Endocrine Therapy Resistance Research Articles

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

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

Abstract 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.

Abstract PO4-05-08: Primary endocrine therapy resistance in patients with de novo HR+/HER2- metastatic breast Cancer: a National Cancer Information Database Analysis in China

Abstract Background: Endocrine therapy resistance was classifed as primary and secondary, guiding the first-line (1L) treatment choices for hormone receptor positive, HER2-negative (HR+/Her2-) metastatic breast cancer (mBC). However, it is unknown whether it is primary ET resistance or not to the stage IV de novo patients. Therefore, We performed a retrospective study to predict primary resistance in advance in de novo patients and have a more appropriate treatment strategy. Methods: We identified newly diagnosed with stage IV de novo HR+/HER2- mBC patients received single-agent endocrine therapy (ET) or ET plus CDK4/6 inhibitors (CDK4/6i) in 1L setting from January 1, 2020 to Jun 30, 2022, using the National Cancer Information Database (NCID) in China. Primary ET resistance (PER) was defined as switching to second-line treatment within 6 months after receiving the 1L ET±CDK4/6i, whereas secondary ET resistance (SER) patients were those who received 1L ET±CDK4/6i treatment more than six months. The Chi-Square test was used to compare the baseline characteristics of PER and SER patients. Result: A total of 217 patients who met the inclusion criteria were included in the analysis, among which 161 patients received single-agent ET and 56 patients received ET plus CDK4/6i. 18.4% of the patients were classified as PER patients, whereas 81.6% showed SER patients. PER patients were younger (median age of 52 years vs. 58 years in SER patients, p=0.01). In terms of clinicopathological characteristics, patients with PER have a higher percentage of liver metastases (14/40 (35%) vs. 20/177 (11.3%) in SER, p< 0.01), distant lymph node metastases (19/40 (34%) vs. 45/177 (25.4%) in SER, p = 0.01), and bone metastases (30/40(75%) vs. 97/177(54.8%) in SER, p = 0.02). Furthermore, the PER rate was 21.7% (35/161) in patients received single-agent ET, compare with 8.9% (5/56) in ET plus CDK4/6i patients, p=0.04. Regarding the following treatment pattern in the PER patients, a high percentage of patients received chemotherapy, accounting for 50%, 30% still received another endocrine therapy, and the remaining 20% patients received CDK4/6i combined with endocrine therapy. Conclusions: There is still a significant proportion of de novo HR+/HER2- mBC patients with primary endocrine resistance, a relatively low proportion of PER in patients receiving endocrine in combination with CDK4/6i. Table. The proportion of primary endocrine resistance across treatment regimens in de novo HR+/HER2- metastatic breast Cancer Citation Format: Zhanhong Chen, Xiying Shao, Yabing Zheng, Wenming Cao, Junqing Chen, Weizhu Wu, Ting Wang, Xiaojia Wang. Primary endocrine therapy resistance in patients with de novo HR+/HER2- metastatic breast Cancer: a National Cancer Information Database Analysis in China [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-05-08.

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 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 PO3-04-11: Unmet clinical need in patients with pre-treated hormone receptor-positive/HER2-negative (HR+/HER2–) metastatic breast cancer in routine care: a targeted literature review

Abstract Background Treatment options remain limited to chemotherapy (CT) for patients with HR+/HER2− metastatic breast cancer (MBC) who have progressed on endocrine therapy (ET) with/without CDK4/6 or PI3K pathway inhibitors (i). We aimed to review the current unmet need in patients who had received ≥ 1 prior systemic therapy in terms of ET resistance as well as CT survival outcomes, tolerability and discontinuation. Methods MEDLINE, Embase and the Cochrane Library were searched via Ovid for observational studies (≥ 50 patients who had received ≥ 1 prior systemic therapy, January 2018 – January 2023). Abstracts from relevant congresses (January 2021 – February 2023) were reviewed. All screening was performed in a single-blind manner by one reviewer and any uncertainties were resolved by a senior reviewer. Results We included 72 full-text publications and 48 congress abstracts (US based 39%, European 28%, Asian 23%). Median treatment duration was 1.9–16.2 months (m) for ET (7 studies) and 2.0–24.5 m for ET-based therapy (5 studies). Exceptional responders (10% of patients treated for the longest time) received ET for ≥ 43.6 m (1 study, N = 4195). The proportion of patients with ET-resistant disease ranged widely (3–100%; 22 studies) and increased by line (L) of therapy: 14–19% of patients treated in 1L had primary resistance, 14–49% in ≥ 2L and 27–68% in ≥ 3L; acquired resistance was reported for 26–38% of patients in 1L, 51–57% in ≥ 2L, 32–60% in ≥ 3L (2 studies). One or more prior lines of CT had a negative impact on median overall survival (mOS; 5 studies) and median progression-free survival (mPFS; 11 studies) in subsequent therapy lines regardless of the chosen treatment; prior CT had no effect on mOS in 3 studies, no effect on mPFS in 4 studies. Consecutive CT cycles (primarily after ET + CDK4/6i) steadily lost clinical benefit (Table), with mPFS with 2 to 5 lines of CT: 7.6 m, 4.8 m, 4.2 m and 3.2 m, and mOS: 13.5 m, 9.1 m, 6.8 m and 7.2 m, respectively (1 study, N = 266). Paclitaxel + bevacizumab as second CT had a mPFS and a mOS of 8.6 m and 19.8 m, respectively (1 study, N = 538). Eribulin use in ≥ 2L had a mPFS of 4.6–10.0 m (2 studies) and a mOS of 10.1–11.2 m (2 studies). Patients who progressed on CDK4/6i as best response and those with a mPFS of < 6 m on ET benefited more from CT than ET in ≥ 2L with a mPFS of 7.1–8.1 m compared with 1.7–3.9 m, respectively (2 studies). Discontinuation due to toxicity was 2–36% (3 studies) for CT in 2L and dose reduction was required in 19–37% of patients (2 studies). Common grade 3/4 adverse events in patients receiving CT were leukopenia (17%), neutropenia (7%), thrombocytopenia (3%) and pneumonitis (1–3%) (3 studies). Conclusions Our review of real-world evidence highlights the limitations of repeated CT for patients with ET-resistant HR+/HER2− MBC, characterized by diminishing clinical benefit and significant toxicity burden. Results are limited by treatment and study population heterogeneity. Clinical outcomes are expected to improve with the use of new therapies, for which real-world evidence has yet to accrue. Table Real-world clinical outcomes with chemotherapy from observational studies CI, confidence interval; CT, chemotherapy; L, line; mOS, median overall survival; mPFS, median progression-free survival; NR, not reported Citation Format: Laura Spring, Simon M Collin, Inderjit K Dhillon, Gráinne Long, Evelina Bertranou, Komal Jhaveri, Aditya Bardia. Unmet clinical need in patients with pre-treated hormone receptor-positive/HER2-negative (HR+/HER2–) metastatic breast cancer in routine care: a targeted literature review [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 PO3-04-11.

Abstract PO2-16-05: ESR1 mutations (ESR1mut) in HR(+)HER2(-)patients with metastatic breast cancer (MBC): prevalence along treatment course and predictive value for endocrine therapy (ET) resistance in real-world practice

Abstract Background: ET combined with CDK4/6 inhibitors (CDKi) is the standard of care for HR(+)HER2(-) patients with MBC. ESR1mut is an acquired resistance mechanism to ET, especially aromatase inhibitors (AI). The prevalence and behavior of ESR1mut in patients receiving the selective estrogen receptor degrader (SERD) fulvestrant remains in question. This study aimed to characterize the prevalence of ESR1mut at start of successive lines of therapy and to evaluate clinical outcomes of ET by ESR1 status in 1st line therapy in real-world practice. Methods: This study included patients with HR(+)HER2(-) MBC who underwent genomic testing using Foundation Medicine tissue or liquid comprehensive genomic profiling (CGP) assays, with specimens collected within 60 days of therapy initiation. Patient clinical data was obtained by the nationwide (US-based) de-identified Flatiron Health and Foundation Medicine real-world clinicogenomic breast database (FH-FMI CGDB), originated from ~280 US cancer clinics (~800 sites of care) between 01/2011 and 04/2023. ESR1mut prevalence was calculated in tissue and liquid samples collected in the 1st, 2nd, and 3rd lines of therapy. Real-world progression free-survival (rwPFS), time to treatment discontinuation (rwTTD), and overall survival (rwOS) were compared between patients who received AI + CDKi and between patients receiving fulvestrant + CDKi 1st line therapy [ESR1mut vs. ESR1 wild-type (wt) by tissue CGP] by Cox models. Multivariable analyses adjusted for adjuvant therapy, age, ECOG, menopausal status, histology, stage at diagnosis, metastatic site, and specific ESR1mut (Y537S, D538G, E380W, or others) were performed. Results: The prevalence of ESR1mut in tissue samples collected in 1st, 2nd, and 3rd lines of therapy was 7.9% (n=159/2004), 26.4% (n=66/250), and 34.0% (n=68/200), respectively. The prevalence of ESR1mut in liquid samples collected in the 1st, 2nd, and 3rd lines of therapy was 11.6% (n=30/259) 34.4% (n=63/183), and 35.9% (n=42/117), respectively. Among patients receiving AI + CDKi 1st line therapy (n=485), those with ESR1mut vs. ESR1wt had less favorable rwPFS [median 6.1 vs. 20.8 months, hazard ratio (HR) 2.36, 95% CI 1.35-4.14, p=0.003]. rwTTD and rwOS assessments were consistent with the magnitude of effect of rwPFS. Among patients receiving fulvestrant + CDKi 1st line therapy (n=302), those with ESR1mut vs. ESR1wt had less favorable rwPFS (median 10.5 vs. 14.5 months, HR 1.5, 95% CI 1.05-2.15, p=0.025), but no difference was observed for rwTTD and rwOS (p >0.05). Notably, the multivariable analyses found the independent association of ESR1mut and less favorable rwPFS (HR 2.18, 95% CI 1.15-4.1, p=0.018), rwTTD (HR 3.34, 95% CI, 1.84-6.1, p< 0.001), and rwOS (HR 2.46, 95% CI 1.04-5.9, p=0.041) in patients receiving AI + CDKi. The multivariable analyses confirmed no association between ESR1mut and outcomes in patients receiving fulvestrant + CDKi (p >0.05). No substantial differential associations were observed between specific ESR1mut and clinical outcomes in the multivariable analyses. Conclusion: In our dataset, ESR1mut is prevalent in about 8-11% of baseline tissue and liquid samples of patients with HR(+)HER2(-) MBC. Baseline ESR1mut is associated with less favorable outcomes in patients receiving AI + CDKi 1st line therapy, but not in patients receiving fulvestrant + CDKi. Specific ESR1mut do not seem to be associated with different outcomes to ET. ESR1mut prevalence increased during treatment course. Further investigation is warranted to determine optimal endocrine partner with CDKi based upon ESR1 status, particularly with development of oral SERDs. Citation Format: Manali Bhave, Julia Quintanilha, Ryon Graf, Takara Scott, Hanna Tukachinsky, Gerald Li, Alexa Schrock, Geoffrey Oxnard, Mia Levy, Kevin Kalinsky. ESR1 mutations (ESR1mut) in HR(+)HER2(-)patients with metastatic breast cancer (MBC): prevalence along treatment course and predictive value for endocrine therapy (ET) resistance in real-world practice [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-16-05.

Abstract PO5-21-05: An Endocrine Resistant-Related Gene Signature Revealing the Tumor Microenvironment to Predict the Prognosis of Hormone Receptor-Positive Breast Cancer Patients

Abstract Key words: breast cancer; hormone receptor-positive; predictive model; prognosis; endocrine therapy; drug resistance Hormone receptor positive (HR+) breast cancer is the most common type of breast cancer, accounting for approximately 70% of all breast cancer, in whom endocrine therapy plays a vital role. However, the limited effectiveness of endocrine therapy due to intricate mechanisms of drug resistance poses a significant challenge in treating certain patients. Therefore, it is imperative to identify novel indicators of endocrine resistance and develop new models for predicting recurrence-free survival (RFS) in patients with HR+ breast cancer. In this study, we successfully established an endocrine resistance-related risk model based on multicenter RNA sequencing data. This model could represent tumor microenvironment characteristics and provide valuable insights into predicting the prognosis of HR+ breast cancer patients. A seven gene risk model reflecting endocrine therapy resistance in 208 early HR+ breast cancer patients from two centers was constructed by LASSO analysis and multivariate cox regression. Based on the median value of risk score, patients were divided into high-risk group and low-risk group. Compared with the low-risk group, the high-risk group exhibited a significantly worse prognosis, which showed consistent across two external validation cohorts (TCGA-BRCA and METABRIC cohort). Time dependent ROC curve showed excellent capacity of gene model in predicting 1,3 and 5-year RFS. The IC50 analysis revealed that patients in low-risk group exhibited greater sensitivity to endocrine drugs, such as tamoxifen, fulvestrant and palbociclib, which strongly suggested that the identified gene signature can effectively differentiate between endocrine-resistant and -sensitive status. Gene-set enrichment analysis revealed regulation of cell killing, positive regulation of inflammatory response and T cell mediated cytotoxicity were enriched in low risk group, suggesting immune activation and a higher immune infiltration status. Conversely, the high-risk group demonstrated associations with collagen metabolic process, extracellular matrix structural constituent and collagen fibril organization, which were the indicative of the location and function of cancer-associated fibroblasts (CAFs). As expected, ssGSEA results revealed elevated expression levels of immune cells (e.g., natural killer cells, tumor-infiltrating lymphocytes, CD8+ T cells) and immune functions (e.g., T cell co-stimulation, inflammation-promoting, cytolytic activity) in the low-risk group. However, three diverse acknowledged methods (TIDE, EPIC and MCP-counter) consistently identified CAFs as predominantly content in the high-risk groups. Furthermore, considering that CD8+ T cell is the primary cell type responsible for exerting cytotoxic effects, we observed a negative correlation between CD8+ T cells and CAFs. This suggests that CAFs may inhibit the infiltration of CD8+ T cells, thereby weakening their cytotoxic capabilities, leading to a shorter RFS in high-risk patient groups. In conclusion, a novel gene signature constructed with 7 endocrine resistance response-related genes can be used for prognostic prediction and reflecting the tumor microenvironment status in HR+ early breast cancer. The low-risk group exhibited enhanced anti-tumor immunity, resulting in a favorable prognosis. In contrast, the high-risk group displayed pronounced infiltration of CAFs, indicating their potential to impede immune cell infiltration and impact the efficacy of endocrine drugs through the formation of physical barriers and recruitment of immunosuppressive factors. Consequently, patients in the high-risk group experienced a worse prognosis. Therefore, inhibiting these genes may be a therapeutic option for overcoming endocrine therapy resistance in HR+ breast cancer patients. Citation Format: Xiyu Kang, Jiaxiang Liu, Xin Wang. An Endocrine Resistant-Related Gene Signature Revealing the Tumor Microenvironment to Predict the Prognosis of Hormone Receptor-Positive Breast Cancer Patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-21-05.

Abstract PO4-04-08: Design and Development of the APIS ESR1 Mutations Kit to Detect Eleven Mutations Relevant to Acquired Endocrine Therapy Resistance

Abstract Introduction: Estrogen receptor 1 (ER/ESR1) mutations have arisen as key biomarkers for endocrine therapy resistance in ER positive (ER+) breast cancer (BC) patients: detecting these mutations is key to guiding researchers to better understand acquired resistance during treatment. Ongoing clinical trials, which explore the prevalence of ESR1 mutations under various treatments, commonly use next-generation sequencing (NGS)-based assays. However, the NGS panels available are costly, can have long turnaround times and require specialist equipment and software. Research use only digital PCR assays are also available but have similar drawbacks to NGS. A targeted qPCR-based assay is more time and cost effective, and does not require specialist equipment. Here, we demonstrate the APIS ESR1 Mutations Kit, a targeted, sensitive, and robust qualitative qPCR assay able to detect eleven ESR1 mutations across ESR1 exons 5 (E380Q), 7 (S463P) and 8 (P535H, L536R, L536Q, L536H, L536P, Y537C, Y537S, Y537N and D538G). The assay is designed to assess circulating-free DNA (cfDNA) samples, reducing the need for invasive procedures in clinical settings and can make use of standard cfDNA extraction kits. Therefore, the APIS ESR1 Mutations Kit provides the flexibility for researchers in any conventional molecular biology laboratory to explore ESR1 mutations with high sensitivity and specificity. Methods: The APIS ESR1 Mutations Kit provides all components and controls required to assess samples. For kit development, mutation-specific DNA fragments were spiked into wildtype background DNA (either cfDNA extracted from pooled healthy donor plasma, human genomic DNA, or DNA fragments); the Limit of Blank (LoB) and Limit of Detection (LoD) were determined using these samples. To determine linearity, a dilution series of DNA fragments ranging from 5 to 10,000 copies per reaction was assessed. All PCR runs were performed using a QuantStudio™5 Dx (ThermoFisher) instrument. Results: The performance studies showed the APIS ESR1 Mutations Kit can detect D538G at 0.4 % mutant allele frequency (MAF) and Y537S at 0.06 % MAF. All other mutations can be detected at ≤ 1.0 % MAF. By using clamp and blocking technologies, each design is mutation specific, enabling identification of specific amino acid mutations. In addition, the assays can perform in high wild-type backgrounds, enabling lower limit of blanks with ≥95% confidence and threshold cut-offs to enhance the sensitivity of the assay. The linearity of each target is within 90-110% from 50 to 10,000 copies per reaction. Conclusions: The ESR1 Mutations Kit demonstrated high sensitivity and performance as a qualitative qPCR assay to detect eleven ESR1 mutations. Table. Mutations detected with the APIS ESR1 Mutations Kit Citation Format: Anna Gasior, Colette Whitfield, Anita Campbell, Christine Hoy, Sally Holdsworth, Jesse Mukose, Ryan Nana, Sahar Shammakhi, Benjamin Scales, Joanna Gorniak. Design and Development of the APIS ESR1 Mutations Kit to Detect Eleven Mutations Relevant to Acquired Endocrine Therapy Resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-04-08.

Abstract PO4-15-09: Unraveling the influence of progesterone receptor status on endocrine therapy sensitivity: insight from epigenetic and fragmentomics ctDNA profiling in estrogen receptor positive, HER2 negative metastatic breast cancer (MBC)

Abstract Background: In patients (pts) with estrogen receptor (ER) positive (pos), HER2 negative (neg) MBC, the absence of progesterone receptor (PR) expression is generally associated with a poorer endocrine therapy (ET) response and an unfavorable prognosis. However, real-time molecular changes during ET according to PR status are still not fully understood. The aim of this study was to examine the prognostic impact of PR and its association with the methylation levels of ESR1 promoters A (promA) and B (promB) at different timepoints in pts with ERpos HER2neg MBC who received first-line ET. Methods: Pts were enrolled in the prospective multicenter MAGNETIC.1 trial (CRO-2018-56) between January 2018 and January 2023. As first-line ET, pts received either fulvestrant or AIs with or without CDK4/6 inhibitors (CDK4/6i) as first-line ET. ctDNA samples were collected at baseline (T0) and at 3-month intervals (T3 and T6) and analyzed through methylation-specific (MS) droplet digital PCR (ddPCR), ddPCR fragmentomics and next generation sequencing (NGS). Clinico-pathological differences across the PR expression spectrum were analyzed using Fisher’s exact test. Matched pairs variations across timepoints (T0, T3, T6) of ACTBfragments distribution and ESR1 promA and promB were tested through Wilcoxon signed rank test. Results: Out of the 111 enrolled pts, 28 had PRneg MBC, of which 20 had an invasive ductal carcinoma (IDC) histotype, and 10 were diagnosed as de novo disease. Bone was the most common metastatic site both in the PRpos and PRneg subgroups (75% vs 73%), most pts received ET+CDK4/6i (96% vs 95%). ESR1 and PIK3CA mutations were respectively detected in 8% and 6%, and in 23% and 25% pts respectively for PRpos and PRneg. The prognostic impact of PR status was evident for both PFS (median 38 mos and 14 mos respectively for PRpos and PRneg, P = 0.0048) and OS (median not reached and median 33 mos respectively for PRpos and PRneg, P= 0.001). A significant increase in promB was observed at T3 vs T0 and at T6 vs T3 in the PRpos subgroup (respectively P=0.0158 and P< 0.001) but not in the PRneg. When comparing T6 to T0, no significant differences were observed regardless of PR status. At T3, significant decreases were observed in both the PRpos and PRneg populations for ACTBshort (P < 0.001 and P=0.03, respectively), ACTBmedium (P=0.0049 and P= 0.035, respectively) and ACTBlong(P< 0.001 and P< 0.001, respectively), compared to T0. In the comparison of T6 to T3, a significant variation in ACTBshort levels was observed in both subgroups (P< 0.001 for PRpos and P=0.012 for PRneg). while a significant reduction in ACTBmedium levels was observed only in the PRpos population when comparing T6 to T0 (P< 0.001). Conclusions: PRneg status was confirmed as an unfavorable prognostic factor with respect to PRpos both for PFS and OS, potentially due to different ET sensitivity. Epigenetic ctDNA profiling revealed different dynamics of ESR1 promB between PR-positive and PR-negative MBC, suggesting a distinct role of ESR1 in the onset of ET resistance within the two subgroups. Citation Format: Lorenzo Foffano, Elisabetta Molteni, Arianna Dri, Alessandra Franzoni, Linda Cucciniello, Lucia Da Ros, Silvia Buriolla, Silvia Bolzonello, Claudia Noto, Stefania Russo, Simon Spazzapan, Elena Nascimbeni, Brenno Pastò, Giada Targato, Serena Della Rossa, Marta Bonotto, Alessandro Marco Minisini, Giuseppe Damante, Barbara Belletti, Lorenzo Gerratana, Fabio Puglisi. Unraveling the influence of progesterone receptor status on endocrine therapy sensitivity: insight from epigenetic and fragmentomics ctDNA profiling in estrogen receptor positive, HER2 negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-15-09.

Abstract PO2-05-08: ISM5043, a novel, selective KAT6 inhibitor for the treatment of advanced and refractory ER+ breast cancer

Abstract Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death among women in the US. Approximately 70% of human breast tumors are estrogen receptor positive (ER+), and endocrine therapy (ET), which inhibits the estrogen signaling pathway, remains the backbone of therapy for ER+ breast cancer. However, ET resistance often develops, and patients eventually experience disease progression. This is a significant clinical challenge and highlights the urgent need for novel therapies that may help overcome the resistance. KAT6A is a histone (lysine) acetyltransferase (HAT) belonging to the MYST family. KAT6A and its paralog KAT6B play important roles in cell cycle progression, neuron stem cell maintenance and hematopoietic development via acetylation of H3K23. Molecular dysregulation of KAT6A, including amplification and fusion, has been reported in many cancers. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in about 10-15% of the ER+ breast cancer population, where it functions as an epigenetic modulator of ER expression, and is associated with a worse clinical outcome. Therefore, inhibition of KAT6A may be a promising therapy for ER+ breast cancer. Here, we evaluated ISM5043, a novel, small molecule KAT6 inhibitor for anti-tumor effects in ER+/HER2- breast cancer cell lines and animal models. ISM5043 demonstrated potent inhibitory activity against KAT6A (IC50, 8 nM) and KAT6B (IC50, 16 nM) with selectivity over KAT7 (IC50, 344 nM) and other HATs (IC50 >2000 nM). The compound showed robust, dose-dependent, anti-proliferation activity (IC50 < 10 nM) in multiple ER+/HER2- breast cancer cell lines with KAT6A amplification, in accordance with its suppression of H3K23 acetylation. Investigation of the mechanism of action revealed that treatment with ISM5043 resulted in the downregulation of the expression of ERα in these breast cancer cells. In the ZR-75-1 (ER+, HER2-) xenograft model, ISM5043 demonstrated robust and dose-dependent anti-tumor activity, as monotherapy, with Tumor Growth Inhibition (TGI) between 80~110% at doses ranging from 0.3 to 10 mg/kg, QD. ISM5043 (0.3 mg/kg, QD), in combination with tamoxifen (20 mg/kg, QD) exhibited synergistic effect against tumor growth, with a Q value of 1.25 based on Jin’s equation. Notably, ISM5043 displayed a strong anti-tumor effect (TGI of 67.7% and 83.6% at 3 and 10 mg/kg QD, respectively) as monotherapy, in a patient-derived ER+/HER2- breast cancer xenograft model representing disease progression following multiple prior lines of treatment (including palbociclib in combination with letrozole). ISM5043 also showed favorable ADME properties as well as good pharmacokinetic and safety profiles. Taken together, these data support the clinical evaluation of ISM5043 as a targeted therapy in patients with advanced and refractory ER+ breast cancer. Citation Format: Xin Cai, Xin Cheng, Luoheng Qin, Man Zhang, Junwen Qiao, Supriya Bavadekar, Sujata Rao, Feng Ren. ISM5043, a novel, selective KAT6 inhibitor for the treatment of advanced and refractory ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-05-08.

Abstract PO2-14-09: Baseline genomic alterations and the activity of lasofoxifene (LAS) plus abemaciclib (Abema) in patients with ER+/HER2- metastatic breast cancer (mBC): the ELAINE 2 study

Abstract Introduction: Estrogen receptor-positive (ER+)/HER2-negative (HER2-) breast cancers commonly acquire constitutively active mutations in the ERα-encoding gene (ESR1), resulting in endocrine therapy (ET) resistance. In the phase 2, ELAINE 2 study (NCT04432454), LAS (a breast ER antagonist) combined with Abema provided a median progression-free survival (PFS) of ~13 months (mos) and clinical benefit rate (CBR) of 66% in patients with endocrine-resistant, ER+/HER2- mBC and ESR1 mutations (mESR1; Damodaran. J Clin Oncol 2023;41:suppl16;1057). Our objective was to describe baseline genomic alterations co-occuring with mESR1 and treatment responses to LAS plus Abema in patients with these concurrent genomic alterations. Methods: Women (age ≥18 yrs) with ER+/HER2- mBC that progressed after prior ET and CDK4/6 inhibitor (CDK4/6i) with an mESR1 detected in blood using the Sysmex-Inostics SafeSeq circulating tumor DNA (ctDNA) test were enrolled in ELAINE 2. Oral LAS 5 mg/day and Abema (provided by Eli Lilly and Co) 150 mg BID were taken until disease progression, death, unacceptable toxicity, or withdrawal. The Guardant360 CDx test was used to identify and describe baseline genomic alterations in ctDNA (including mESR1). Median PFS and CBR were assessed in patients with mESR1 and co-existing baseline genomic alterations. For patients who withdrew before disease progression, their last observation time was used as the progression time for PFS estimation. Data were summarized descriptively with no formal hypothesis testing. Results: Of the 29 patients (median age 60 yrs) enrolled, mutations in ESR1 were identified in 26 by Guardant360; all these 26 patients had received prior CDK4/6i, 21 (81%) prior fulvestrant, and 12 (46%) prior chemotherapy for mBC. Alterations in 39 genes were concurrently identified with mESR1, including TP53 mutations (n=11), PIK3CA mutations (n=8), CCND1 amplifications (n=6), and FGFR1 amplifications (n=5); co-amplification of CCND1 and FGFR1 was identified in 3 patients. LAS plus Abema treatment was associated with a CBR of 73% and a median PFS of 12.9 mos in the 26 patients with mESR1 (Table). CBRs were 63% in the co-occurring PIK3CA mutation subgroup and 64% in the TP53 mutation subgroup; median PFS times were 7.8 and 8.3 mos, respectively. All patients in the CCND1 and FGFR1 amplification subgroups achieved clinical benefit (CBR 100%) and the median PFS was 16.6 mos for both subgroups. In patients with mESR1 and co-alterations in ≥2 other genes of interest (PIK3CA, FGFR1, CCND1, TP53, ERBB2, CCNE1, or RB1), CBR was 83% and median PFS was 16.6 mos. Conclusion: Using Guardant360 ctDNA profiling from patients in ELAINE 2, we demonstrate that other baseline genomic alterations are frequently detected concurrently with mESR1 in the endocrine resistant setting, but without apparent compromise on the efficacy of LAS plus Abema. These results should be interpreted with caution considering the small numbers of patients and the exploratory nature of the analysis. The ELAINE 2 study suggests the potential of LAS plus Abema for treating ESR1-mutated, ER+/HER2- mBC in the post-CDK4/6i setting. This will be further evaluated in the larger, registrational, phase 3, ELAINE 3 study (NCT05696626) that compares LAS plus Abema with fulvestrant plus Abema. Table. Treatment response by genomic alterations concurrent with ESR1 mutations (n=26). aFor patients who withdrew before disease progression, the last observation time was used as the progression time for PFS estimation. bOther genes of interest include PIK3CA, FGFR1, CCND1, TP53, ERBB2, CCNE1, or RB1. CBR, clinical benefit rate; mPFS, median progression-free survival. Citation Format: Senthil Damodaran, Massimo Cristofanilli, Matthew Goetz, Ciara O'Sullivan, Gary Riordan, Hope Rugo, Daniel Stover, Seth Wander, Dominic Carroll, Paul V. Plourde, David J Portman, Einav Nili-Gal Yam. Baseline genomic alterations and the activity of lasofoxifene (LAS) plus abemaciclib (Abema) in patients with ER+/HER2- metastatic breast cancer (mBC): the ELAINE 2 study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-09.

Abstract PO5-24-06: Genomic findings in metastatic breast cancer

Abstract Introduction: Although metastatic breast cancer is the second leading cause of cancer death in women, the genomic findings in metastatic breast cancer are not as well characterized as those of primary breast cancers. Metastatic tumors are genetically heterogenous, enriched for resistance mutations, and often lack targetable alterations. Improved understanding of the molecular mechanisms that drive metastatic disease of all receptor subtypes is required to identify possible molecular targets and to develop new targeted therapies in an effort to reduce the mortality. Methods: A cohort of 50 metastatic breast cancer cases (n=33 ER+/PR+, n=3 ER+/HER2+, n=3 ER-/HER2+ n=11 TNBC) sequenced at our medical center between 2018-2023 was evaluated, and the molecular findings of the major subtypes were analyzed. Genomic data included mutations, copy number alterations, tumor mutation burden and microsatellite instability. Results: The most frequent somatic alteration were PIK3CA mutations, involving 17/33 of ER+/PR+/HER- tumors, 5/11 TNBC tumors and 5/6 HER+ cases. TP53 mutations (frameshift and missense) were more frequently identified in TNBC and HER2+ cases (p< 0.05). Recurrent CCND1, FGFR1 and MYC copy number amplifications were seen almost exclusively in hormone receptor positive/HER2- tumors (p >0.05). ESR1 mutations involved 15% of the ER positive cancers and were frequently associated with co-occurring CCND1 and FGFR1 copy number gains. Significant genomic losses that drive tumorigenesis were also detected. Both CDKN2a and PTEN deletions were found in ER/PR positive disease, as well as triple negative tumors. Genomic deletions associated with worse outcomes such as TP53 and DICER1 were also detected. ERBB2 amplification was 100% concordant with our immunohistochemical and FISH results for the 6 HER2 positive cases. All 50 cases demonstrated a low tumor mutation burden and were microsatellite stable. Conclusion: We detected driver alterations that involve multiple pathways including estrogen receptor signaling, PI3K/AKT/MTOR, the cell cycle, and receptor tyrosine kinases. The genomic alterations are enriched for resistance mechanisms, like ESR1 mutations and FGFR1 gains that mediate resistance to endocrine treatment, as well as PIK3CA, also implicated in both endocrine and HER2-targeting therapy resistance. Of particular interest, were the amplifications detected in the ER+/PR+ tumors, such as CCND1 on chromosome 11q, FGFR1 on 8p and MYC on 8q, which were detected in almost half (45%) of our hormone receptor positive cohort. Some of the identified driver amplifications also define a subset of the Integrated Molecular Subtypes (Curtis et al., Nature; 486(7403): 346–352), i.e., Clusters 2, 6 and 9 that are typically hormone receptor positive but associated with either a very poor prognosis (Cluster 2) or an intermediate prognosis (Clusters 6 and 9), unlike the copy neutral ER positive tumors or those with the classic 1p and 16q alterations. Therefore, copy number profiling of advanced-stage breast cancers may be a useful tool for determining prognosis, identifying possible targetable driver pathways and selecting appropriate patients for molecularly guided clinical trials. Citation Format: Kimberly Cole, Melissa Tjota, Jeremy Segal, Peng Wang, Susanne Crespo-Ramos. Genomic findings in metastatic 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 PO5-24-06.

Abstract PO1-23-11: Differential m6A modification identified by direct RNA sequencing in endocrine- resistant and sensitive breast cancer cells

Abstract A major limitation of current adjuvant treatments for the ~ 70% of breast cancer patients with estrogen receptor α–expressing (ER+) primary breast tumors is the development of acquired and adaptive endocrine therapy (ET) resistance and metastatic spread. Chemical modifications of transcribed RNAs alters their cellular location, stability, and, for mRNAs, translation into proteins. N-6 methyladenosine (m6A) is the most common modification of mRNA. We tested the hypothesis that m6A epitranscriptomic changes alters the transcriptome resulting in changes in targets in pathways contributing to the metastatic cascade in ET-resistant breast cancer cells. We used an unbiased approach of direct mRNA sequencing using the Oxford Nanopore MinION platform on polyA-selected mRNA from MCF-7 luminal A breast cancer cells and ER+, ET-resistant LCC9 breast cancer cells treated with vehicle control or 100 nM 4-hydroxytamoxifen (4-OHT) for 24 h. In addition, MCF-7 cells were treated with the METTL3 (the catalytic methyltransferase that adds the methyl group from S-adenosylmethionine (SAM) to the adenosine on the N6 position) inhibitor STM2457 to identify m6A positions and transcripts directly regulated by METTL3 activity. Computational analysis directly identified m6A locations in transcripts. We examined the differential modification rate (DMR) between MCF-7 cells +/STM2457 treatment, between MCF-7 and LCC9 cells with vehicle control treatment or with 4-OHT treatment and within each cell line with 4-OHT treatment. We identified 183 and 3,097 genes with m6A DMRs between vehicle-treated and 4-OHT-treated MCF-7 and LCC9 cells, respectively. For 4-OHT-treated cells, we identified 2,401 and 2,638 genes with m6A DMRs in MCF-7 and LCC9 cells respectively with 1,760 genes in common. We identified many genes with multiple m6A modifications, e.g., AMIGO2 appeared to have 14 m6A sites at DRACH motifs in MCF-7 cells, but none were m6A modified in LCC9 cells. Enrichment by Pathway Maps, Process Networks, and GO Processes were performed. Overall, our data suggest a role for altered m6A positions in transcripts and pathways involved in ET-resistance in breast cancer. Citation Format: Belinda Petri, Bailey Valdes, Kellianne Piell, Eric Rouchka, Carolyn Klinge. Differential m6A modification identified by direct RNA sequencing in endocrine- resistant and sensitive breast cancer cells [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 PO1-23-11.

Abstract GS01-08: CDK4/6 inhibition is a potential vulnerability in NF1-depleted ER+ breast cancer

Abstract CDK4/6 control a key checkpoint by phosphorylating Rb, leading to Rb degradation and subsequent S-phase entry. CDK4/6 inhibition, together with endocrine therapy, is standard of care for advanced ER+ breast cancer; however, CDK4/6 inhibition typically is cytostatic. This study aims to identify breast cancer patients who may better respond to treatments targeting CDK4/6. NF1/neurofibromin is a key tumor suppressor that we have shown can represses not only RAS as a GAP but also ER as a transcriptional co-repressor. Dual activation of RAS and ER promotes endocrine therapy resistance. We computed kinase activity scores from phosphor-proteomic data in the CPTAC breast cancer cohort and found that both CDK4/6 activity scores and Rb-pS780 levels negatively correlated with NF1 protein levels. Furthermore, NF1 mutations co-occur with mutations in CDK4/6 and CCND1-3 in breast tumors. These results suggest a functional dependency between NF1 loss and activation of the CDK4/6-Rb pathway. To delineate how NF1 and CDK4 interact molecularly, we depleted NF1 in ER+ breast cancer cells and found an increase in Rb-pS780 and in phosphorylation in CDK4’s activation loop (CDK4-pT172). Cyclin-D binding is required for CDK4 activation. CCND1 expression is under the direct control by ER, and upon NF1-depletion, Cyclin D1 expression was increased, partly due to enhanced ER recruitment to an ERE in CCND1. Cyclin-D-bound CDK4 must be phosphorylated at T172 to be fully active; however, what kinase is responsible for this is largely unknown. We have evidence that CDK4-T172 phosphorylation is RAF, but not ERK, dependent. When the NF1-depleted ER+ cells were seeded in the presence of fulvestrant together with either palbociclib or abemaciclib, apoptosis readily occurred. Remarkably, fulvestrant plus palbociclib efficiently caused durable tumor regression in two ER+ PDX models, in which NF1 is undetectable by IHC. In contrast, no tumor regression was seen in a PDX model with detectable NF1. Finally, greater growth inhibition and apoptosis were detected in NF1-low tumors in the neoadjuvant NeoPalAna trial when palbociclib was added after anastrozole. These data support a model whereby ER and RAS signaling converge upon CDK4/6, and CDK4/6 activation is a key survival mechanism when ER signaling is attenuated by treatment in NF1-depleted ER+ breast cancer cells. This apparent addiction for CDK4/6 activity makes NF1-depleted ER+ breast tumors vulnerable to CDK4/6 inhibition, thus creating a potential therapeutic opportunity to match CDK4/6 inhibition with patients who can benefit the most. Citation Format: Ze-Yi Zheng, Anran Chen, Eric Jaehnig, Meenakshi Anurag, Jonathan Lei, Chenwei Wang, Long Feng, Purba Singh, Hilda Kennedy, Jin Cao, Ghazal Yadav, Jill Tsai, Xi Chen, Charles Foulds, Bora Lim, Matthew Ellis, Bing Zhang, Eric Chang. CDK4/6 inhibition is a potential vulnerability in NF1-depleted ER+ 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 GS01-08.

Abstract PO3-24-09: May ESR1 mutant breast cancer cells influence fibroblast phenotype?

Abstract For almost 50 years, it has been postulated that carcinogenesis relies on the intrinsic genetic abnormalities of tumor cells, but in the last decades, it has been reported that their functional interactions with the tumor microenvironment (TME) might affect therapy response and contribute to the disease progression. Based on these observations, the aim of the present study was to investigate the functional interactions between one of the essential components of TME, the fibroblasts, and breast cancer (BC) cells, expressing mutations in the hormone-binding domain (HBD) of the gene encoding for the estrogen receptor alpha (ESR1). Thus, we employed estrogen receptor-positive MCF-7 BC cells CRISPR engineered to express the Y537S-HBD mutation. Y537S is the most commonly detected mutation harbored in HBD-ESR1 and is a major contributor to endocrine therapy resistance and poor clinical outcome in BC. We collected conditioned media (CM) from BC cells and fibroblasts isolated from primary human tissues (normal fibroblasts, NFs, and cancer-associated fibroblasts, CAFs) in order to assess in vitro co-culture systems that can mimic the complex in vivo TME. It was interesting to observe that CAFs displayed a significant increase in their proliferation and migration when exposed to CM of mutant BC cells with respect to the parental ones. These latter findings fit with the immunofluorescence analysis showing a more evident formation of actin stress fibers. However, proteomic analysis demonstrated, in the same circumstances, more significant changes in NFs than in CAFs. Among the up-regulated proteins revealed by NFs upon Y537S-CM exposure, most of them overlap the up-regulated proteins featuring the intrinsic phenotype of CAFs, mainly involved in the cellular ultrastructural organization (i.e. cytoskeleton, focal adhesion, vesicle, and organelle) as evidenced by Metacore functional analysis. On the other side, we demonstrated that mutant clones showed more aggressive behavior in terms of proliferation, growth, migration, and invasion, compared to the parental counterpart upon exposure to the CM-derived from NFs and CAFs. This led us to further investigate the intrinsic properties of the mutated clones through their genomic and proteomic profiles that evidenced, by using Metacore software, an enrichment of insulin-like growth factor 1 receptor (IGF1R) pathway. The relevance of the latter datum was confirmed by the higher content of the IGF-1 ligand found in the secretome of mutant cells. Thus, the short autocrine loop maintaining IGF1/IGF1R signaling activation in mutant clones might reasonably play an important role in mediating the autocrine and paracrine effects between mutant cells and fibroblasts. For instance, the pharmacological blockade of IGF1R signaling interfered with the reciprocal interactions between fibroblasts and BC cells that sustained tumor growth and progression as evidenced in both “in vitro” and “in vivo” experiments. In conclusion, our study addressed the IGF1R pathway as a potential druggable target helpful to disconnect mutant breast cancer cell-fibroblast crosstalk that potentiates breast tumor growth and progression. Citation Format: Luca Gelsomino, Amanda Caruso, Rocco Malivindi, Adel Elisabetta Leonetti, Emine Tasan, Salvatore Panza, Giuseppina Daniela Naimo, Ines Barone, Cinzia Giordano, Daniela Bonofiglio, Loredana Mauro, Guowei Gu, Suzanne Fuqua, Stefania Catalano, Sebastiano Andò. May ESR1 mutant breast cancer cells influence fibroblast phenotype? [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 PO3-24-09.

Abstract PS17-03: Capivasertib and fulvestrant for patients with aromatase inhibitor-resistant HR positive/HER2-negative advanced breast cancer: exploratory analysis of PFS by AKT pathway gene from the Phase 3 CAPItello-291 trial

Abstract Background: In the Phase 3 randomized, double-blind CAPItello-291 trial, the addition of capivasertib (a potent, selective pan-AKT inhibitor) to fulvestrant in patients with aromatase inhibitor-resistant, hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative (HER2− defined as immunohistochemistry [IHC] 0, or 1-positive or IHC2-positive/in situ hybridization-negative) advanced breast cancer (ABC) significantly improved progression-free survival (PFS) versus placebo + fulvestrant (hazard ratio [HR] 0.60, 95% confidence interval [CI] 0.51–0.71; p< 0.001). PFS benefit was observed in patients with detectable AKT pathway alterations (HR 0.50, 95% CI 0.38–0.65; p< 0.001) and without (0.70; 95% CI; 0.56–0.88). Here, we report PFS by gene within the AKT pathway-altered population. Methods: Patients were randomized 1:1 to receive fulvestrant (500 mg intramuscularly on days 1 and 15 of cycle 1, and day 1 of each subsequent 28-day cycle) with either placebo or capivasertib (400 mg twice daily; 4 days on, 3 days off). AKT pathway-alteration status (at least one qualifying alteration in the genes PIK3CA, AKT1, or PTEN) was determined post-randomization, using next-generation sequencing in tumor tissue. HRs were calculated using Cox proportional hazards models. Data cut-off Aug 15, 2022. Results: Of the 708 patients randomized to treatment, 289 (41%) had AKT pathway-altered tumors (capivasertib-fulvestrant n=155; placebo-fulvestrant n=134). In the AKT pathway-altered population, 43% had liver metastases and 40% primary endocrine therapy resistance. Prior therapy for advanced disease included: 89% of patients with ≥1 line of prior treatment, 71% with a prior cyclin-dependent kinase 4 and 6 inhibitor, and 18% with prior chemotherapy. Baseline characteristics were broadly balanced between treatment groups. Most patients with an AKT pathway-altered tumor had only one detectable alteration (272/289, 94%). Thirteen patients (capivasertib-fulvestrant n=4; placebo-fulvestrant n=9) had co-occurring PIK3CA and PTEN alterations, and four patients (capivasertib-fulvestrant n=2; placebo-fulvestrant n=2) had co-occurring PIK3CA and AKT1 alterations. Consistent PFS benefit of capivasertib-fulvestrant over placebo-fulvestrant was observed across all alterations (Table). The safety profile of capivasertib-fulvestrant in the AKT pathway-altered population was consistent with the overall population. Conclusions: Compared with fulvestrant alone, the addition of capivasertib to fulvestrant provided a consistent PFS benefit across alterations in all three key genes within the AKT pathway in patients with HR-positive/HER2-negative ABC. https://clinicaltrials.gov/: NCT04305496 Funding: CAPItello-291 is sponsored by AstraZeneca. Editorial acknowledgment: AstraZeneca-funded medical writing support was provided by Suzanne Patel, Ph.D., from BOLDSCIENCE Inc. Capivasertib was discovered by AstraZeneca subsequent to a collaboration with Astex Therapeutics (and its collaboration with the Institute of Cancer Research and Cancer Research Technology Limited). Table. Citation Format: Sacha Howell, Hope Rugo, Mafalda Oliveira, Florence Dalenc, Javier Cortés, Henry Gómez, Xichun Hu, Komal Jhaveri, Petr Krivorotko, Sibylle Loibl, Serafin Morales Murillo, Meena Okera, Yeon Park, Joo Hyuk Sohn, Masakazu Toi, Eriko Tokunaga, Lyudmila Zhukova, Andrew Lloyd, Elza de Bruin, Coumaran Egile, Celina D’Cruz, Nicholas Turner. Capivasertib and fulvestrant for patients with aromatase inhibitor-resistant HR positive/HER2-negative advanced breast cancer: exploratory analysis of PFS by AKT pathway gene from the Phase 3 CAPItello-291 trial [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 PS17-03.

Estrogen Receptor Alpha Mutations, Truncations, Heterodimers, and Therapies.

Annual breast cancer (BCa) deaths have declined since its apex in 1989 concomitant with widespread adoption of hormone therapies that target estrogen receptor alpha (ERα), the prominent nuclear receptor expressed in ∼80% of BCa. However, up to ∼50% of patients who are ER+ with high-risk disease experience post endocrine therapy relapse and metastasis to distant organs. The vast majority of BCa mortality occurs in this setting, highlighting the inadequacy of current therapies. Genomic abnormalities to ESR1, the gene encoding ERα, emerge under prolonged selective pressure to enable endocrine therapy resistance. These genetic lesions include focal gene amplifications, hotspot missense mutations in the ligand binding domain, truncations, fusions, and complex interactions with other nuclear receptors. Tumor cells utilize aberrant ERα activity to proliferate, spread, and evade therapy in BCa as well as other cancers. Cutting edge studies on ERα structural and transcriptional relationships are being harnessed to produce new therapies that have shown benefits in patients with ESR1 hotspot mutations. In this review we discuss the history of ERα, current research unlocking unknown aspects of ERα signaling including the structural basis for receptor antagonism, and future directions of ESR1 investigation. In addition, we discuss the development of endocrine therapies from their inception to present day and survey new avenues of drug development to improve pharmaceutical profiles, targeting, and efficacy.

Epigenetic Control of Cancer Cell Dormancy and Awakening in Endocrine Therapy Resistance.

Rosano, Sofyali, Dhiman, and colleagues show that epigenetic-related changes occur in endocrine therapy (ET)-induced dormancy in estrogen receptor positive (ER+) breast cancer, as well as in its reawakening. Targeting these epigenetic changes blocks the entrance to dormancy and reduces the persister cancer cell population, enhancing the cytotoxic effects of ET in vitro. See related article by Rosano et al., p. 866 (9).

The prognostic value of MEK pathway-associated estrogen receptor signaling activity for female cancers.

Other than for breast cancer, endocrine therapy has not been highly effective for gynecologic cancers. Endocrine therapy resistance in estrogen receptor positive gynecologic cancers is still poorly understood. In this retrospective study, we examined the estrogen receptor (ER) signaling pathway activities of breast, ovarian, endometrial, and cervical cancers to identify those that may predict endocrine therapy responsiveness. Clinical and genomic data of women with breast and gynecological cancers were downloaded from cBioPortal for Cancer Genomics. Estrogen receptor alpha (ESR1) expression level and sample-level pathway enrichment scores (EERES) were calculated to classify patients into four groups (low/high ESR1 and low/high EERES). Correlation between ESR1/EERES score and survival was further validated with RNAseq data from low-grade serous ovarian cancer. Pathway analyses were performed among different ESR1/EERES groups to identify genes that correlate with endocrine resistance, which are validated using Cancer Cell Line Encyclopedia gene expression and Genomics of Drug Sensitivity in Cancer data. We identified a novel combined prognostic value of ESR1 expression and the corresponding estrogen response signaling (EERES score) for breast cancer. The combined prognostic value (ESR1/EERES) may be applicable to other gynecologic cancers. More importantly, we discovered that ER signaling can cross-regulate MEK pathway activation. We identified downstream genes in the MEK pathway (EPHA2, INAVA, MALL, MPZL2, PCDH1, and TNFRSF21) that are potential endocrine therapy response biomarkers. This study demonstrated that targeting both the ER and the ER signaling activity related MEK pathway may aid the development of endocrine therapy strategies for personalized medicine.