Mechanisms Of Endocrine Therapy Resistance Research Articles

Abstract PO1-23-10: Multi-omics analysis identifies the mechanism in cross-resistance to endocrine therapy and CDK4/6 inhibitor in breast cancer

Abstract Background In advanced/metastatic breast cancer (MBC) or recurrent breast cancer on prior endocrine therapy (ET), selective estrogen receptor downregulator (SERD) of fulvestrant and CDK4/6i are the mainstream therapy regimen. In the clinical, fulvestrant combined with CDK4/6i is the second-line therapy for breast cancer (BC) that had progressed during previous endocrine therapy. The alternation of backbone from aromatase inhibitor or tamoxifen (endocrine sensitive) to fulvestrant (endocrine resistance) partially comes from the mechanism of ET resistance. Moreover, in the PALOMA-2 trial, approximately 30% of patients developed into recurrence after two years of CDK4/6i treatment, indicating that the ET backbone may affect CDK4/6i efficacy, bringing an emerging clinical issue of cross-resistance. However, whether the cross-resistance of ET would be more sensitive to CDK4/6i or impair the efficacy of CDK4/6i is still an ambiguous and heterogeneous issue. Also, in CDK4/6i resistance, whether the mechanism of ET resistance is associated with the dysregulation of the cell cycle or activation of other “bypass” signaling pathways is not fully understood. Methods We use a 3D and 2D culture of ET-sensitive cell S0.5, tamoxifen-resistant cell TAM, and fulvestrant-resistant cell 182R6 as our model. The 3D culture was performed by using the Tools 3d culture plus kit. Drug responses to fulvestrant and ribociclib were conducted by using CCK-8 assay. The potential target and pathway involved in the mechanisms of cross-resistance were analyzed by using multi-omics analysis of sequential window acquisition of all theoretical mass spectra (SWATH-MS), RNA-seq, micro-western array (MWA), traditional western blots, and open access database. Results In comparison with S0.5, the sensitivity to fulvestrant was indeed reduced in 182R6 cell; however, we found the sensitivity to ribociclib was also reduced in 182R6 cell, which was in contrast to the ribociclib-sensitive TAM cell. The different response to ribociclib between 182R6 and TAM, indicating that cross-resistance between fulvestrant and ribociclib existed in the model of 182R6 and TAM would be another control for 182R6 to find the target with or without cross-resistance. With multi-omics analysis of SWATH-MS, RNA-seq, MWA, western blots, and open access database, we found some unique or high expression of targets and pathways including EGFR, HER2, CDK6, CDK2, MAPK, and TNF-α pathways in cross-resistance R6 cell than in parental and TAM cell. We reconfirmed the above targets by 3D culture and found MAPK signaling pathway of p-ERK was not affected under ribociclib treatment, indicating the important role of the MAPK signaling pathway in ribociclib resistance. Conclusion To our knowledge, these results provide the first preliminary mechanism of cross-resistance between fulvestrant and ribociclib. We found fulvestrant resistant cell R6 exhibited the phenomenon of cross-resistance to ribociclib, which was in contrast to ribociclib-sensitive TAM cell. We also found some unique or high expression of target and pathway in cross-resistance R6 cells than in parental and TAM cells. In the future, with compound library screening and in vivo animal models, we expect to discover an FDA-approved drug or potential compound to overcome the cross-resistance between fulvestrant and ribociclib. Citation Format: Ming-Feng Hou, Chung-Liang Li, Chih-Po Chiang. Multi-omics analysis identifies the mechanism in cross-resistance to endocrine therapy and CDK4/6 inhibitor in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-23-10.

Abstract PS17-04: Clinical and Genomic Features of ER-Positive/HER2-negative Metastatic Breast Cancer in AURORA Molecular Screening Initiative (BIG 14-01): Mechanisms of Endocrine Therapy Resistance and Implications for Adjuvant Approaches

Abstract Background: Up to 30% of patients (pts) with ER-positive early breast cancers develop metastatic relapse. The molecular differences between endocrine therapy (ET)-sensitive and ET-resistant relapses, as well as the impact of specific adjuvant ET-based therapies, remain unclear. Methods: The AURORA program (NCT02102165) analyzed multi-omics data of paired primary (prim) and metastatic (meta) tumor tissue, along with plasma samples, from 1,156 patients with metastatic breast cancer (MBC). Targeted genome sequencing (TGS), RNA sequencing, and circulating tumor DNA analysis were performed. ET-resistance at MBC diagnosis was defined following the 5th ESO-ESMO ABC Guidelines. Results: We studied 628 pts with metastatic ER+/HER2- disease. Patient´s median age was 56 years, 133 (21%) were premenopausal, 314 (50%) had ET-resistance at recurrence (9% primary, 41% secondary), 132 (21%) had ET-sensitive recurrence, and the rest were ET-naive recurrences or de novo MBC. Adjuvant treatment was aromatase inhibitor (AI) +/- ovarian function suppression (OFS) in 288 (46%) pts, while 159 (25%) had tamoxifen only (+/- OFS). Gene expression correlation significantly differed (p < 0.005) based on ET-resistance in 92 paired samples. ET-sensitive (n=11) and de-novo tumors (n=41) showed higher correlation than primary (n=7) and secondary ET-resistance (n=32), regardless of adjuvant ET type. Prim and meta showed concordance in 91% of IHC subtypes and 62% of intrinsic subtypes. Intrinsic subtype showed 9% of prim luminal tumors switching to meta non-luminal, with 66% of Luminal A switching to Luminal B. Neither IHC nor intrinsic subtype switching was associated with the type of adjuvant ET or ET resistance. Pts with meta non-luminal intrinsic subtype (18%) had worse PFS on CDK4/6i (HR 4.0, 95% CI 1.9-8.7) and OS (HR 3.6, 95% CI 2.0-5.7) than luminal subtypes. TGS was performed in 534 meta (365 before 1L, 305 pairs). In meta before 1L, the dN/dS algorithm revealed selection in 17 genes, including TP53, PIK3CA, and ESR1. Mutations (mut) in ESR1, ERBB2, ERBB3, and RB1 among others, were specifically identified in meta. The incidence of ESR1mut was 3% in prim, 12.6% before 1L, 23.4% after 1L, and 4.7% in meta from ET-naive tumors (Table). Before 1L, ESR1mut was higher after adjuvant AI vs tamoxifen (21% vs. 4%, p< 0.001). ESR1mut incidence varied with ET-resistance (16% primary, 19% secondary, 9% sensitive, 4% ET-naïve/de novo, p< 0.01). Paired samples showed that ESR1mut were mainly acquired events and associated with higher ER mRNA signaling (hallmark estrogen response) compared to ESR1wt (p < 0.05). The agreement between ESR1mut detected in ctDNA and tissue was high before 1L (90%), and slightly lower after 1L (84%). In a multivariate model with relevant clinical factors, ESR1mut were associated with worse OS (HR 1.76, 95% CI 1.2-2.5, p=0.003), independently of TP53 and PIK3CA mutational status. Conclusion: AURORA study sheds light on metastatic tumor alterations acquired under anti-cancer therapy, in ER+/HER2- MBC. We observed a high prevalence of acquired ESR1mut prior to the initiation of first-line therapy, particularly in tumors exposed to adjuvant AI or with primary or secondary ET_resistance. The association of ESR1muts with poorer OS underscores the importance of implementing effective adjuvant ET strategies to prevent the emergence of these mutations. Frequency of driver gene mutations in primary and metastastic tumors before 1st-line treatment according to adjuvant ET and type of ET resistance Table. Citation Format: Angel Guerrero-Zotano, Matteo Benelli, Alexandre Irrthum, David Cameron, Lorenzo Ferrando, Dario Romagnoli, Marta Paoli, Arnau Llinas, Maya Dadiani, Danai Fimereli, Mafalda Oliveira, Carmela Caballero, Thayane Crestani, Elisa Agostinetto, Diogo Martins-Branco, Florentine Hilbers, Einav Gal-Yam, Marija Balic, Fatima Cardoso, Jorge Reis-Filho, Christos Sotiriou, Giuseppe Curigliano, Barbro Linderholm, Evandro de Azambuja, Susan Knox, Cristina Rotaru, Eva Ciruelos, Nancy E Davidson, Giuseppe Viale, Donatienne Taylor, Jean-Luc Canon, Lourdes Calvo, Joan Albanell, Marco Colleoni, Caroline Duhem, Nadia Harbeck, Catherine Herremans, Sibylle Loibl, Sandrine Marreaud, Elsemieke Scheepers, Alice Raimbault, Theodora Goulioti, Andrea Vingiani, Chiara Biagioni, Sebastian Vosberg, Gabriele Zoppoli, Jose Seoane, David Venet, Philippe Aftimos, Martine Piccart. Clinical and Genomic Features of ER-Positive/HER2-negative Metastatic Breast Cancer in AURORA Molecular Screening Initiative (BIG 14-01): Mechanisms of Endocrine Therapy Resistance and Implications for Adjuvant Approaches [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS17-04.

Abstract PO3-23-11: Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy

Abstract Background: 80% of all breast cancers (BCs) are ER-positive (ER+). A significant number develop endocrine therapy resistance (ETR) . Clones develop in lymph nodes (LNs) which differ from the primary and these are implicated in ETR. A multiomics analysis of primary ER+ breast cancers, matched LN metastasis (LN+), if present, and matched recurrences (primary, nodal or distant) has been performed. Patients and Methods: The patients included are: Cohort A: 95 patients with matched primary and recurrent ER+ BC on different endocrine therapies. 35% had involved LNs at diagnosis. Recurrences: local in 59/95, concurrent local and nodal in 33/95, LN-only in 3/95. Median time to recurrence 4.5 years. Cohort B: 18 patients with ER+ LN+ BC who developed multiple recurrences (local and/or nodal, >3 consecutive recurrences/patient) on different lines of sequential endocrine therapy. Cohort C: 7 patients with ER+ breast cancer with multiple involved LNs treated with 3-6 months of neoadjuvant ET before surgery. Biopsies of primary and matched LNs performed at diagnosis and throughout treatment (3-4 timepoints/patient). Clinical response by serial ultrasound. Primary and LN samples (mean 12 samples/patient) were analysed together with normal DNA. Targeted RNAseq and DNA exome sequencing and whole-genome expression analysis was performed. Somatic mutations and copy number alterations (CNA) were determined, and differential gene expression analysis was performed. Validation of pathways implicated in ETR was by spatial NanoString GeoMx. All patients have long term follow-up. Results: Cohort A: A mean of 6 potential driver mutations identified by DNA sequencing in each primary BC (range 1-16). Most commonly mutated genes were PIK3CA, TP53, ERBB4, MAP3K1 and ERBB3. Multiple aberrations and a highly diverse mutational landscape were observed in all recurrences. In the majority of recurrences significant changes in variant allele frequency (VAF) were identified across several ETR-implicated genes and in over 50% new gains/losses were identified. Most frequently, losses were seen in ERBB3, ARID1A, HRAS and FOXA1 with gains in CDH1, NF1, PIK3CA, ESR1, TBX3 and GATA3 in recurrences vs primaries. Transcriptomic analysis revealed differentially expressed pathways including ER, HER2, GATA3, AKT, RAS and p63 signalling. Integrated analysis revealed several driver mutations with corresponding changes in upstream pathway activity. 33 patients had concurrent local and nodal recurrences and 52% had similar somatic mutation profiles in both with only minor variance in VAF. In 48%, 1 or more new potential driver mutations was identified in axillary nodes compared to recurrent primary cancers with losses in AKT1, CDH1 and TP53 being most common. Multiomics profiling of Cohorts B and C is complete and analysis underway. Discussion: This multiomics study is the largest cohort to-date of matched early and recurrent ER+/HER2- cancers. It sheds new light on the complex somatic and transcriptomic changes in local and nodal recurrent disease. The mechanisms of ETR are diverse and underlying mechanisms and clinically meaningful biomarkers including potentially actionable mutations and targets have been identified. Cohort C will uniquely capture and compare clonal evolution of breast cancer in primary and multiple matched lymph during neoadjuvant ET. Citation Format: Carlos Martinez-Perez, Charlene Kay, Rebecca Swan, Lorna Renshaw, J Michael Dixon, Arran K Turnbull. Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-11.

ESR1 Gene Mutations and Liquid Biopsy in ER-Positive Breast Cancers: A Small Step Forward, a Giant Leap for Personalization of Endocrine Therapy?

The predominant forms of breast cancer (BC) are hormone receptor-positive (HR+) tumors characterized by the expression of estrogen receptors (ERs) and/or progesterone receptors (PRs). Patients with HR+ tumors can benefit from endocrine therapy (ET). Three types of ET are approved for the treatment of HR+ BCs and include selective ER modulators, aromatase inhibitors, and selective ER downregulators. ET is the mainstay of adjuvant treatment in the early setting and the backbone of the first-line treatment in an advanced setting; however, the emergence of acquired resistance can lead to cancer recurrence or progression. The mechanisms of ET resistance are often related to the occurrence of mutations in the ESR1 gene, which encodes the ER-alpha protein. As ESR1 mutations are hardly detectable at diagnosis but are present in 30% to 40% of advanced BC (ABC) after treatment, the timeline of testing is crucial. To manage this resistance, ESR1 testing has recently been recommended; in ER+ HER2- ABC and circulating cell-free DNA, so-called liquid biopsy appears to be the most convenient way to detect the emergence of ESR1 mutations. Technically, several options exist, including Next Generation Sequencing and ultra-sensitive PCR-based techniques. In this context, personalization of ET through the surveillance of ESR1 mutations in the plasma of HR+ BC patients throughout the disease course represents an innovative way to improve the standard of care.

Development of a Detection System for ESR1 Mutations in Circulating Tumour DNA Using PNA-LNA-Mediated PCR Clamping.

Although circulating tumour DNA (ctDNA)-based next-generation sequencing (NGS) is a less invasive method for assessing ESR1 mutations that are essential mechanisms of endocrine therapy resistance in patients with oestrogen receptor-positive breast cancer, adequate amounts of DNA are required to assess polyclonal ESR1 mutations. By combining a peptide nucleic acid and locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamping assay, we have developed a novel detection system to screen for polyclonal ESR1 mutations in ctDNA. A validation assay was prospectively performed on clinical samples and compared with the NGS results. The PNA-LNA PCR clamp assay was validated using six and four blood samples in which ESR1 mutations were detected by NGS and no mutations were detected, respectively. The PNA-LNA assay results were comparable with those of NGS. We prospectively assessed the concordance between the PNA-LNA PCR clamp method and NGS. Using the PNA-LNA PCR clamp method, ESR1 mutations were detected in 5 out of 18 samples, including those in which mutations were not detected by NGS due to small amounts of ctDNA. The PNA-LNA PCR clamping method is a highly sensitive and minimally invasive assay for polyclonal ESR1 mutation detection in the ctDNA of patients with breast cancer.

OR30-5 Androgen-Regulated Secreted Factors Promote Tumorigenesis and Immune Suppression in Breast Cancer

Abstract Background Breast cancer (BC) is the most diagnosed cancer and the second-leading cause of cancer-associated death in women in the United States. BCs that express the estrogen and/or progesterone receptor (ER/PR) are treated with ER-targeting endocrine therapies that effectively reduce disease burden and extend lifespan. However, up to 50% of patients with ER+BCs will experience recurrence, often at a metastatic site, within 10 years of diagnosis. Novel targeted therapies are needed to treat both metastatic ER+BC resistant to current treatment strategies and chemoresistant ER-BCs.We identified chitinase 3–like-1 (CHI3L1/BRP-39/YKL-40) and prolactin inducible protein (PIP/GCDFP-15) as androgen receptor (AR)-regulated in BC. PIP is reported to be detectable in >90% of BCs, but its function in BC is not well-characterized. CHI3L1 is a glycosyl hydrolase family 18 protein member with reported roles in aging and numerous pathologies. During mammary gland involution, CHI3L1 contributes to a pro-tumorigenic wound-healing-like environment and increases macrophage infiltration. Further, CHI3L1 correlates with tumor-suppressive M2 macrophage infiltration in ER-BC. We previously reported that ER+ cells and patient-derived xenografts with activating mutations in the ESR1 gene encoding ER—a known mechanism of endocrine therapy resistance—had elevated AR and CHI3L1 gene and protein expression. Based on the above and our finding that metastatic BCs biopsies harboring ESR1 mutations exhibited increased AR expression, we hypothesized that AR regulation of CHI3L1 and PIP promote progression to metastatic disease in part through immune suppressive effects. Methods A ConTra(V3) transcription factor binding site analysis was performed on CHI3L1 and PIP promoters. CHI3L1 and PIP were measured via immunohistochemistry in xenograft tumors in mice treated with or without the androgen dihydrotestosterone (DHT). Immunoblotting, immunohistochemistry, and immunofluorescence approaches were also utilized to assess CHI3L1 protein expression in DHT-treated, tamoxifen resistant (TAMR), and long term E2 deprived (LTED) ER+BC cells. CIBERSORT analysis was conducted on the Firehouse Legacy TCGA dataset. Macrophage efferocytosis assays were performed on an IncuCyte Live Cell Imaging system. Results We identified highly conserved AR binding sites in the CHI3L1 and PIP promoters. CHI3L1 protein was elevated in TAMR and LTED BC cells, which was further elevated in BC cells with ESR1 mutations, as well as DHT-treated cells. Further, CHI3L1 and PIP were increased in xenograft tumors after DHT treatment. CIBERSORT analysis revealed that CHI3L1 and PIP high tumors had altered immune profiles, including differential macrophage levels. Finally, CHI3L1 and PIP treatment significantly reduced macrophage efferocytosis of BC cells (p ≤ 0.0001). Conclusions Our work suggests that AR-mediated regulation of CHI3L1 and PIP may alter immune cell infiltration/function in BC and may be important in endocrine therapy resistant ER+BCs. Further characterization and manipulation of these AR-regulated proteins is underway to test the potential to target and reverse their immune suppressive functions. Presentation: Tuesday, June 14, 2022 10:45 a.m. - 11:00 a.m.

Comprehensive Transcriptomic and Proteomic Analyses Identify a Candidate Gene Set in Cross-Resistance for Endocrine Therapy in Breast Cancer.

Endocrine therapy (ET) of selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs), and aromatase inhibitors (AIs) has been used as the gold standard treatment for hormone-receptor-positive (HR+) breast cancer. Despite its clinical benefits, approximately 30% of patients develop ET resistance, which remains a major clinical challenge in patients with HR+ breast cancer. The mechanisms of ET resistance mainly focus on mutations in the ER and related pathways; however, other targets still exist from ligand-independent ER reactivation. Moreover, mutations in the ER that confer resistance to SERMs or AIs seldom appear in SERDs. To date, little research has been conducted to identify a critical target that appears in both SERMs/SERDs and AIs. In this study, we conducted comprehensive transcriptomic and proteomic analyses from two cohorts of The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) to identify the critical targets for both SERMs/SERDs and AIs of ET resistance. From a treatment response cohort with treatment response for the initial ET regimen and an endocrine therapy cohort with survival outcomes, we identified candidate gene sets that appeared in both SERMs/SERDs and AIs of ET resistance. The candidate gene sets successfully differentiated progress/resistant groups (PD) from complete response groups (CR) and were significantly correlated with survival outcomes in both cohorts. In summary, this study provides valuable clinical implications for the critical roles played by candidate gene sets in the diagnosis, mechanism, and therapeutic strategy for both SERMs/SERDs and AIs of ET resistance for the future.

Targeting ESR1 mutation-induced transcriptional addiction in breast cancer with BET inhibition.

Acquired mutations in the ligand-binding domain (LBD) of the gene encoding estrogen receptor α (ESR1) are common mechanisms of endocrine therapy resistance in patients with metastatic ER+ breast cancer. The ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1,200 Federal Drug Administration–approved (FDA-approved) drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib + fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with WT cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant–induced endocrine therapy resistance in breast cancer.

Oral Selective Estrogen Receptor Degraders (SERDs) in Breast Cancer: Advances, Challenges, and Current Status.

Several endocrine therapies are currently available for the treatment of estrogen receptor (ER) positive breast cancer, but the clinical benefit of these agents is limited by endocrine therapy drug resistance. A common mechanism of endocrine therapy resistance is ESR1 mutations. The first-generation selective estrogen receptor degrader (SERD) fulvestrant has activity against ESR1 mutant tumors but requires intramuscular injection and has poor bioavailability that precludes optimal drug dosing. This led to the development of second-generation SERDs which are potent and have improved oral bioavailability and pharmacokinetics. Several of these oral SERDs are now in phase III trials in both the early and advanced ER positive breast cancer settings. This review summarizes the background of oral SERD development, the current status and future perspectives.

Abstract P2-01-08: Esr1 Y537 mutations are associated with increased baseline circulating tumor cells enumeration for patients with estrogen receptor positive metastatic breast cancer

Abstract Introduction: Estrogen Receptor (ER)-positive breast cancer is the most frequent breast cancer subtype. Endocrine therapy (ET) targeting the ER pathway including CDK4/6 inhibitors represents the main initial therapeutic approach. However, clinical resistance associated with progression of disease caused by ESR1 mutations is a recognized an important mechanism of ET resistance. ESR1 mutations, most often detected from liquid biopsies, have been consistently associated with a worse outcome and are being currently evaluated as a potential biomarker to guide therapeutic decisions. Here we reported a new finding on the association between ESR1 amino acid Y537 mutations in ctDNA and circulating tumor cells (CTC) in metastatic breast cancer (MBC). Methods: The study included 158 ER positive MBC patients enrolled under an IRB-approved trial (NU16B06) at Lurie Cancer Center, Northwestern University. The patients received systemic treatments in 2016-2020. Whole blood samples (7.5ml/each) were collected in EDTA tubes from patients who were longitudinally characterized for CTCs before therapy (baseline). CTCs enumeration were performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CXC Kit contains antibodies targeting EpCAM antigen for capturing CTCs, anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI for staining the cell nucleus, anti-CD45-APC is specific for leukocytes. The CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. Plasma ctDNA was isolated using a Qiagen circulating nucleic acid kit, and then was analyzed using the Guardant360 next-generation sequencing (NGS)-based assay. In this study, only patients with ESR1 mutations were included for statistical analyses of correlation between the hotspot mutation with the endocrine therapy resistance by using Causal Inference approach. All statistical analyses were conducted Mann-Whitney U test by IBM SPSS version 23.0. Results: ESR1 mutations were detected in 40 out of 158 patients at baseline. ESR1 Y537(N/C/S) mutations were detected in 13 patients, among which there were 9 patients who only had the Y537(N/C/S) mutations and no other ESR1 hotspot mutations (Group 1) and there were 4 patients who had polyclonal ESR1 mutations. In the latter cohort, there were 17 ESR1 amino acid mutations detected in 31 patients (Group 2) including Q314, T347T, N359I, K362N, E380Q, V392I, G442R, F461I, S463P, S464, I487M, K520K, M528V, L536H, D538G, D545D and Q565. There were no any ESR1 mutations detected in the other 118 patients (Group 3). The CTC>5/7.5mL (Stage IV aggressive) were found in 66.7%, 38.7% and 29.3% patients in Group 1, Group 2 and Group 3 respectively. The median total CTCs was 26.0/7.5mL in MBC patients with ESR1 Y537(N/C/S) mutations (Group 1). The median of total CTCs was 2.0/7.5mL in the MBC patients with polyclonal ESR1 mutations but not on Y537 (N/C/S) (Group 2), and it was 1.0/7.5mL in the group of metastatic breast cancer without ESR1 mutations (Group 3). The median total CTCs in Group 1 was significantly higher than Group 2 (Mann-Whitney U, P<0.05) and Group 3 (P=0.009), which indicated that Y537(N/C/S) mutations were correlated with higher baseline CTCs. Conclusions: In this study, we demonstrated a correlation between presence of ESR1 Y537 mutations and increased CTCs (higher Stage IV aggressive cases) in patients with HR+ MBC, These preliminary data may suggest a critical role of ESR1 Y537 mutations in the metastatic process, also indicating that different ESR1 amino acids mutations may play different roles on disease progress. Larger validation studies are needed to confirm that evaluation of ESR1 Y537 variant together with CTCs numeration is an accurate tool to identify endocrine-refractory disease. Citation Format: Qiang Zhang, Jianhua Jiao, Paolo D'Amico, Andrew A. Davis, Weijun Qin, Lorenzo Gerratana, Saya L. Jacob, Youbin Zhang, Jeannine Donahue, Wenan Qiang, Ami N. Shah, Lisa Flaum, William Gradishar, Leonidas C. Platanias, Massimo Cristofanilli. Esr1 Y537 mutations are associated with increased baseline circulating tumor cells enumeration for patients with estrogen receptor positive metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-08.

Abstract OT1-15-01: SGNTUC-019: Phase 2 basket study of tucatinib and trastuzumab in previously treated solid tumors with HER2 alterations: HER2-mutated breast cancer cohort (ongoing clinical trial)

Abstract Background: Tucatinib (TUC), approved in multiple regions for HER2+ metastatic breast cancer, is a highly selective HER2-directed tyrosine kinase inhibitor with minimal EGFR inhibition. TUC is being developed as a novel therapy for patients with HER2+ metastatic breast cancer, colorectal cancer, and gastric cancer. In xenograft models of HER2+ and HER2-mutated tumors, dual targeting of HER2 with TUC and trastuzumab showed superior activity to either agent alone. Somatic HER2 mutations occur in approximately 3% of breast cancers, mostly in patients with hormone receptor-positive disease and the lobular sub-type. HER2 mutations lead to enhanced tyrosine kinase activity and tumorigenesis in preclinical models and have been postulated as a mechanism of endocrine therapy resistance. The SGNTUC-019 basket study (NCT04579380) is evaluating TUC in combination with trastuzumab in patients with HER2+ or HER2-mutated solid tumors, including a cohort of patients with locally advanced unresectable or metastatic breast cancer that is HER2-mutated and not overexpressed/amplified. Trial Design: SGNTUC-019 is a multi-cohort, open-label, international Phase 2 study evaluating patients with previously treated solid tumors displaying HER2 overexpression/amplification or activating mutations. Eligible breast cancer patients must have HER2-mutated, locally advanced unresectable or metastatic disease with progression on or after ≥1 prior line of therapy (chemotherapy, endocrine or targeted therapy) in the locally advanced or metastatic disease setting. Additionally, an ECOG Performance Status of ≤1; adequate hepatic, hematological, renal, and cardiac functions; and no previous HER2-directed therapy are required for enrollment. For eligibility, HER2 mutations can be demonstrated in a previous or on-study next-generation sequencing (NGS) assay of circulating tumor DNA or a previous tissue NGS assay. The breast cancer cohort will enroll 30 response-evaluable patients with HER2-mutated disease. Patients with HER2+ (overexpression/amplification) breast cancer will not be enrolled. The primary objective in each cohort is antitumor activity. The primary endpoint is a confirmed objective response rate, and secondary endpoints are the disease control rate, duration of response, progression-free survival, and overall survival. Safety and efficacy endpoints will be summarized descriptively. For response rates, the 2-sided exact interval using the Clopper-Pearson method will be calculated. Patients will receive TUC 300 mg orally twice a day and trastuzumab 8 mg/kg intravenously on Cycle 1 Day 1 and 6 mg/kg every 21 days from Cycle 2 Day 1. Hormone receptor-positive HER2-mutated breast cancer patients will also receive fulvestrant 500 mg intramuscularly every 4 weeks and on Cycle 1 Day 15. Disease assessments per RECIST v1.1 are every 6 weeks for 24 weeks, then every 12 weeks. Patients in the breast cancer cohort will undergo baseline brain magnetic resonance imaging, and those patients with brain metastases may be eligible for the study. Quality of life will be evaluated using EQ-5D-5L every 2 cycles. Enrollment began in December 2020 and is ongoing globally. Citation Format: Alicia Okines, Paula R. Pohlmann, Jorge Ramos, Luke Walker, Erika Hamilton. SGNTUC-019: Phase 2 basket study of tucatinib and trastuzumab in previously treated solid tumors with HER2 alterations: HER2-mutated breast cancer cohort (ongoing clinical trial) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-15-01.

In vitro breast cancer models for studying mechanisms of resistance to endocrine therapy.

The development of endocrine resistance is a common reason for the failure of endocrine therapies in hormone receptor-positive breast cancer. This review provides an overview of the different types of in vitro models that have been developed as tools for studying endocrine resistance. In vitro models include cell lines that have been rendered endocrine-resistant by ex vivo treatment; cell lines with de novo resistance mechanisms, including genetic alterations; three-dimensional (3D) spheroid, co-culture, and mammosphere techniques; and patient-derived organoid models. In each case, the key discoveries, different analysis strategies that are suitable, and strengths and weaknesses are discussed. Certain recently developed methodologies that can be used to further characterize the biological changes involved in endocrine resistance are then emphasized, along with a commentary on the types of research outcomes that using these techniques can support. Finally, a discussion anticipates how these recent developments will shape future trends in the field. We hope this overview will serve as a useful resource for investigators that are interested in understanding and testing hypotheses related to mechanisms of endocrine therapy resistance.

Management of hormone receptor-positive, human epidermal growth factor 2-negative metastatic breast cancer.

Estrogen receptor (ER) is the major driver of most metastatic breast cancers (mBCs). Endocrine therapy (ET) is the most effective treatment for ER + mBC, but its effectiveness is limited by high rates of de novo and acquired resistance. A growing understanding of the biological characteristics and complexity of the ER pathway and the mechanisms of ET resistance has led to the development of a new generation of targeted therapies. One such mechanism is the cell cycle signaling pathways, which lead to the development of cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) that have, in turn, transformed the management of such tumors. Another important mechanism is the alteration of the phosphatidylinositol 3'-kinase/AKT/mammalian target of rapamycin pathway. Drugs targeting each component of these pathways are currently used in clinical practice, and several more are in development. As a result, a myriad of new targeted therapies are consistently being added to the clinical oncologist armamentarium. Navigating the evolving and highly complex treatment landscape of HR + /HER2- mBC remains both an art and a challenge. In this review, we discuss the biological features of HR + /HER2- mBC and the different mechanisms of resistance to ET. We also discuss the management of mBC as the disease changes from endocrine-sensitive to endocrine-resistant.

Mechanisms of endocrine therapy resistance in breast cancer

The most commonly diagnosed breast cancer (BC) subtype is characterized by estrogen receptor (ER) expression. Treatment of this BC subtype typically involves modalities that either suppress the production of estrogen or impede the binding of estrgen to its receptors, constituting the basis for endocrine therapy. While many patients have benefitted from endocrine therapy with clear reduction in mortality and cancer recurrence, one of the clinical hurdles that remain involves overcoming intrinsic (de novo) or acquired resistance to endocrine therapy driven by diverse and complex changes occurring in the tumor microenvironment. Moreover, such resistance may persist even after progression through additional antiestrogen therapies thus demonstrating the importance of further investigation of mechanisms of ER modulation. Here, we discuss a number of advances that provide a better understanding of the complex mechanistic basis for resistance to endocrine therapy as well as future therapeutic maneuvers that may break this resistance.

Abstract SP092: Special topics in metastatic ER+ breast cancer

Abstract About 70% of all diagnosed breast cancers express hormone receptors (estrogen [ER+], progesterone [PR+] or both [HR+]). Despite an overall good prognosis, the clinicopathological and molecular heterogeneity of these tumors account for a diverse natural history of disease, with several patients still developing distant metastatic recurrence at virtually any point during the disease trajectory, regardless of endocrine therapy. Improved risk stratification and treatment individualization accounts for longer disease-free and overall survival, but distant metastatic recurrences still occur and about half of these occur beyond 6 years from the original diagnosis. A better understanding of breast cancer biology and mechanisms of endocrine therapy resistance has resulted in the approval of novel therapeutic strategies for patients with HR+ metastatic breast cancer (MBC), which now often include addition of targeted treatment, such as CDK4/6 or PI3K pathway inhibitors, to first, second or third line endocrine therapy. Improvement in progression-free survival and sometimes even overall survival, as well as a delay in chemotherapy use in the metastatic setting and maintenance of a good quality of life have been important achievements with these targeted therapies. However, more refined clinical and molecular biomarkers are lacking to determine which patients truly need (or not) the (early or at all) addition of these targeted treatments to conventional endocrine therapy, the timing and order of targeted therapy addition and even the timing to introduce chemotherapy. Currently, the amount of ER/PR expression in the tumor, presence of tumor actionable genomic alterations (such as PIK3CA, ESR1, BRCA mutations to name a few), the patient’s disease-free interval between original (early) and metastatic breast cancer diagnosis, metastatic burden and sites of metastasis, absence or presence of symptoms or visceral crisis, previous treatments, among others, are some of the main variables that clinicians take into account to customize HR+ MBC treatment. Several research efforts are under way, focusing on important questions addressing the optimal sequence and administration of drugs to circumvent current endocrine therapy with or without CDK4/6 inhibition strategies, such as (a) timing and order of CDK4/6 inhibitor addition to endocrine therapy, (b) if CDK4/6 inhibitors should be continued with a different endocrine therapy partners beyond progression of disease on endocrine therapy and CDK4/6 inhibitors, (c) novel combination of endocrine therapy with or without CDK4/6 inhibitors with targeted treatments or immunotherapy, (d) new endocrine therapy agents such as novel selective ER downregulators (SERDs) and selective ER covalent agonists (SERCAs), (e) serial collection of tumor tissue and blood for profiling at different timepoints during the metastatic setting treatment in an effort to identify new mechanisms of resistance, and (f) novel chemotherapy combinations with targeted treatment or immunotherapy. In conclusion: many therapeutic and diagnostic advances have contributed to better outcomes in HR+ MBC, but recognition of differences between clinicopathological and molecular characteristics of patients and their tumors is key in promoting optimization of treatment choices that would maximize survival, palliation of symptoms and quality of life for patients with HR+ MBC in the years to come. Citation Format: I Mayer. Special topics in metastatic ER+ 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 SP092.

Overcoming oncogene addiction in breast and prostate cancers: a comparative mechanistic overview

Prostate cancer (PCa) and breast cancer (BCa) are both hormone-dependent cancers that require the androgen receptor (AR) and estrogen receptor (ER, ESR1) for growth and proliferation, respectively. Endocrine therapies that target these nuclear receptors (NRs) provide significant clinical benefit for metastatic patients. However, these therapeutic strategies are seldom curative and therapy resistance is prevalent. Because the vast majority of therapy-resistant PCa and BCa remain dependent on the augmented activity of their primary NR driver, common mechanisms of resistance involve enhanced NR signaling through overexpression, mutation, or alternative splicing of the receptor, coregulator alterations, and increased intracrine hormonal synthesis. In addition, a significant subset of endocrine therapy-resistant tumors become independent of their primary NR and switch to alternative NR or transcriptional drivers. While these hormone-dependent cancers generally employ similar mechanisms of endocrine therapy resistance, distinct differences between the two tumor types have been observed. In this review, we compare and contrast the most frequent mechanisms of antiandrogen and antiestrogen resistance, and provide potential therapeutic strategies for targeting both advanced PCa and BCa.

ESR1 Mutation Status as a Biomarker for Radiation Response in ER-Positive Breast Cancer

Acquired mutations in oncogenic drivers in response to systemic therapies is a common mechanism of treatment resistance. How such mutations alter response to radiation therapy is poorly understood. Acquired mutations in the gene encoding for Estrogen Receptor (ESR1) are one of the most common mechanisms of endocrine therapy resistance in breast cancer. The goal of this study is to define the role of ESR1 mutations in altering response to therapeutic radiation in breast cancer and to develop therapeutic approaches to overcome such resistance. To test if ESR1 mutations influence response to radiation therapy, we employed ER-positive MCF7 and T47D cell lines which have been genome edited to knock in two of the most common ESR1 mutations found in breast cancer patients (Y537S and D538G). Clonogenic survival assays were carried out to characterize differences in radiation sensitivity of these cells. γ-H2AX and Comet assays were employed to study the differences in residual unrepaired DNA damage following radiation in these cell lines. MCF7 cell line-derived xenografts harboring the wild type or mutant ESR1 were employed to study differences in radiation resistance of these tumors in vivo. ER-positive breast cancer cell lines harboring ESR1 mutations exhibit resistance to radiation compared to the corresponding isogenic cell lines with the wild type ESR1. Following exposure to radiation, the ESR1 mutant cells have significantly less residual, unrepaired DNA damage. We also show an endogenous interaction between mutant ER proteins and BRD4, a bromodomain and extraterminal domain (BET) family protein. Recent studies have established a key role for BRD4 in repairing DNA double strand breaks via non-homologous end joining pathway. Pharmacologic BET inhibition with OTX015 overcomes ESR1 mutation-induced resistance to therapeutic radiation in breast cancer, both in vitro and in vivo. ESR1 mutations, which confer resistance to endocrine therapies, also confer resistance to radiation therapy. Recent studies show that ESR1 mutations are also enriched in non-metastatic, ER-positive breast cancer patients receiving neoadjuvant endocrine therapy. Our findings suggest that patients who develop ESR1 mutations under selective pressure of neoadjuvant endocrine therapy may respond poorly to subsequent radiation therapy and may experience increased risk of locoregional failure. In fact, a recent study reported enrichment of ESR1 mutations in locoregionally recurrent breast cancers. Our findings support prospective clinical studies for personalization of radiation treatments in breast cancer based on ESR1 mutation status.

Endocrine therapy combined with targeted therapy in hormone receptor-positive metastatic breast cancer.

Increasing numbers of targeted drugs are used in hormone receptor (HR)-positive metastatic breast cancer (MBC) to overcome or delay resistance to endocrine therapy. This study will systemically review the progress made in endocrine therapy combined with targeted therapy in the treatment of HR-positive MBC. From the “AI (aromatase inhibitor) era” represented by aromatase inhibitors, we have gradually entered the “post-AI era” represented by fulvestrant. Under the guidance of research on the molecular mechanism of endocrine therapy resistance, the “combination of endocrine therapy and targeted therapy” era is approaching. The development of drugs that target endocrine therapy resistance has concentrated on cyclin-dependent kinase 4/6 inhibitors, histone deacetylase inhibitors, and inhibitors of drug targets in the phosphatidylinositol 3 kinase-protein kinase B-mammalian target of rapamycin (PI3K-AKT-mTOR) pathway, providing new strategies for HR-positive MBC. Exploring biomarkers to guide the more precise use of targeted drugs in endocrine therapy for MBC is the focus of current and future research.

Targeting STAT3 signaling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer

Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX‐01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX‐01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho‐STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months’ endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

Abstract P6-04-24: Transcription factor dynamics in endocrine therapy resistance models using a novel chromatin landscape analysis algorithm based on ATAC-Seq

Abstract Endocrine therapy resistance is one of the major challenges in breast cancer therapy today. As the causes are not well understood, therapeutic options are limited. To investigate the molecular mechanisms of endocrine therapy resistance, we established in vitro Long-Term Estrogen Deprivation (LTED) models using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression with upregulation in some cell lines and downregulation in others. Although all cells continued to grow, none acquired mutations in the ESR1 ligand binding domain, reported to be responsible for endocrine therapy resistance. Current data using gene expression and DNA modification profiles yield limited information for molecular mechanisms of resistance and potential for developing novel therapies. The chromatin landscape is a potential identifier of disease state, and can provide specific information on transcriptional regulation by enhancers. Thus, changes in chromatin open/closed sites could provide important information to predict disease progression and can be used to identify potential therapeutic targets. We analyzed the time-course of chromatin landscape transitions during acquisition of endocrine resistance by the Assay for Transposase Accessible Chromatin (ATAC-seq), and combined these studies with RNA-seq and genomic DNA sequence analysis. ATAC-seq permits the analysis of all sites potentially accessible to the transcriptional machinery. Furthermore, digital footprinting of protected transcription factor motifs within the accessible regions can evaluate the occupancy of hundreds of different transcription factors at one time. We performed bioinformatic analysis of open and closed chromatin sites and selected relevant enhancer signatures. Because transcription factors play a primary role in gene expression and in cell-fate changes, including response to therapies, we derived digital footprints from transcription factor occupancy to correlate this information with phenotypes of LTED cells. Comparing changes in the chromatin landscape with RNA-seq, we created a high throughput algorithm that integrates the information from open/closed chromatin sites, transcription factors occupancy, and gene expression. We found key transcription factors for acquisition of LTED cells; Myc-Max is associated with ESR1 overexpressed LTEDs, whereas Oct1 and Hox genes are associated with LTEDs with absence of ESR1 expression. These methods establish novel mechanisms of endocrine therapy resistance in breast cancer that can also be applied to other tumor types. Our studies provide a rationale for using chromatin landscape and transcription factor networks to expand the range of translational research in the search for novel therapies for breast cancer. These studies will potentially have high impact on development of important novel therapeutic approaches in cancer and many other diseases. Citation Format: Saori Fujiwara. Transcription factor dynamics in endocrine therapy resistance models using a novel chromatin landscape analysis algorithm based on ATAC-Seq [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 P6-04-24.