient data was prospectively collected through the breast unit database. Additional data regarding endocrine therapy, adherence, weight, height and concomitant medications were obtained from case note review. Recurrence data was obtained from the hospital medical records system, as well as the breast unit database. Mammograms were obtained and mammographic breast density readings undertaken by a single reader using Cumulus. Percentage breast densities were obtained and statistical analysis undertaken to investigate changes in mammographic density on endocrine therapy, at switch of therapy, and cessation of therapy and correlation with disease free and overall survival. Results 1942 eligible patients were identified. 417 were premenopausal at time of diagnosis, 148 perimenopausal, 1328 postmenopausal and the remainder unknown status. 12 declined adjuvant endocrine therapy, 520 received both at least 1 aromatase inhibitor and tamoxifen during follow-up, 1189 tamoxifen only, 56 tamoxifen plus goserelin, and the remainder either aromatase inhibitor only or aromatase inhibitor with ovarian suppression. Over 10,000 mammograms were obtained for analysis. Currently results are available from 4301 mammograms from 689 patients. Mean density change between baseline scan and subsequent imaging after between 11-24 months of patient-reported endocrine adherence was -6.0%, with mean reduction of -11% in patients who were premenopausal at baseline and -4.5% in those who were postmenopausal at baseline. Kaplan Meier analysis showed late separation of overall survival curves favouring those with reduction in mammographic breast density however there was no statistically significant difference in the curves Conclusion Reduction in mammographic breast density was greatest in those who were premenopausal at baseline. Further multivariate analysis and assessment of the additional mammograms in this data set is required to assess the association between mammographic breast density and outcome in this cohort. Citation Format: Redfern AD, Martin HL, Stone J, Davidson JA, Yap F, Chung K. Mammographic breast density as a predictor of hormone receptor positive breast cancer recurrence: A single centre longitudinal analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD1-06.

Background: Aromatase inhibitors (AIs) have an established role in the treatment of estrogen receptor alpha positive post-menopausal breast cancer. Recently we have developed and validated a microarray-derived 4-gene test (Edinburgh EndoResponse4) to predict response to AIs in the neoadjuvant setting. We have also demonstrated the translational potential of this test in predicting accurately clinical response when mRNA is measured for these genes by polymerase chain reaction (PCR) or the gene protein is measured by immunohistochemistry (IHC). There is a major clinical need for biomarkers to predict which patients are likely to recur on adjuvant endocrine therapy so alternative or additional treatments can be provided to reduce recurrence and improve outcome. The aim of this study was to determine if Endoresponse4 and IHC of these gene proteins could do this. Methods: The original microarray assay used pre- and on-treatment (14-days) biopsies from 73 post-menopausal women with ER-rich breast cancer receiving 3 months of neoadjuvant letrozole prior to surgery with 10 years follow-up after adjuvant letrozole. Matched formalin-fixed paraffin embedded (FFPE) tissue sections from 42 of these patients were used for IHC and antibodies were optimised against 3 of the 4 proteins (where validated antibodies were available) using Envision technology. The ability of our test to estimate recurrence-free (RFS) and breast cancer specific overall survival (OS) using both PCR and IHC was then tested in a unique validation cohort of 140 post-menopausal women with ER-rich breast cancer treated with 2 weeks of neoadjuvant letrozole or anastrozole prior to surgery followed by adjuvant endocrine therapy and 10 years of follow up.. Results: Within our training cohort (n=73) using Kaplan-Meier analysis our 4-gene test predicted neoadjuvant clinical response and demonstrated a significant association with both RFS (P=0.029) and OS (P=0.009). This approach predicts outcomes within 2-weeks rather than 4-months of treatment required in other studies such as P024. Using IHC in the training cohort (n=42), two gene markers in combination (IL6ST at diagnosis and MCM4 after 2-weeks treatment) predicted both RFS (P=0.017) and OS (P=0.009) with great accuracy. The 140 patient group is being analysed and the findings are so far are consistent with the initial training cohort and indicate a significant association with outcomes. Conclusion: • A 4 gene model with clinical potential has been developed and validated to predict response to neoadjuvant aromatase inhibitors. • This 4 gene model predicts for response and also predicts relapse free and overall survival. • Proteins encoded by 2 of these 4 genes measured by IHC in an initial test set of 42 patients predict accurately both EFS and OS • A validation cohort (n=140) with over 10-years of follow-up will be available at SABCS 2015 to determine if this 2 biomarker test can predict outcome on adjuvant endocrine therapy. Citation Format: Turnbull AK, Arthur LM, Webber V, Thomas J, Uddin S, Webb H, Dunbier A, Dowsett M, Renshaw L, Sims AH, Dixon JM. A validated test for neoadjuvant clinical response to endocrine therapy in breast cancer that estimates accurately recurrence-free and overall survival. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-20.

Introduction: The association between smoking and breast cancer prognosis has been investigated in several studies but remains unclear. To our knowledge, no study has investigated whether the response to different endocrine treatments differs between smokers and non-smokers. Smoking can suppress aromatase activity, but also increase inflammation, which may lead to higher activity. The aim was to investigate whether preoperative smoking was associated with risk of breast cancer events in endocrine-treated patients. Patients and methods: This population based cohort consisted of 1026 female breast cancer patients with invasive tumors and no preoperative treatment who were enrolled in an ongoing prospective cohort study at Skåne University Hospital in Lund between October 2002 and June 2012. Pre- and postoperative questionnaires regarding lifestyle factors, including smoking status, and treatments were completed. Information on tumor characteristics, treatments, and dates for new breast cancer events or deaths was obtained from pathology reports, patients' charts and population registers. A breast cancer event was defined as local or regional recurrence, contralateral breast cancer, or distant metastasis. Results: For the survival analyses, two patients were excluded due to missing information on smoking and eight patients were excluded due to metastatic spread within 0.3 years of inclusion, leaving 1016 patients of which 206 (20.3%) reported preoperative smoking. Less than 1% of the 810 preoperative non-smokers reported smoking at either the 3-6-months or 1-year postoperative visits, while about 10% of the patients who smoked preoperatively reported not to smoke during the follow-up visits. Thus, the majority of the patients did not switch smoking status. Patients were followed for up to 11 years (median 5.1 years for patients still at risk). Overall, there was no significant association between smoking and risk of breast cancer events (adjusted hazard ratio (HR) 1.45: 95% CI 0.95-2.20; P=0.08) adjusted for patient and tumor characteristics. For the 408 tamoxifen-treated patients aged 50 years or older with estrogen receptor positive tumors, smoking was not significantly associated with risk for early events (adjusted HR 1.58: 95% CI 0.76-3.30; P=0.22). However, for the 309 aromatase inhibitor-treated patients aged 50 years or older with estrogen receptor positive tumors, smoking was significantly associated with a 3-fold increased risk of breast cancer events (adjusted HR 2.97: 95% CI 1.44-6.12; P=0.003). Some patients had been treated with sequential tamoxifen and aromatase inhibitor therapy. Conclusions: Preoperative smoking was associated with a significantly increased risk for breast cancer events among patients treated with aromatase inhibitors, but not among tamoxifen-treated patients. If confirmed, smoking status should be taken into consideration when selecting endocrine therapy. Citation Format: Jernström H, Persson M, Simonsson M, Markkula A, Rose C, Ingvar C. Tamoxifen or aromatase inhibitors: Should smoking status impact on selection of endocrine therapy in breast cancer patients?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-29.

VT-464 is a lyase-selective inhibitor of the dual-activity CYP17A1 enzyme that is required for the synthesis of androgens and estrogens in the gonads, adrenals, and tumors. In addition to its role as a CYP17A1 lyase inhibitor, we previously showed that VT-464 also functions as a direct and effective AR antagonist in prostate cancer models. In our current study, we evaluated the therapeutic potential of VT-464 in breast cancer by analyzing its effectiveness in several AR-positive breast cancer cell lines, including those that are ER-positive and ER-negative. Through in vitro assays and xenograft analysis, we compared the activity of VT-464 to enzalutamide, a second generation AR antagonist that is approved for the treatment of castration resistant prostate cancer and is in multiple Phase 2 breast cancer studies. Our results showed that VT-464 was highly effective in preventing proliferation of both ER-positive and ER-negative breast cancer cell lines in vitro. Importantly, significant inhibition was also observed in soft agar assays that assesses anchorage-independent growth. In mechanistic studies, VT-464 and enzalutamide both inhibited induction of AR target genes and recruitment of AR to target promoters, verifying direct AR antagonistic activity observed previously in prostate cancer cells. Furthermore, we evaluated the ability of VT-464 and enzalutamide to inhibit tumor formation in a tamoxifen-resistant model of breast cancer. Oral administration of either enzalutamide or VT-464 significantly decreased tumor growth in mice, with VT-464 achieving greater growth inhibition than enzalutamide. Together, these data provide further rationale for the future study of the AR as a viable therapeutic target in breast cancer, and importantly, suggest that AR inhibition can impact tamoxifen-resistant tumor growth. The dual effects of CYP17A1 lyase inhibition and AR antagonism that are achieved with VT-464 further supports its development as an effective oral therapy option for AR-positive breast cancer. A phase 1 / 2 clinical study of oral VT-464 in women with AR+ triple-negative breast cancer or ER+ cancer resistant to aromatase inhibitors will commence in 2015. Citation Format: Ellison SJ, Norris JD, Wardell S, Eisner JR, Hoekstra WJ, Stagg DB, Alley HM, Moore WR, McDonnell DP. Effects of the dual selective CYP17 lyase inhibitor and androgen receptor (AR) antagonist, VT-464, on AR+ and ER+ tumor models in vitro and in vivo. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-04.

Introduction: Obesity is associated with poor breast cancer outcomes in postmenopausal women in response to aromatase inhibitor therapy. Our prior studies have shown an association between reduced recurrence rate and use of cyclooxygenase-2 (COX-2) inhibiting non-steroidal anti-inflammatory drugs (NSAIDs) in obese breast cancer patients. The mechanism proposed was a decrease in prostaglandin E2 (PGE2) and reduced activation of the aromatase promote locally in the breast. Methods: Postmenopausal women of varying body habitus were recruited at the CTRC in San Antonio and underwent randomized assignment to 1 of 3 arms: ASA 81mg daily, 1500mg of docosahexaenoic acid (DHA) and 2500mg eicosapentaenoic acid (EPA) given daily, or combined ASA and DHA/EPA. Sera were collected prior to and following 28 days of exposure, and cytokines including prostaglandin E2 were assessed via enzyme-linked immunosorbant assay (ELISA). Conditioned media was generated by exposing macrophages to patient sera in order to see if the patient sera induced PGE2 concentration in vitro. Results: Thirty of the planned 120 subjects have completed assessment. No toxicity has been noted. In 71% of the patients, serum PGE2 levels decreased, but only 60% demonstrated concurrent decrease in serum PGE2 levels as well as macrophage PGE2 production, while almost all (88%) of the patients whose serum did not demonstrate a decrease in PGE2 levels also demonstrated no decrease in induced levels. Conclusion: NSAIDs appear to effectively decrease circulating levels of PGE2 in most obese women. However, one third of the subjects did not demonstrate concurrent suppression of induced PGE2 from macrophages. These data suggest that circulating levels of PGE2 may not be reflective of local tumor microenvironment levels, and other pro-inflammatory circulating factors may be responsible for regulating local inflammatory responses. Final analysis will be completed and presented at the SABCS meeting. Citation Format: Lengfelder L, Brenner A, Bowers L, Apte S, Galván G, Kist K, deGraffenried L. Phase 0 study evaluating COX2 inhibition on circulating PGE2 levels from obese subjects. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-12.

Background. The estrogen receptor (ER)/ GATA3/ Forkhead box A1 (FOXA1) network is necessary for the ERα functional signature specific to luminal type breast cancers. High expression of FOXA1 indicates a good prognosis in ER-positive breast cancer. However, little is known about the association between the expression of FOXA1 and GATA3, and the efficacy of neoadjuvant endocrine therapy (NAE). This study investigated their predictive potential for NAE and the changes of their expression after NAE. Methods. The expression of ER, progesterone receptor (PgR), Ki67, FOXA1, and GATA3 were analyzed by immunohistochemistry in 66 patients with hormone receptor-positive/ human epidermal growth factor receptor 2 (HER2)-negative breast cancer who had been treated with NAE between March 2003 and December 2012 at Kyushu University Hospital, National Kyushu Cancer Center, and Sagara Hospital. The association between the expression of biological marker and the efficacy of NAE, and their expression changes after NAE were evaluated. Results. The median age of the patients was 60 years (range, 30–84 years). Pre- and post-menopausal patients were 24 (36.4%) and 42 (63.6%). Endocrine agents that were administered are as follows: aromatase inhibitors (AIs) for 42 patients (63.6%), luteinizing hormone-releasing hormone (LHRH) agonist plus AI for 10 patients (15.2%), LHRH agonist plus tamoxifen for 13 patients (19.7%). NAE yielded a partial response (PR) in 21 patients (31.8%) and stable disease (SD) in 45 patients (68.2%). Breast conserving surgery was performed in 56 patients (84.8%) and mastectomy was performed in 10 patients (15.2%). Preoperative Endocrine Prognostic Index (PEPI) score was 0 in 10 patients (15.2%) and 1 or greater (score 1 ≤) in 56 patients (84.8%). Pre-treatment FOXA1 expression was positively correlated with GATA3 (P = 0.0003) and PgR (P = 0.0138). Post-treatment Ki67 expression was significantly lower in tumors, which achieved PR compared with those with SD (P = 0.0007). The expression of PgR, Ki67, and FOXA1 was significantly lower in post-treatment tumors compared with those in pre-treatment samples (p < 0.0001, p < 0.0001 and p < 0.0001, respectively). The expression of PgR, Ki67, and FOXA1 was significantly reduced in both tumors with PR and those with SD (PR: P = 0.0004, P < 0.0001, and P = 0.0417, respectively; SD: P < 0.0001, P = 0.0001, and P < 0.0001, respectively). The expression of PgR, Ki67, and FOXA1 was significantly decreased in post-treatment tumors in both patients with the PEPI score 0 and those with score 1 ≤ (score 0: P = 0.0078, P = 0.0059, and P = 0.0098, respectively; score 1 ≤: P < 0.0001, P < 0.0001, and P = 0.0002, respectively). In tumors with PgR > 20%, the expression of Ki67 and FOXA1 were significantly lower in post-treatment tumors (P < 0.0001 and P < 0.0001, respectively). Conclusions. FOXA1 expression correlated with PgR expression, and was reduced significantly after NAE. These results suggest that blocking the effect of estrogen might reduce FOXA1 expression. Citation Format: Tanaka K, Tokunaga E, Inoue Y, Ueo H, Yamashita N, Sagara Y, Ohi Y, Taguchi K, Ohno S, Okano S, Kitao H, Oki E, Oda Y, Maehara Y. The relationship between the expression of FOXA1 and GATA3 and the efficacy of neoadjuvant endocrine therapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-13-05.

Chemoprevention is a risk reduction option for women who have increased risk of breast cancer. Selective Estrogen Receptor Modulators (SERMs) have been extensively tested, and alternative agents are being evaluated. Long-term adherence to chemoprevention is critical to obtaining the drug's full benefit. We systematically reviewed articles reporting uptake rates and adherence among healthy adult women, who were prescribed medication to prevent primary breast cancer. We also extracted data on the clinical, socio-demographic and psychological predictors of uptake and adherence. Searches were performed in PubMed, CINAHL, EMBASE, and PsychInfo, yielding 3851 unique articles. Title, abstract and full text screening left 53 articles that met inclusion criteria, and a further 4 studies were identified from reference lists, giving a total of 57. The mean quality score using the Mixed Methods Appraisal Tool was 3 out of 4. Thirty-one articles reported uptake, of which 14 tested predictors, and 23 reported adherence of which 11 tested predictors. Seven studies reported qualitative data. Most studies (50) involved SERMs, but 5 tested Aromatase Inhibitors, 1 tested Aspirin, 1 tested a statin. Twenty studies included data from a clinical setting, 35 reported trial data, and 2 reported both. Twenty-four studies reporting 26 instances of uptake in 21,423 women were included in a meta-analysis. The pooled uptake estimate was 16.3% (95% CI, 13.6-19.0), with high heterogeneity (I^2=98.9%, p Chemoprevention uptake for the prevention of breast cancer is low, and long-term adherence is often insufficient for the full preventive effect. Uptake rates were higher in trials than in clinical settings, suggesting further work should focus on implementing chemoprevention within routine patient care. Further research is warranted to identify factors amenable to modification and to improve informed decision-making surrounding chemoprevention. Citation Format: Smith SG, Sestak I, Forster A, Partridge A, Side L, Horne R, Wardle J, Cuzick J. Factors affecting uptake and adherence to breast cancer chemoprevention: A systematic review and meta-analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD1-08.

[][1] On June 7, 2017, the cancer research community lost Professor Angela MH Brodie, PhD, FAACR, the “Mother of Aromatase Inhibitors.” The discovery of specific aromatase inhibitors (AI) is a major advance in targeted therapeutics. Three cheap orally active agents, anastrozole

The phosphoinositide 3-kinase (PI3K) pathway is a key driver of hormone receptor (HR)–positive breast cancer growth and survival. It is estimated that 40-45% of HR+ breast cancers harbor oncogenic mutations in the PIK3CA gene, which encodes the p110α isoform of PI3K. Taselisib (GDC-0032) is a mutant-selective PI3K inhibitor that demonstrates enhanced potency in PIK3CA mutant breast cancer cells and is being developed as a treatment for metastatic breast cancer that targets PIK3CA-mutant, HR-positive, HER2-negative patients. Activating mutations in the ESR1 gene were recently described in metastatic breast cancer. These mutations confer hormone independent growth and may be associated with resistance to aromatase inhibitors. Drugs that selectively bind and antagonize the Estrogen Receptor alpha (ERα) protein and target it for degradation, such as fulvestrant, are referred to as selective estrogen receptor degraders (SERDs). Preclinical activity of the orally bioavailable SERD, GDC-0810, has not been well characterized in ESR1 mutant PDX models. Therefore, our aim was to evaluate the efficacy and pharmacodynamic responses to agents that target ERα and PI3K as monotherapies and in combination, in ESR1 and PIK3CA mutant HR+ breast cancer patient-derived xenograft (PDX) models. We hypothesized that mutational status of ESR1 and PIK3CA may predict the responsiveness of HR+ PDX models to SERDs and PI3K inhibitors in vivo. Characterization of seven PDX models included authentication of hormone receptor status by immunohistochemistry (IHC) and determination of ESR1 and PIK3CA genotype and allele frequency by exome sequencing. For a subset of models that utilize estrogen for growth, mice were supplemented with 17β-estradiol, and cells or tumor fragments were implanted into the fat pad of intact female NOD-SCID or NOD-SCID-IL2Rgamma null mice and treated with fulvestrant, GDC-0810, or taselisib. Both fulvestrant and GDC-0810 were efficacious in ESR1 wild type (WT) and mutant PDX models but to variable degrees ranging from tumor stasis to growth delay, with GDC-0810 resulting in superior single agent activity at relevant clinical exposure in the WHIM20 and WHIM43 ESR1 mutant models. PIK3CA mutations (E542K, E545K, M1004V, and H1047R) were confirmed in six PDX models and PI3K pathway activation verified by strong pS6RP IHC staining. Taselisib induced tumor growth inhibition and tumor regressions in models harboring PIK3CA mutations, and models with no detectable expression of WT p110α were the most sensitive. In the WHIM43 (ESR1 D538G, PIK3CA M1004V), HCI-011 (ESR1 WT, PIK3CA E545K) and HCI-013 (ESR1 Y537S, PIK3CA H1047R) PDX models, combining fulvestrant and taselisib treatment further enhanced tumor growth inhibition with respect to either treatment alone. Our studies demonstrate the diverse anti-tumor responses of HR+ PDX models to SERDs and the PI3K inhibitor taselisib in the context of clinically relevant ESR1 and PIK3CA mutations. Pharmacological and genomic characterization of additional PDX models may aid in strengthening associations between genotype, drug sensitivity and predictive biomarkers of response. Citation Format: Young A, Crocker L, Cheng E, Lacap J, Hamilton P, Oeh J, Ingalla E, Arrazate A, Hager J, Nannini M, Friedman L, Daemen A, Giltnane J, Sampath D. Treatment of ESR1 mutant and PIK3CA mutant patient-derived breast cancer xenograft models reveals differential anti-tumor responses to estrogen receptor degraders and PI3K inhibitors in vivo [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 P4-06-05.