Endocrine Resistance Research Articles

Novel Endocrine Therapeutic Opportunities for Estrogen Receptor-Positive Ovarian Cancer—What Can We Learn from Breast Cancer?

Low-grade serous ovarian cancer (LGSOC) is a rare ovarian malignancy primarily affecting younger women and is characterized by an indolent growth pattern. It exhibits indolent growth and high estrogen/progesterone receptor expression, suggesting potential responsiveness to endocrine therapy. However, treatment efficacy remains limited due to the development of endocrine resistance. The mechanisms of resistance, whether primary or acquired, are still largely unknown and present a significant hurdle in achieving favorable treatment outcomes with endocrine therapy in these patients. In estrogen receptor-positive breast cancer, mechanisms of endocrine resistance have been largely explored and novel treatment strategies to overcome resistance have emerged. Considering the shared estrogen receptor positivity in LGSOC and breast cancer, we wanted to explore whether there are any parallel mechanisms of resistance and whether we can extend endocrine breast cancer treatments to LGSOC. This review aims to highlight the underlying molecular mechanisms possibly driving endocrine resistance in ovarian cancer, while also exploring the available therapeutic opportunities to overcome this resistance. By unraveling the potential pathways involved and examining emerging strategies, this review explores valuable insights for advancing treatment options and improving patient outcomes in LGSOC, which has limited therapeutic options available.

MICAL-L2, as an estrogen-responsive gene, is involved in ER-positive breast cancer cell progression and tamoxifen sensitivity via the AKT/mTOR pathway

Endocrine treatment, particularly tamoxifen, has shown significant improvement in the prognosis of patients with estrogen receptor-positive (ER-positive) breast cancer. However, the clinical utility of this treatment is often hindered by the development of endocrine resistance. Therefore, a comprehensive understanding of the underlying mechanisms driving ER-positive breast cancer carcinogenesis and endocrine resistance is crucial to overcome this clinical challenge. In this study, we investigated the expression of MICAL-L2 in ER-positive breast cancer and its impact on patient prognosis. We observed a significant upregulation of MICAL-L2 expression in ER-positive breast cancer, which correlated with a poorer prognosis in these patients. Furthermore, we found that estrogen-ERβ signaling promoted the expression of MICAL-L2. Functionally, our study demonstrated that MICAL-L2 not only played an oncogenic role in ER-positive breast cancer tumorigenesis but also influenced the sensitivity of ER-positive breast cancer cells to tamoxifen. Mechanistically, as an estrogen-responsive gene, MICAL-L2 facilitated the activation of the AKT/mTOR signaling pathway in ER-positive breast cancer cells. Collectively, our findings suggest that MICAL-L2 could serve as a potential prognostic marker for ER-positive breast cancer and represent a promising molecular target for improving endocrine treatment and developing therapeutic approaches for this subtype of breast cancer.

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 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 PO3-05-05: The effect of histological breast cancer subtype on progression free survival in patients with CDK4/6 inhibitors

Abstract Background: Multiple trials have proven that adding CDK4/6 inhibitors to endocrine treatment increases progression free (PFS) and overall survival (OS) of patients with metastatic estrogen receptor (ER)-positive HER2 negative breast cancer. However, only limited evidence is available of treatment efficacy of CDK4/6 inhibitors for invasive lobular carcinoma (ILC). This retrospective study aims to compare treatment duration of the three FDA/EMA approved CDK4/6 inhibitors in patients with no special type (NST) breast cancer versus patients with ILC. Methods: All patients with metastatic ER-positive HER2-negative breast cancer who were treated with a CDK4/6 inhibitor (1st, 2nd; 3rd line) in University Hospitals Leuven between December 2014 and February 2023, were included. A comparison of PFS and OS was made between patients with NST and ILC by use of the Kaplan Meier method. Other histological subtypes as well as mixed subtypes were excluded. Uni- and multivariable cox regression models were performed to quantify the association of histological subtype with PFS and OS. Results: A total of 418 patients were included of which 119 (28.5%) patients with ILC (median age at primary diagnosis 59 years, range 36 – 89 years) and 299 (71.5%) patients with NST (median age at primary diagnosis 55 years, range 23 – 90 years). Median follow-up was 26.8 months (range 1.1 – 88.1 months). Median PFS was 15.2 months (range 1.0 – 74.6 months) and 14.7 months (range 1.0 – 89.3 months) for patients with ILC and NST respectively. The OS rate after 60 months follow up was 39.2 % (CI 26.8 – 51.4) for ILC and 40.1% (CI 32.2 – 47.8) for NST. As shown in table 1, clear differences were observed in PFS rates after 12, 24 and 60 months between patients with NST that received CDK4/6 inhibitors in first line versus second or third line. These differences were less apparent for patients with ILC. For both NST and ILC, endocrine resistance at start of CDK4/6 inhibition impacted PFS rates negatively (table 2). In multivariable analyses, histological subtype was not predictive for CDK4/6 inhibition outcome (PFS: hazard ratio (HR) 0.996, CI 0.728 – 1.364, p-value 0.981; OS: HR 0.872, CI 0.602 – 1.263, p-value 0.468). Endocrine sensitivity vs. resistance at the time CDK4/6 inhibitor was started, was proven to be predictive for both PFS (HR 0.478, CI 0.337 – 0.678, p-value < 0.001) and OS (HR 0.528, CI 0.347 – 0.804, p-value 0.003). Conclusion: In our center, the histological subtype of breast cancer did not seem to impact PFS and OS significantly after treatment with CDK4/6 inhibition. Patients with NST seemed to have an increased benefit in PFS from treatment with CDK4/6 inhibition in first line as compared to later lines. While for patients with ILC, no difference between treatment lines were observed. Therefore, clinicians might be able to safely postpone CDK4/6 inhibitors to second- or third-line treatment in patients with metastatic ILC. Table 1: PFS rates by histological subtype and line of CDK4/6 treatment Table 2: PFS rates by histological subtype and endocrine sensitivity at start of CDK4/6 inhibition Citation Format: Cedric Bauters, Karen Van Baelen, Hans Wildiers, Giuseppe Floris, Sileny Han, Patrick Berteloot, Thaïs Baert, Ann Smeets, Ines Nevelsteen, Yannick Van Herck, Anne Deblander, Chantal Remmerie, Lieke Dullens, Maxime Van Houdt, Annouschka Laenen, Christine Desmedt, Patrick Neven. The effect of histological breast cancer subtype on progression free survival in patients with CDK4/6 inhibitors [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 PO3-05-05.

Abstract PO2-27-04: Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer

Abstract Fibroblast Growth Factor Receptor 1 (FGFR1) has emerged as a critical player in various cancer types, including breast cancer. FGFR1 alterations, including gene amplification, overexpression, and activating mutations, have been identified in subsets of breast cancers, contributing to aberrant signaling pathways that promote tumor growth, angiogenesis, and metastasis. Our current research endeavors are directed towards unravelling the intricate mechanistic underpinnings of FGFR1's dual impact on breast tumor progression, specifically its contribution to endocrine resistance and its regulatory role in innate immune sensing and subsequent induction of Type 1 interferons (IFNs) Our investigative efforts have previously established a direct association between FGFR1 and endocrine resistance in hormone receptor positive T47D breast cancer cells. Moreover, a remarkable and distinct inverse correlation linking FGFR1 expression to the Type 1 IFN response was observed. We noticed that in vitro stimulation of T47D cells with Type 1 interferons induces the expression of FGFR1 and ESR1, thereby suppressing the interferon-stimulated genes. Interestingly, we observed that the FGFR1-mediated suppression of the Type 1 interferon response is associated with intracellular nucleic acid sensors, specifically toll-like receptors TLR7 and TLR9. Furthermore, we found that the inhibition of FGFR kinase enhances the expression of IFN-alpha, IFN-beta, TLR7, and TLR9 in T47D cells when stimulated with the TLR7 agonist loxoribine. These results strongly suggest that FGFR1 functions as an innate checkpoint and may be exploited by tumor cells to dampen Type 1 interferon responses, thereby inhibiting antitumor immunity in hormone receptor-positive breast cancer. Citation Format: Sushil Dhakal, Torbjørn Amundsen Lien, Jens Henrik Norum, Helga Bergholtz, Helene Midtun Flatekvål, Therese Sørlie. Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance 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 PO2-27-04.

Abstract PO3-26-06: A self-replicating RNA Precision Immunotherapeutic for Overcoming Resistance to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer (ER+BC)

Abstract Advanced or metastatic ER+HER2- breast cancer (ER+BC) is an incurable disease. Standard 1L therapy (SOC) utilizes endocrine blockade with a nonsteroidal aromatase inhibitor (NSAI) combined with a CDK4/6 inhibitor (CDK4/6i). While most patients respond and disease remains controlled for over 2 years (mPFS ~25-28 months), virtually all patients progress over time, requiring more toxic therapies with decreasing periods of disease control. Prolonging the time that the tumor remains under endocrine control is therefore of significant medical value. The most common molecular drivers of endocrine resistance are well characterized and include mutations in ESR1 and bypass mechanisms in select signaling pathways. RBI-1000 is a self-replicating RNA (srRNA) precision immunotherapeutic to generate robust immunity directed against acquired resistance mutations that develop in ER+ BC in response to endocrine blockade by NSAI. RBI-1000 includes 6 on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in PI3K kinase or amplifications of HER2/HER3. Precision immunotherapy (PIO), combined with SOC targeted therapy, educates the patient’s immune system to eliminate tumor cells that express acquired resistance mutations. Thus, the tumor is in a lose-lose situation where it is either eliminated by the targeted therapy, or if it develops acquired resistance mutations to the targeted therapy, it is killed by the PIO. Specifically for endocrine resistance, RBI-1000 PIO offers an attractive therapeutic approach to prevent/delay endocrine resistance specifically, by generating an effective immune response against those tumor cells that express the resistance-associated molecules that arise while on NSAI+CDK4/6i. The ability to encode multiple targets in a single therapeutic srRNA molecule, alongside the prolonged expression of srRNA in host cells (>49 days) creates a versatile and powerful cancer drug development platform. srRNA precision immunotherapeutics are delivered intramuscularly (i.e. a jab in the non-dominant deltoid) and generate an effective immune response via the same pathways and mechanisms as a traditional vaccine. We have demonstrated that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells and antibody responses, leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutations. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. RBI-1000 is anticipated to enter clinical studies in the first half of 2024. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shiegeki Miyake-Stoner, Zachary C Hartman, Christopher Rabiola, Erika Crosby, Herbert Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA Precision Immunotherapeutic for Overcoming Resistance to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer (ER+BC) [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 PO3-26-06.

Abstract PO2-05-09: Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer

Abstract Purpose: All patients with metastatic breast cancer (MBC) expressing estrogen receptor-α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of patients, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Accumulating clinical evidence indicate adverse outcomes for these patients, beyond that expected by resistance to endocrine therapy. We hypothesized that ESR1 mutations may also confer resistance to chemotherapy. Experimental Design: MCF-7 cells harboring Y537S and D538G ESR1 mutations (mut-ER) were employed to study response to chemotherapy using viability and apoptotic assay in vitro, and tumor growth in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter and ChIP assays. MDR1 expression was analyzed in clinical samples using IHC. Results: Cell harboring ESR1 mutations displayed relative chemoresistance, evidenced by higher viability and reduced apoptosis as well as resistance to paclitaxel in vivo. To elucidate the underlying mechanism, MDR1 expression was examined and elevated levels were observed in mut-ER cells, and in clinical BC samples. MDR1 is regulated by the JNK/cJun pathway, and indeed, we detected higher JNK/cJun expression and activity in mut-ER cells, as well as increased occupancy of c-Jun in MDR1 promoter. Importantly, JNK inhibition decreased MDR1 expression, particularly of D538G-cells, and reduced viability in response to chemotherapy. Conclusions: Taken together, these data indicate that ESR1 mutations confer chemoresistance in BC through activation of the JNK/MDR1 axis. Targeting this pathway may restore sensitivity to chemotherapy and serve as a novel treatment strategy for MBC patients carrying ESR1 mutations. Citation Format: Marwa Taya, Keren Merenbakh-Lamin, Asia Zubkov, Zohar Honig, Ori Mayer, Noam Shomron, Wolf Ido, Tami Rubinek. Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway 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 PO2-05-09.

Abstract PO2-23-08: High intratumor heterogeneity induced PSME2 confers endocrine resistance via ERα PARylation in hormone receptor-positive and HER2-negative breast cancer

Abstract Background: Hormone receptor-positive and HER2-negative breast cancer, characterized by its significant intratumor heterogeneity (ITH), poses increased recurrence, metastasis risk, and endocrine resistance. A comprehensive understanding of the genomic and biological characteristics underlying ITH is crucial for the development of effective therapeutic strategies. Methods: We leveraged the multiomics data from our large cohort of 381 hormone receptor-positive and HER2-negative breast tumors, supplemented with single-cell sequencing (scRNA-seq) data from 9 individual samples, to investigate ITH and its associated molecular features. Mechanistic investigations were conducted using Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) proteomics, co-immunoprecipitation (Co-IP), Chromatin Immunoprecipitation followed by quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assays. We specifically focused on the role of Proteasome activator subunit beta (PSME2), a potential regulator of drug resistance and metastasis. Results: We quantified the degree of ITH at sample level using multi-omics and scRNA-seq data and observed a correlation between higher ITH levels, increased genomic instability, upregulation of the STING type-I-interferon-dependent pathway, and poor efficacy of endocrine therapy. Both bulk and single-cell data revealed an enrichment of PSME2, associated with type-I interferon, in groups exhibiting high ITH. Patients with elevated PSME2 expression manifested a poorer prognosis and diminished responsiveness to endocrine therapy. Within the context of ITH, we discovered that IFN-I transcriptionally activated PSME2 via STAT1. Further investigation revealed a complex interplay among PSME2, PARP1, and EWS, wherein PSME2-mediated degradation of EWS relieved the transcriptional repression of PARP1, resulting in its overexpression. This series of events, orchestrated by PSME2, led to ERα PARylation and promoted tamoxifen resistance. In subsequent clinical translation studies employing mouse xenograft, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models, we demonstrated that PARP1 inhibitors could effectively mitigate tamoxifen resistance induced by high PSME2 expression, providing new insights into therapeutic strategies. Conclusions: Our study highlights the importance of understanding ITH in hormone receptor-positive and HER2-negative breast cancer and identifies PSME2 as a key player in endocrine resistance and patient prognosis. The application of PARP1 inhibitors shows promise in improving treatment outcomes in tumors with elevated ITH and PSME2 expression. Working Modle Citation Format: Li-Ping Ge, Zi-Yu Wang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. High intratumor heterogeneity induced PSME2 confers endocrine resistance via ERα PARylation in hormone receptor-positive and HER2-negative 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 PO2-23-08.

Abstract PO3-14-01: Heterogeneity of tumor immune microenvironment to predict everolimus efficacy in premenopausal women with hormone receptor-positive/human epidermal growth factor receptor 2-negative adavanced breast cancer

Abstract Objective Everolimus (EVE), a mammalian target of rapamycin (mTOR) inhibitor, has been shown to prolong survival of patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-), advanced breast cancer (ABC) with endocrine resistance. Preclinical studies have suggested that mTOR is closely associated with the tumor immune microenvironment (TME). We aimed to explore the tumor heterogeneity of TME between patients with different response to everolimus (EVE), and identify potential markers to predict efficacy of EVE in premenopausal patients with HR+/HER2−, advanced breast cancer (ABC) suffering endocrine resistance. Methods Patients were divided into two groups according to the best response to EVE: the responsive subgroup (RS) and the non-responsive subgroup (NRS). Postoperative tumor tissue from patients who received EVE was collected for whole exome sequencing. Transcript abundance of 126 distinct tumor regions of interest (ROIs) were quantitated using digital spatial profiling, including 42 tumor cell zones (TCZs), 42 immune cell zones (ICZs), and 42 stroma cell zones (SCZs). Based on investigation in multiple discrete areas, differences in cell components, function and pathway, gene expression and immune cell infiltration between RS and NRS were investigated. Results Totally, 93 differentially expressed genes (DEGs) were identified in the ICZs, 174 DEGs in the TCZs, and 197 DEGs in the SCZs, using p< 0.05 and |log2FC >1| as the screening criterion. Among all the subgroup analyses on different ROIs, the statistical significance of PIP gene was the highest (ICZs: p=2.6E-12, TCZs: p=1.25E-20, SCZs: p=2.0E-18) in the comparison of RS and NRS. Moreover, we found that PIP was highly expressed in NRS and patients with a progression-free survival < 1 year. The receptor-legend analysis showed that, the intra-and inter-cellular communication in RS was mainly mediated by interaction between CCL-CCR family, while NRS mainly by COL family complex. Comparing all cell components extracted from each ROI, no statistical differences were found between RS and NRS, only a statistical trend in the SCZs (p=0.098). Further cell classification analysis showed fibroblasts highest in TCZs and SCZs, macrophages in ICZs. Compared to NRS, in TCZs, fibroblasts were significantly increased (p=0.01), and plasma cells (p=0.02) and neutrophils (p=0.03) were decreased in RS. In SCZs, the downregulated fibroblasts (p=0.04) and enhanced plasma cells (p=0.01) were observed in NRS. In ICZs, pDCs (p=0.048) and neutrophils (p=0.006) were significantly elevated in NRS compared with RS. Conclusion The utility of digital spatial gene expression profiling might provide some references to predict the efficacy of EVE in premenopausal patients with HR+/HER2- ABC with endocrine resistance. Citation Format: Yujing Tan, Fei Ma, Jiayu Wang, Pin Zhang, Binghe Xu, Liyan Xue, Ying Fan. Heterogeneity of tumor immune microenvironment to predict everolimus efficacy in premenopausal women with hormone receptor-positive/human epidermal growth factor receptor 2-negative adavanced 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 PO3-14-01.

Abstract PO5-04-12: Comparison of chemotherapy efficacy in metastatic lobular vs. ductal breast cancer

Abstract Comparison of chemotherapy efficacy in metastatic lobular vs. ductal breast cancer Background: Invasive lobular carcinomas (ILC) are thought to be less chemo-sensitive than invasive ductal carcinomas (IDC), as reflected by lower rates of pathological complete response following neoadjuvant therapy. In the metastatic setting, evidence regarding chemotherapy (Cx) efficacy is limited. Methods: A retrospective review of prospectively collected data for consecutive patients (pts) with metastatic ILC (mILC) treated at a single institution between 2000 and 2023 was included. Pts with mILC were matched on a 1:2 ratio with a cohort of pts with metastatic IDC (mIDC) by age, era of diagnosis and initial metastatic burden. Primary outcome was efficacy of Cx in mILC vs. mIDC pts as measured by time to next treatment - TTNTc (months (m) between start date of first & second Cx). Key secondary endpoints included differences in efficacy of endocrine therapy between the two groups (as measured by TTNT and outcomes in pts with endocrine resistance (EnR) as defined by ESMO consensus guideline) and overall survival (time from diagnosis of metastatic breast cancer (BC) to death or last follow-up [OS]). Results: 376 pts were included, mILC (122) and mIDC (254) with median age 64y and 62y, respectively. A significantly higher proportion of mILC pts had hormone receptor positive/HER2 negative or lower proportion of triple negative disease (p< 0.0001). Compared to mIDC, mILC pts were more likely to have de novo disease (29% vs. 18%; p=0.02), lower grade (p< 0.0001), received endocrine therapy only (p=0.03) and were less likely to have visceral disease at diagnosis or any time after metastatic diagnosis (20% vs. 45% p< 0.0001, 35% vs. 51% < 0.0004, respectively). Median time to commencing chemotherapy from metastatic diagnosis was longer in mILC pts (5.8 v 2.0 m, p=0.03). There was no significant differences in TTNTc for patients with mILC and mIDC except for those with visceral disease at diagnosis (Table 1). There was no significant difference in TTNTc by histological subtype of mILC (p=0.46) or by choice of first line chemotherapy used (p=0.91). In the entire population, no significant OS difference observed in mILC and mIDC (Table 1). BC outcome was assessed in pts receiving endocrine therapy as first-line treatment (mILC 65, mIDC 102). There was no difference in TTNT (mILC 14.6m vs. mIDC 14.8m). There were no differences in incidence of EnR between the groups. OS was shorter in mILC pts with primary EnR. The presence of primary, secondary or no resistance within each histological group was significantly associated with OS (Table 1). Conclusion: Pts with mILC have a longer time to commencing chemotherapy, but efficacy of chemotherapy is similar to pts with mIDC; irrespective of lobular histologic subtype, first line chemotherapy regimen used and sites of disease, except for those mILC with visceral disease. There was no significant difference in OS between mILC and mIDC pts treated in this single centre study. Efficacy of endocrine treatment when given as first line treatment was similar in both groups with no significant difference when pts were evaluated by presence or absence of endocrine resistance, except for primary EnR, but pt numbers were small in this group. Our results demonstrate equivalent chemotherapy efficacy in pts with mILC as compared to mIDC.

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 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 PO3-05-04: Characteristics and clinical outcome of patients with HR positive HER2 low metastatic breast cancer treated with CDK 4/6 inhibitors

Abstract Introduction Breast cancer (BC) is a disease characterized by significant intra- and intertumoral heterogeneity. Hence, it is not surprising that new subtypes with distinct biological features are being discovered, even among previously well-defined BC groups. Recently, HER2-low BC emerged as a new entity with specific clinical behavior, response to treatment and prognosis. HER2-low is a subset of HER2-negative BC, with HER2 immunohistochemical (IHC) score of 1+ or 2+, without HER2 gene amplification measured by in situ hybridization (ISH). As new therapeutic options become available for HER2-low patients, the best treatment sequence is yet to be determined. Furthermore, it is important to distinguish whether there is a difference in the response to standard treatment lines, such as CDK 4/6 inhibitors in metastatic HR positive BC patients, depending on HER2-low status. Methods A retrospective study of 369 metastatic BC (mBC) cases who started CDK 4/6 inhibitor therapy from January 2018 through December 2022 at University Hospital Centre Zagreb was conducted, with prior Ethics Committee approval. All patients with HR positive HER2 negative mBC, determined by standard IHC and ISH, were included in the research. Patient demographics and clinical presentation, tumor characteristics and treatment information were collected. Progression-free survival (PFS) analysis was done with the final data cut-off date being June 1st, 2023. Type 1 right censoring was performed. The data was analyzed using the Kaplan-Meier method and Cox proportional-hazards regression for clinically relevant covariates (age, line of treatment, de novo metastatic disease, endocrine resistance, liver metastases, and detected PIK3CA mutation). Results Median follow-up was 23 months. Of the 283 patients included, 146 (51.59%) had HER2-low disease. A change in HER2 expression between primary tumor and metastasis was found in 16.96% (N=48) patients. Of them,10.25% (N=29) who were initially HER2-low, were found to be HER2-0 in metastatic disease. Meanwhile, 6.71% (N=19) of patients had a change in HER2 expression from 0 to low upon becoming metastatic. In the HER2-low group, 47.06% (N=45) patients had a PIK3CA mutation as opposed to 33.33% (N=30) in the HER2-0 group. Odds ratio for a PIK3CA mutation in HER2-low patients was 1.86 (95% confidence interval (CI): 1.01-3.43, p-value 0.046). Median PFS in the HER2-low group was 18 months (95% confidence interval (CI): 14-24) versus 23 (95% CI: 18-30) in the HER2-0 group. Using multivariable analysis an adjusted hazard ratio of 1.15 (95% CI:0.84-1.57; p-value  0.389) was calculated. Covariates associated with a statistically significant increased risk of disease progression were a higher line of therapy (HR 1.39, 95% CI 1.36-1.71, p-value 0.002) and the presence of liver metastases (HR 2.17, 95% CI 1.42-3.32, p-value 0.0004). A covariate associated with a statistically significant longer PFS was de novo metastatic disease (HR 0.63, 95% CI 0.41-0.97, p-value 0.034). Conclusion There was a trend toward worse PFS in HER2-low mBC that did not reach statistical significance. HER2-low patients were more likely to harbor PIK3CA mutations than HER2-0 patient group. Longer follow-up and a larger cohort are needed in order to make definitive conclusions. Citation Format: Katarina Čular, Kristina Kanceljak, Ana Magdalena Glas, Dora Gudelj, Marija Križić, Marina Popović, Natalija Dedić Plavetić, Maja Sirotković-Skerlev, Stjepko Pleština, Tajana Silovski. Characteristics and clinical outcome of patients with HR positive HER2 low metastatic breast cancer treated with CDK 4/6 inhibitors [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 PO3-05-04.

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 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 PO5-13-02: BRCA mutational profiles of endocrine-resistant breast cancer

Abstract Background Around 75% of breast cancer (BC) patients have tumors expressing the treatment-predictive biomarker estrogen receptor α (ER) and are consequently offered endocrine therapy. One-third of these patients develop endocrine resistance, a majority with tumors that retain ER expression. For most cases, the resistance mechanism is still unknown, but mutational activity is a suggested mechanism. Germline BRCA mutations are well-known in BC and are found in approximately 23% of patients with triple-negative BC and in 5% of patients with ER+ BC. However, less is certain about the somatic variants, though studies have shown prevalences of around 3% in BC. This study aimed to examine the somatic BRCA mutational profiles of endocrine-resistant paired primary and relapse tumors. Methods A retrospective cohort of verified endocrine-resistant BC patients was collected at Karolinska University Hospital in Stockholm, Sweden. Patients diagnosed in 2008-2012 with an ER+ and human epidermal growth factor receptor 2 (HER2) negative primary tumor and a following ER+ and HER2- relapse tumor within five years of ongoing endocrine therapy were included (N=63). DNA was extracted from archived formalin-fixed paraffin-embedded relapse and primary tumor tissue and analyzed by panel sequencing. Tumor-free lymph nodes were used as germline normal samples. Results The analysis showed somatic BRCA mutational activity in 38.1% of the patients. Variants were present in 20.3% of primary tumors and 30.4% of relapse tumors. BRCA1 mutations were present in 11.9% of primary tumors and 21.4% of relapse tumors. BRCA2 variations were, in turn, seen in 11.9% of primary tumors and 14.3% of relapse tumors. The frequency of patients that had alterations in both the primary and relapse tumors was 3.2% for BRCA1 and BRCA2 respectively. Conclusion This study of a unique endocrine-resistant BC cohort shows a high presence and varying distribution of somatic BRCA mutations in both primary and even higher in relapse tumors. These results suggest that mutational testing for possible further treatment decisions may be relevant for patients exhibiting endocrine resistance, especially in the advanced setting. Citation Format: Emelie Karlsson, Caroline Schagerholm, Stephanie Robertson, Hosein Toosi, Emmanouil Sifakis, Johan Hartman. BRCA mutational profiles of endocrine-resistant 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 PO5-13-02.

Abstract PS09-08: Genomic characterization of endocrine resistance in ER+HER2+ breast cancers in the POETIC Trial

Abstract Background: Mechanisms of resistance to endocrine therapy are not well understood within ER+HER2+ breast cancer (BC). Our prior work suggested that intrinsic HER2-Enriched (HER2E) molecular subtype predicts early resistance to aromatase inhibitors (AI) (Bergamino eBioMedicine 2022) and high on-treatment (on-Txt) Ki67 levels predict poor survival (Smith Lancet Oncol 2020). Improved early detection of persistent proliferating tumor cells with endocrine resistance pathways could be targeted by pre-emptive personalized therapy and reduction in recurrence. In this study, we proposed to further identify additional alterations/features from genomic and spatial data to provide unprecedented new insight into intrinsic and adaptive resistant pathways in tumor cells that may assist to identify molecular targets for treatment. Materials: POETIC was a phase III trial of post-menopausal patients with ER/PR+ invasive BC (n = 4480) randomized 2:1 to 2 weeks of peri-operative AI (POAI) vs control, followed by standard-of-care treatment. Ki67 was assessed by IHC and intra-tumor heterogeneity was evaluated (5-15 regions) for all the POETIC POAI samples (N = 2487). ER+HER2+ samples were classified as good responders (GR) or poor responders (PR) based on a reduction in Ki67 between pre-treatment (pre-Txt) and 2-week on-Txt samples. Tumor-infiltrating lymphocytes were assessed; multiplex Immunofluorescence (mIF) was performed to measure immune cell densities in tumor and stroma compartments (CD3, CD20, CD68, FOXP3, and CD3 FOXP3 co-expression). Gene expression profiles by BC360™ (Nanostring) on all 210 pairs of POAI treated ER+/HER2+; whole exome sequencing (WES, 100X) were performed on pre-Txt tumor and blood samples from 13 GR, 17 PR, and 9 HER2E GR. We performed GeoMx Whole Transcriptome on 4 pairs (pre-Txt and on-Txt) of GR and GeoMx Proteins (77 including IO proteins) on 6 pairs of GRs and 6 pairs of PRs. Results: The most frequently mutated genes were TP53, PIK3CA, GATA3, and CHD4. Only TP53 was associated with PR (Fisher’s exact p=0.01). TP53 mutated cases had higher expression of TP53 mutant-like gene expression signature compared to wild-type cases (Wilcoxon test p=0.001), mIF FOXP3 (Wilcoxon test p = 0.0005), and CD68 (Wilcoxon test p = 0.019) density score. However, within the HER2-E subset, we found that TP53 mutations were associated with GR (Fisher’s exact p=0.02). We found spatial heterogeneity of Ki67 IHC levels across POAI samples. Examining IHC, while there was higher heterogeneity of Ki67 in the ER+HER2- samples (n = 2264) with 3% of pre-Txt and 9% on-Txt, 6% of ER+HER2+ samples (13/223, 6 LumA, 5 LumB, and 2 HER2E) showed heterogeneity of Ki67 exclusively on-Txt. Even in GR tumors with Ki67 < 10% on-Txt, we identified hotspots with retained proliferating Ki67+ cells after 2 weeks of POAI. The lobular tumors were GR and had characteristic CDH1 mutations. Importantly, cases with persistent areas of Ki67+ cells, regardless of Her2 status, were associated with late relapse. To further explore intratumoral heterogeneity, we performed spatial whole transcriptomics profiling on 95 regions from 4 pairs of GR samples (Ki67 > 10% at baseline and Ki67 < 10% on-Txt) and found low intratumoral heterogeneity in the pre-Txt samples that increased at 2 weeks on-Txt. In a larger set of samples including both GR and PR with the GeoMx protein method, we found increased intratumoral heterogeneity in the PR vs GR. Conclusion: While TP53 mutation was generally a predictor of poor response; in HER2-E it paradoxically was associated with a good early response to aromatase inhibitor which warrants further investigation. Ki67 levels in ER+HER2+ showed higher intratumoral heterogeneity in a subset of patients on treatment suggesting the potential of persistent, proliferating cells leading to later recurrence. Our spatial RNA and protein data further observe the intratumoral heterogeneity that identifies pathways for use as potential spatial biomarkers. Citation Format: Maggie Chon U Cheang, Xixuan Zhu, Orsolya Sipos, Anastasia Alataki, Mikayla Feldbauer, Elena López-Knowles, Holly Tovey, Lucy Kilburn, Milana Bergamino Sirvén, Dhrusti Patel, Hui Xiao, Perry Maxwell, Anthony Skene, Chris Holcombe, Manuel Salto-Tellez, Nicholas Turner, Andrew Dodson, Ian Smith, John Robertson, Judith Bliss, Gene Schuster, Roberto Salgado, Mitch Dowsett, Katherine A Hoadley. Genomic characterization of endocrine resistance in ER+HER2+ breast cancers in the POETIC 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 PS09-08.

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 PO3-23-11: 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.