Endocrine-resistant Metastatic Breast Cancer Research Articles

Molecular profiling of serial liquid biopsy specimens utilizing cell free DNA (cfDNA) and circulating tumor cells (CTCs) in TBCRC041: A phase II study of alisertib in endocrine resistant metastatic breast cancer (MBC).

1037 Background: TBCRC041, a randomized phase 2 trial of alisertib (A) ± fulvestrant (F) in women with HR+/HER2- MBC demonstrated promising clinical activity for alisertib (1). Interrogation of baseline (PreC1) and end of cycle 1 (EOC1) cfDNA and CTCs may identify biomarkers associated with response to A±F. Methods: Plasma cfDNA was sequenced using the Guardant INFINITY platform, which includes genomic and methylated tumor fraction (mTF) analysis. Pathogenic variants were determined using publicly available databases and Mayo Clinic annotation pipelines. CTCs were identified as nucleated, EpCAM+/ cytokeratin+/ CD45- cells and assessed for ER/HER2 staining (RareCyte, Seattle). PFS was defined as the time from randomization to progression or death. EOC1-PFS differs from PFS as time starts at C2D1. Stratified log-rank tests and conditional Cox modeling were used to assess whether PFS or EOC1-PFS differed by mutations in ESR1 or PIK3CA, pre-CTC ≥ 5, or mTF>1%. Results: Of the 91 eligible pts, 84 progressed, and 73 were deceased. In the 86 pts with PreC1-cfDNA results, variants were identified in ESR1 (42; 48.8%), PIK3CA (36; 41.9%), AKT1 (7; 8.1%), PTEN(7; 8.1%), BRCA1 (4; 4.7%), and BRCA2 (7; 8.1%). Pts with a PIK3CAmut had significantly decreased PFS (HR 1.8; 95%CI: 1.1 -2.9, p=0.014) but not by ESR1mut status (p=0.583). PreC1 and EOC1-mTF were determined for 78 and 76 pts, respectively. PreC1-mTF was 0-1% in 21; 1.1-10.0% in 27; and 10.1-100% in 30. mTF levels remained between 0-1% in 17 pts fell to 0-1% in 11 pts, and was 0-1% in 5 pts without a preC1-mTF determination. PFS did not differ with respect to PreC1-mTF (p=0.210), but EOC1-PFS was worse in pts with mTF >1% (HR 3.0; 95%CI: 1.6 -5.2, p<0.001). PreC1 and EOC1-CTC were determined for 78 and 76 pts. 36 (46.2%) pts had a PreC1 CTC count ≥ 5. EOC1-CTC levels remained at (n=17) or increased to (n=1) ≥ 5 and was ≥ 5 in 2 pts without a preC1-CTC determination. EOC1-PFS was not significantly different for EOC1-CTC ≥ 5 (p=0.065), but was significantly decreased in pts with PreC1-CTC≥5 (HR=1.8; 95%CI: 1.1-3.0; p=0.018). Broader cfDNA NGS panel and CTC-derived ER/HER2 staining results will be presented at the meeting. Conclusions: Among patients receiving A±F, PFS varied based on the PIK3CAmutation status. cfDNA and CTC assessment PreC1 and EOC1 provided complementary information. Further validation in larger datasets is needed. 1. JAMA Oncol 2023;9:815. Clinical trial information: NCT02860000 .

Abstract PO2-16-02: RBsig gene-expression signature in patients with endocrine resistant metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial

Abstract Background: We have previously identified RBsig, a gene-expression signature of resistance to CDK4/6 inhibitors composed of E2F1/E2F2 dependent genes, which is associated with genetic loss of RB1. Although RBsig has been previously tested in vitro and in clinical trials with CDK4/6 inhibitors, further clinical validation is needed. Additionally, data on the correlation of RBsig with other prognostic/predictive biomarkers for CDK4/6 inhibitors such as serum Thymidine Kinase activity (sTKa), is lacking. Here, we aimed to investigate RBsig in patients (pts) treated with palbociclib (P) + fulvestrant (F) within the PYTHIA trial (IBCSG 53- 14/BIG 14-04; NCT02536742), a downstream trial of the AURORA molecular program (BIG 14-01; NCT02102165). Methods: PYTHIA is a biomarker discovery phase II trial which enrolled 122 pts with endocrine-resistant ER+ and HER2 - metastatic breast cancer (MBC) who received P+F at standard schedule and dose. We analyzed a subset of 38 pts enrolled in PYTHIA and successfully included in AURORA who had available RNAseq from tumor tissue samples (24 primary, 24 metastatic, 10 both). Ki67 by IHC was also available. The PYTHIA biomarker assessment included sTKa at baseline and during treatment (D15 and D28) measured using DiviTumÒ TKa (Biovica International). Evaluation of the prognostic role of RBsig was a pre-specified primary objective of PYTHIA. RBsig is composed of 87 genes and was calculated as previously described. Wilcoxon rank sum test was used to compare continuous RBsig values between groups defined by sTKa. Median progression free survival (mPFS) was estimated using Kaplan-Meier method for groups defined by dichotomizing RBsig at zero. Results: Median RBsig was higher in metastatic than in primary samples; in paired samples, RBsig increased in metastatic for 7/10 pts vs primary. When evaluated as continuous variables, RBsig correlated with tissue Ki67 both in primary (Spearman R 0.8) and in metastatic (R 0.68) samples. Additionally, RBsig was higher in Basal and Luminal B intrinsic subtypes in metastatic samples. RBsig was significantly higher in pts with high baseline sTKa (median cut-off) (Wilcoxon test, p=0.04 and p=0.03 in primary and metastatic samples respectively), with a similar direction, albeit not statistically significant, observed in the group of pts with no sTKa clearance at D15 (below limit of detection cut-off). When evaluated in the combined cohort, where RBSig values were taken from metastatic samples when available and from primary samples otherwise, higher RBsig values were associated with poorer PFS as compared to lower RBsig values [mPFS 5.5 months (95% CI 3.7, 14) vs 13 months (95% CI 8.6, 28); Log-rank p-value=0.004]. Conclusions: Higher RBsig was associated with higher tumor proliferation (measured by both tissue ki67 and sTKa), luminal B status, and shorter PFS on treatment with P+F. This analysis reinforces prior data on RBsig prognostic significance in pts treated with CDK4/6 inhibitors. Further investigation on the added value of RBsig as a novel biomarker for patients’ stratification, including in the early setting, is warranted. Citation Format: Matteo Benelli, Svitlana Tyekucheva, Thayane Crestani, Michail Ignatiadis, Patrick Neven, Marco Colleoni, Stéphanie Henry, Konstantinos Papadimitriou, Antonio Bernardo, Elena Seles, François Duhoux, Iain Macpherson, Alastair Thomson, David Mark Davies, Ilenia Migliaccio, Gabriele Zoppoli, Roswitha Kammler, Heidi De Swert, Barbara Ruepp, Philippe Aftimos, Jose Seoane, Dario Romagnoli, David Venet, Theodora Goulioti, David Cameron, Sherene Loi, Martine Piccart, Meredith Regan, Luca Malorni. RBsig gene-expression signature in patients with endocrine resistant metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA 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 PO2-16-02.

Proton Pump Inhibitors and Cyclin-Dependent Kinase 4/6 Inhibitors in Patients With Breast Cancer.

Proton pump inhibitors (PPIs) reduce the bioavailability of several anticancer drugs. The impact of PPIs co-administered with cyclin-dependent kinase 4 and 6 inhibitors is controversial. We aimed to clarify whether the concomitant use of PPIs impacts palbociclib and abemaciclib effectiveness in breast cancer treatment. This multicenter, retrospective, observational study, conducted across 4 medical institutions in Japan, consecutively included patients with endocrine-resistant metastatic breast cancer, receiving palbociclib or abemaciclib between December 2017 and August 2022. Propensity score-matched analyses were performed. Treatment efficacy and safety with and without PPIs were compared. Progression-free survival and overall survival were estimated using the Kaplan-Meier method and compared using a log-rank test. A Cox proportional hazards model was used to estimate the hazard ratio. The study included 240 patients. After 1:1 matching, 112 patients were treated with and without PPIs. The median progression-free survival period was 1.2 years in the PPI group and 1.3 years in the non-PPI group (hazard ratio, 1.19; 95% CI, 0.70-2.02). The median overall survival period was 3.6 years in the PPI group, whereas it was not reached in the non-PPI group (hazard ratio, 1.23; 95% CI, 0.61-2.47). Consistent results were obtained for subgroups receiving palbociclib (n = 177) and abemaciclib (n = 63) without propensity score matching. Adverse event incidence and severity were similar in both groups. The effectiveness of cyclin-dependent kinase 4/6 inhibitors is unlikely to be affected by concomitant PPI use. Future prospective pharmacokinetic studies are warranted.

P4EBP1 staining predicts outcome in ER-positive endocrine-resistant metastatic breast cancer patients treated with everolimus and exemestane.

To identify patients most likely to respond to everolimus, a mammalian target of rapamycin (mTOR) inhibitor, a prospective biomarker study was conducted in hormone receptor-positive endocrine-resistant metastatic breast cancer patients treated with exemestane-everolimus therapy. Metastatic tumor biopsies were processed for immunohistochemical staining (p4EBP1, PTEN, pAKT, LKB1, and pS6K). ESR1, PIK3CA and AKT1 gene mutations were detected by NGS. The primary endpoint was the association between the p4EBP1 expression and clinical benefit rate (CBR) at 6 months of everolimus plus exemestane treatment. Of 150 patients included, 107 were evaluable for the primary endpoint. p4EBP1 staining above the median (Allred score ≥6) was associated with a higher CBR at 6 months (62% versus 40% in high-p4EBP1 versus low-p4EBP1, χ2 test, p = 0.026) and a longer progression-free survival (PFS) (median PFS of 9.2 versus 5.8 months in high-p4EBP1 versus low-p4EBP1; p = 0.02). When tested with other biomarkers, only p4EBP1 remained a significant predictive marker of PFS in multivariate analysis (hazard ratio, 0.591; p = 0.01). This study identified a subset of patients with hormone receptor-positive endocrine-resistant metastatic breast cancer and poor outcome who would derive less benefit from everolimus and exemestane. p4EBP1 may be a useful predictive biomarker in routine clinical practice. NCT02444390.

Upfront Taxane Could Be Superior to Pegylated Liposomal Doxorubicin (PLD): A Retrospective Real-World Analysis of Treatment Sequence Taxane-PLD versus PLD-Taxane in Patients with Metastatic Breast Cancer.

Background: Patients with endocrine-resistant metastatic breast cancer (MBC) require cytostatic therapy. Single-agent taxanes and anthracyclines, including pegylated liposomal doxorubicin (PLD), are standard treatment options. There are no prospective data regarding optimal treatment sequences, and real-world data regarding both treatment options are limited. Methods: We analyzed electronic records of all patients with Her2-negative MBC treated with either first-line PLD or first-line taxane and subsequent crossover at the University Hospital Basel between 2003 and 2021. The primary endpoint was time to next chemotherapy or death (TTNC). Secondary endpoints were overall survival (OS), progression-free survival (PFS), and objective response rate (ORR). We used the Kaplan-Meyer method and logrank test to compare time-to-event endpoints and the Fisher exact test to compare discrete variables. Results: We retrospectively identified 42 patients with Her2-negative MBC who have received either single-agent PLD or single-agent taxane as first-line chemotherapy with subsequent crossover, including 23 patients who received first-line PLD and 19 patients who received first-line taxane. Baseline characteristics were similar between treatment groups. Treatment sequence PLD-taxane was significantly inferior to taxane-PLD regarding all endpoints: median TTNC 4.9 vs. 9.9 months (p = 0.006), median OS 17.8 vs. 24.6 months (p = 0.05), median PFS 4.4 vs. 9.0 months (p = 0.005), and ORR 13% vs. 53% (p = 0.01). Conclusions: Here, we report a first retrospective head-to-head comparison of the treatment sequence PLD-taxane versus taxane-PLD in patients with MBC, showing a substantial advantage of using taxanes first, followed by PLD. An inherent treatment bias in favor of first-line taxanes cannot be excluded, thus calling for prospective validation.

Genomic Alterations Associated with Estrogen Receptor Pathway Activity in Metastatic Breast Cancer Have a Differential Impact on Downstream ER Signaling.

Mutations in the estrogen receptor gene (ESR1), its transcriptional regulators, and the mitogen-activated protein kinase (MAPK) pathway are enriched in patients with endocrine-resistant metastatic breast cancer (MBC). Here, we integrated whole genome sequencing with RNA sequencing data from the same samples of 101 ER-positive/HER2-negative MBC patients who underwent a tumor biopsy prior to the start of a new line of treatment for MBC (CPCT-02 study, NCT01855477) to analyze the downstream effects of DNA alterations previously linked to endocrine resistance, thereby gaining a better understanding of the associated mechanisms. Hierarchical clustering was performed using expression of ESR1 target genes. Genomic alterations at the DNA level, gene expression levels, and last administered therapy were compared between the identified clusters. Hierarchical clustering revealed two distinct clusters, one of which was characterized by increased expression of ESR1 and its target genes. Samples in this cluster were significantly enriched for mutations in ESR1 and amplifications in FGFR1 and TSPYL. Patients in the other cluster showed relatively lower expression levels of ESR1 and its target genes, comparable to ER-negative samples, and more often received endocrine therapy as their last treatment before biopsy. Genes in the MAPK-pathway, including NF1, and ESR1 transcriptional regulators were evenly distributed. In conclusion, RNA sequencing identified a subgroup of patients with clear expression of ESR1 and its downstream targets, probably still benefiting from ER-targeting agents. The lower ER expression in the other subgroup might be partially explained by ER activity still being blocked by recently administered endocrine treatment, indicating that biopsy timing relative to endocrine treatment needs to be considered when interpreting transcriptomic data.

NAD+ Metabolism Generates a Metabolic Vulnerability in Endocrine-Resistant Metastatic Breast Tumors in Females.

Approximately 70% of human breast cancers express estrogen receptor-α (ERα), providing a potential target for endocrine therapy. However, 30% to 40% of patients with ER+ breast cancer still experience recurrence and metastasis, with a 5-year relative overall survival rate of 24%. In this study, we identified nicotinamide phosphoribosyltransferase (NAMPT), an important enzyme in nicotinamide adenine dinucleotide (NAD+) metabolism, to be increased in metastatic breast cancer (MBC) cells treated with fulvestrant (Fulv). We tested whether the blockade of NAD+ production via inhibition of NAMPT synergizes with standard-of-care therapies for ER+ MBC in vitro and in vivo. A synergistic effect was not observed when KPT-9274 was combined with palbociclib or tamoxifen or when Fulv was combined with other metabolic inhibitors. We show that NAMPT inhibitor KPT-9274 and Fulv works synergistically to reduce metastatic tumor burden. RNA-sequencing analysis showed that NAMPT inhibitor in combination with Fulv reversed the expression of gene sets associated with more aggressive tumor phenotype, and metabolomics analysis showed that NAMPT inhibition reduced the abundance of metabolites associated with several key tumor metabolic pathways. Targeting metabolic adaptations in endocrine-resistant MBC is a novel strategy, and alternative approaches aimed at improving the therapeutic response of metastatic ER+ tumors are needed. Our findings uncover the role of ERα-NAMPT crosstalk in MBC and the utility of NAMPT inhibition and antiestrogen combination therapy in reducing tumor burden and metastasis, potentially leading to new avenues of MBC treatment.

Abstract P4-08-15: Identification of target kinases whose inhibition enhances antitumor efficacy of sacituzumab-govitecan in metastatic HER2-negative breast cancer

Abstract BACKGROUND HER2-negative (HER2−) breast cancer (BC) subtypes, such as luminal A and triple-negative breast cancer (TNBC), express human trophoblast cell-surface antigen 2 (Trop-2). A high level of Trop-2 expression is associated with a poor prognosis in solid tumors. Sacituzumab-govitecan (SG), a Trop-2–directed antibody conjugated with SN-38 (topoisomerase Ⅰ inhibitor) significantly improves survival in patients with metastatic TNBC. Also, the IMMU-132-01 phase Ⅰ/Ⅱ study revealed that SG has antitumor activity in previously treated hormone receptor (HR)+/HER2− metastatic BC. In a phase Ⅲ trial (TROPiCS-02), SG demonstrated statistically significant progression-free survival in patients with heavily pre-treated HR+/HER2− endocrine-resistant metastatic BC. To augment the efficacy of SG, we sought to identify potential kinase targets whose inhibition enhances the antitumor efficacy of SG to formulate an SG-based combination therapy. MATERIALS AND METHODS Fluorescence-activated cell sorting analysis and Western blotting were used to evaluate the expression of Trop-2 in 24 TNBC and 8 HR+ BC cell lines. Non-biased high-throughput kinome-wide RNAi screening was performed with BT-20 (TNBC) and T47D tamoxifen/abemaciclib double-resistant (HR+) cell lines to identify target kinases whose inhibition produces synergistic antitumor efficacy of SG. To validate the combination antitumor effect of SG and selected kinase target inhibitors, sulforhodamine B staining proliferation assays, cell cycle analysis, and caspase 3/7 activity assays were performed in Trop-2+ TNBC and HR+ BC cell lines, including cell lines resistant to tamoxifen and double-resistant to tamoxifen and CDK4/6 inhibitor (palbociclib or abemaciclib). RESULTS In vitro proliferation assays revealed that the half-maximal inhibitory concentration (IC50) of SG in tested TNBC cell lines ranged from 10 nM to 84 nM, and that of HR+ cell lines ranged from 12.5 nM to >250 nM. We identified 69 kinase targets in TNBC cell line BT-20 and 44 kinase targets in HR+ BC cell line T47D (tamoxifen/abemaciclib double-resistant) by the sensitivity index analysis (>0.15) of RNAi screening results, and further pathway analysis revealed DNA damage response (DDR), (drug: BAY1895344), PI3k/Akt/mTOR (drug: copanlisib), and MAPK (drug: trametinib) as potential target canonical pathways whose targeting enhanced the cytotoxic effect of SG in both TNBC and HR+ BC. Among these strategies, inhibition of the DDR pathway with ATR inhibitor (BAY1895344) yielded a synergistic antiproliferative effect in combination with SG compared to SG or BAY1895344 alone in all tested TNBC and HR+ BC cell lines. Growth inhibition by SG combined with BAY1895344 averaged 97.1% in TNBC cell lines and ranged from 30.7% to 81.3% in HR+ BC cell lines, with P< 0.001. Other combinations with SG showed partially synergistic antiproliferative effects (copanlisib: effective in 3 of 5 cell lines, trametinib: effective in 2 of 5 cell lines). CONCLUSIONS RNAi screening uncovered DDR, PI3k/Akt/mTOR, and MAPK pathways as potential targets for combination with SG. Among inhibitors of these targets, DDR pathway inhibitor BAY1895344 had the most robust synergy with SG, compared to two other target inhibitors, against Trop-2+ TNBC and HR+ BC cell lines. These in vitro data warrant future in vivo validation studies of the synergistic antitumor effect of SG and BAY1895344 in metastatic HER2– breast cancer. Citation Format: Nakyung Oh, Jon A. Fuson, Huey Liu, Debu Tripathy, Naoto T. Ueno, Jangsoon Lee. Identification of target kinases whose inhibition enhances antitumor efficacy of sacituzumab-govitecan in metastatic HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-15.

Health-related quality of life and estimation of the minimally important difference in the Functional Assessment of Cancer Therapy-Endocrine Symptom score in postmenopausal ER+/HER2- metastatic breast cancer with low sensitivity to endocrine therapy.

The HORSE-BC study previously demonstrated that second-line endocrine therapy (ET) for patients with acquired endocrine-resistant metastatic breast cancer (MBC) still provided a clinically meaningful benefit. Herein, we investigated the health-related quality of life (HRQOL) in the HORSE-BC study. Patients with acquired endocrine-resistant MBC who were scheduled for second-line ET were recruited. The HRQOL was assessed at baseline, and 1 and 3 months after second-line ET initiation. To investigate the minimally important difference (MID) in the Functional Assessment of Cancer Therapy-Endocrine Symptoms (FACT-ES), we evaluated the means and standard deviations for the distribution-based method, and differences in the change in HRQOL for the anchor-based method. We also investigated the association between FACT-ES total scores and clinical benefit. Overall, 56 patients were enrolled. Of these, 47 were analyzed. When defined as 1/3 standard deviation estimates based on the distribution method, the calculated MID was 5.9. The MIDs of the FACT-ES total scores based on the anchor method were 7.7 for decline and 4.1 for improvement. The MID decline proportions were 6.1% and 14.7% lower in patients who experienced clinical benefits than in those who did not at 1 and 3 months, respectively. The ratios of MID improvement in patients who experienced clinical benefits were 18.3% and 3.2% higher, respectively; the mean change in the FACT-ES total score from baseline improved in patients who experienced clinical benefits. Maintaining the HRQOL as determined by FACT-ES may be associated with clinical benefits in patients with acquired endocrine-resistant MBC treated with ET.

Sacituzumab Govitecan in Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Metastatic Breast Cancer.

Hormone receptor-positive (HR+) human epidermal growth factor receptor 2-negative (HER2-) endocrine-resistant metastatic breast cancer is treated with sequential single-agent chemotherapy with poor outcomes. Sacituzumab govitecan (SG) is a first-in-class antibody-drug conjugate with an SN-38 payload targeting trophoblast cell-surface antigen 2, an epithelial antigen expressed in breast cancer. In this global, randomized, phase III study, SG was compared with physician's choice chemotherapy (eribulin, vinorelbine, capecitabine, or gemcitabine) in endocrine-resistant, chemotherapy-treated HR+/HER2- locally recurrent inoperable or metastatic breast cancer. The primary end point was progression-free survival (PFS) by blinded independent central review. Patients were randomly assigned to receive SG (n = 272) or chemotherapy (n = 271). The median age was 56 years, 95% had visceral metastases, and 99% had a prior cyclin-dependent kinase 4/6 inhibitor, with three median lines of chemotherapy for advanced disease. Primary end point was met with a 34% reduction in risk of progression or death (hazard ratio, 0.66 [95% CI, 0.53 to 0.83; P = .0003]). The median PFS was 5.5 months (95% CI, 4.2 to 7.0) with SG and 4.0 months (95% CI, 3.1 to 4.4) with chemotherapy; the PFS at 6 and 12 months was 46% (95% CI, 39 to 53) v 30% (95% CI, 24 to 37) and 21% (95% CI, 15 to 28) v 7% (95% CI, 3 to 14), respectively. Median overall survival (first planned interim analysis) was not yet mature (hazard ratio, 0.84; P = .14). Key grade ≥ 3 treatment-related adverse events (SG v chemotherapy) were neutropenia (51% v 38%) and diarrhea (9% v 1%). SG demonstrated statistically significant PFS benefit over chemotherapy, with a manageable safety profile in patients with heavily pretreated, endocrine-resistant HR+/HER2- advanced breast cancer and limited treatment options.

P01 The landscape of intra-tumour heterogeneity in endocrine resistant metastatic breast cancer

Abstract Introduction Breast cancer is the most frequently diagnosed malignancy in women worldwide. Heterogeneity is a characteristic of tumour aggression and metastatic progression in breast cancer. With the power of single-cell analysis we uncovered key subpopulations within this heterogeneous landscape and specific progressive metastatic characteristics. Methods Single cell RNA sequencing was carried on three endocrine resistant ER positive xenograft tumours with varying levels of metastatic burden. Data analysis was performed using the Seurat pipeline for filtering, horizontal data integration, cell cycle regression and unsupervised clustering. Individual clusters were characterized through differentially expressed markers and PAM50 molecular subtypes distribution. Results Data integration of scRNA sequencing from primary tumours revealed seven unsupervised clusters, six of which are present in all three mice, highlighting the strong heterogeneity of breast cancer independent of metastatic outcome. PAM50 classification showed a higher distribution of luminal A cells associated with good metastatic outcome. We observed a significant difference in PAM50 distributions between the individual clusters. One cluster diverges significantly from all others and shows a decrease in luminal subtype and an increase in basal like cells. Further analysis of this cluster shows an association of selected differentially expressed markers to metastatic progression. Co-occurrence and decreased expression of several EMT markers was observed with disease progression. Conclusion High tumour heterogeneity is independent of metastatic outcomes. Downregulation of EMT related genes can be essential in metastatic progression when selected markers are co-occurring in a subpopulation. Take-home message High tumour heterogeneity is present in breast cancer independent of metastatic outcomes. Downregulation of EMT related genes can be essential in metastatic progression when selected markers are co-occurring in a subpopulation.

Mutual exclusivity of ESR1 and TP53 mutations in endocrine resistant metastatic breast cancer

Both TP53 and ESR1 mutations occur frequently in estrogen receptor positive (ER+) metastatic breast cancers (MBC) and their distinct roles in breast cancer tumorigenesis and progression are well appreciated. Recent clinical studies discovered mutual exclusivity between TP53 and ESR1 mutations in metastatic breast cancers; however, mechanisms underlying this intriguing clinical observation remain largely understudied and unknown. Here, we explored the interplay between TP53 and ESR1 mutations using publicly available clinical and experimental data sets. We first confirmed the robust mutational exclusivity using six independent cohorts with 1,056 ER+ MBC samples and found that the exclusivity broadly applies to all ER+ breast tumors regardless of their clinical and distinct mutational features. ESR1 mutant tumors do not exhibit differential p53 pathway activity, whereas we identified attenuated ER activity and expression in TP53 mutant tumors, driven by a p53-associated E2 response gene signature. Further, 81% of these p53-associated E2 response genes are either direct targets of wild-type (WT) p53-regulated transactivation or are mutant p53-associated microRNAs, representing bimodal mechanisms of ER suppression. Lastly, we analyzed the very rare cases with co-occurrences of TP53 and ESR1 mutations and found that their simultaneous presence was also associated with reduced ER activity. In addition, tumors with dual mutations showed higher levels of total and PD-L1 positive macrophages. In summary, our study utilized multiple publicly available sources to explore the mechanism underlying the mutual exclusivity between ESR1 and TP53 mutations, providing further insights and testable hypotheses of the molecular interplay between these two pivotal genes in ER+ MBC.

Real World Experience of Long Term Treatment Outcome in Hormone Receptor-positive Metastatic Breast Cancer with or without Everolimus and Exemestane after Prior Aromatase Inhibitor

Background: Everolimus/exemestane has been shown to improve progression-free survival in patients with endocrine-resistant metastatic breast cancer. The regimen has been well-accepted despite lack of survival benefit. In real-life setting, patients were not well-selected and hence benefit of such treatment may not be as robust. Method: This is a retrospective review of 143 hormone receptor (HR) positive, HER-2 negative MBC patients who progressed on nonsteroidal aromatase inhibitors. Patients who received everolimus/exemestane in any treatment lines (EE group) were compared to patients who never received everolimus (NE group). Primary end point was survival adjusted to prognostic factors. Results: There were 52 patients in EE group and 91 in NE group with mean age of 58.6 years. Median follow-up time was 51 months. Unadjusted median OS was significantly longer in EE [33 vs 25 months, HR 0.66 (95%CI 0.44-0.998); p = 0.049]. In univariate analysis, factors affecting survival included numbers of metastatic sites, bone metastasis, EE treatment and numbers of treatment lines. Independent factors that remained significant in multivariate analysis were treatment lines [HR 0.71 (95%CI 0.63-0.79); p < 0.05] and numbers of metastatic sites. Median numbers of treatment line after NSAI failure was 5.2 vs 3.6 lines in EE and NE, respectively.Conclusion: In this real-life practice data, pts with HR positive, HER-2 negative MBC who had progressed on NSAI, sequential use of multiple treatment regimens of endocrine and chemotherapy is essential to longer survival. Everolimus/exemestane may have contributed, to a lesser extent, to this improvement in survival.

Abstract PS17-31: Investigating the estrogen receptor Y537S mutation in transgenic models of luminal B breast cancer

Abstract Breast cancer is the most prevalent cancer in women and over two-thirds of cases express the estrogen receptor (ER). Significantly, metastatic ER positive breast cancer is the leading cause of breast cancer mortality. Despite the success of endocrine therapy to treat ER-positive breast cancers, resistance to treatment is a major problem and relapse commonly occurs though patients initially respond well to treatment. The ER Y537S mutant represents one of the most common gain-of-function mutations detected in the metastatic biopsies of patients with ER-positive breast cancer. Previous in-vitro analyses have shown that the ER Y537S mutant exhibits enhanced ER transcriptional activity by adopting a ligand-independent agonist confirmation. In our study, we seek to provide a deeper understanding of the role of this mutant by modeling the human ER Y537S mutant (ER Y541S in mice). Our lab has generated a Cre-inducible knock-in mouse model expressing the activating point mutation driven by the endogenous ER promoter, and it has been shown that when expressed ubiquitously the mutated ER leads to dramatic developmental effects. In order to further our observations in the context of endocrine resistant metastatic breast cancer, we characterize mamary tumour growth of the ER Y541S mutant in combination with luminal B models of mammary tumourigenesis. Consistent with the notion that these ER mutations occur seldom in primary tumours, preliminary data shows that tumour onset and tumour outgrowth is unaffected by the activating point mutation. When analysing tumours from both ER mutant and wildtype ER control animals, it was found that tumours with mutant ER display more basal-like cytokeratins at the RNA level via RNA sequencing and at the protein level via immunohistochemistry. This expression pattern is often associated with poorer prognosis in the clinic. Through ongoing analyses, we also seek to better understand our model through tumour regression studies (doxycycline withdrawal), treatment with tamoxifen and blocking the main source of estrogens via ovariotomies. By studying the ER Y537S mutation in models of luminal B breast cancer, we hope to investigate the factors leading to tumour recurrence and provide a deeper understanding of potential escape pathways to therapy. Citation Format: Gabriella Johnson, Joshua Roccamo, Cynthia Lavoie, Dongmei Zuo, Virginie Sanguin-Gendreau, Adrian Lee, Zheqi Li, Steffi Oesterreich, William Muller. Investigating the estrogen receptor Y537S mutation in transgenic models of luminal B breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-31.

Abemaciclib, a third CDK 4/6 inhibitor for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer

ABSTRACT Introduction The field of metastatic luminal breast cancer (hormone receptor positive, HER-2 negative) is dynamic and evolving, harboring some of the most significant therapeutic advances in medical oncology. Over the last decade, many pivotal trials showed excellent results with drastic improvements in survival as well as the quality of life of metastatic luminal breast cancer patients. Areas covered The successful inhibition of the cyclinD/cyclin-dependent kinases 4 and 6 (CDK4/6)–retinoblastoma protein (RB) pathway with potent CDK4/6 inhibitors improved the outcome of advanced luminal breast cancers. Abemaciclib is the third CDK 4/6 inhibitor arriving to the market after palbociclib and ribociclib. Here, we describe the biology of the CDK4/6 pathway and summarize clinical data of previously published pivotal trials emphasizing the efficacy and toxicity of abemaciclib. The aim was to define its place in the current guidelines and to make a brief comparison with other available drugs of same class in the absence of cross trials comparison. Expert opinion As there are no available biomarkers to predict response or resistance to abemaciclib, the promising overall survival data of MONARCH-2 could possibly impact the clinician’s choice to optimize treatment for endocrine-resistant metastatic breast cancer.

Point-activated ESR1Y541S has a dramatic effect on the development of sexually dimorphic organs.

Mutations in the estrogen receptor α (ERα) occur in endocrine-resistant metastatic breast cancer. However, a major gap persists with the lack of genetically tractable immune competent mouse models to study disease. Hence, we developed a Cre-inducible murine model expressing a point-activated ESR1Y541S (ESR1Y537S in humans) driven by its endogenous promoter. Germline expression of mutant ESR1Y541S reveals dramatic developmental defects in the reproductive organs, mammary glands, and bones of the mice. These observations provide critical insights into the tissue-specific roles of ERα during development and highlights the potential use of our model in further developmental and cancer studies.

The efficacy of first-line chemotherapy in endocrine-resistant hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer.

Combinations of endocrine therapy (ET) and targeted therapy (CDK4/6 or mTOR inhibitors) are standard of care for HR+/HER2- metastatic breast cancer (MBC). When ET is not effective, chemotherapy is commonly used. However, clinical outcomes of chemotherapy in the endocrine-resistant setting are limited. The purpose of this study was to identify predictive factors and the compare efficacies of chemotherapy agents in endocrine-resistant MBC. We conducted a retrospective study of patients with HR+/HER2- MBC who received chemotherapy after progression on ET with or without targeted therapy at MD Anderson Cancer Center from 1999 to 2017. We collected baseline clinicopathological and all treatment data. Primary endpoint was time to treatment failure (TTF) of first-line chemotherapy for MBC. For the 1258 patients analyzed, mean age was 55.3years (range 21-91). Previous treatment with targeted therapy was recorded for 390 patients (31%): 264 with CDK4/6 inhibitor, 205 with mTOR inhibitor, and 79 treated with both. The most frequent chemotherapy agents were capecitabine (48.9%) and taxanes (28.6%). After adjustment for all factors in a multivariate model, previous treatment with a CDK4/6 inhibitor had the strongest negative effect on TTF regardless of ET duration (hazard ratio [HR] 1.84; 95%CI 1.49-2.27; p < 0.001). Conversely, capecitabine had significantly longer median TTF than taxanes regardless of whether patients had prior exposure to taxanes in primary setting (6.1 vs 4.9months; HR 0.64; 95%CI 0.55-0.75; p < 0.001). Previous exposure to CDK4/6 inhibitor had a negative predictive effect for the efficacy of chemotherapy. Capecitabine had the best efficacy against endocrine-resistant breast cancer.

The efficacy of first-line chemotherapy in endocrine-resistant hormone receptor-positive (HR+), human epidermal growth factor receptor 2- negative (HER2-) metastatic breast cancer (MBC).

1056 Background: Combinations of endocrine therapy (ET) and targeted therapy (CDK4/6 or mTOR inhibitors) are standard of care for HR+/HER2- MBC. When ET is not effective, chemotherapy is commonly used. However, clinical outcomes of chemotherapy in the endocrine-resistant setting are limited. We hypothesized that clinicopathological baseline and prior ET factors determine chemotherapy’s efficacy. We sought to identify predictive factors and the compare efficacies of chemotherapy agents in endocrine-resistant MBC. Methods: We conducted a retrospective study of patients with HR+/HER2- MBC who received chemotherapy after progression on ET with or without targeted therapy at MD Anderson Cancer Center from 1999-2017. We collected baseline clinicopathological and all treatment data. The primary endpoint was time to treatment failure (TTF) of first-line chemotherapy for MBC. We performed univariate and multivariate analyses using the Cox proportional hazard model. Kaplan-Meier methods were used to analyze TTF. Results: In the 1,258 patients analyzed, the mean age was 55.3 years (range 21-91). Forty-five patients (3.6%) had inflammatory breast cancer (IBC). Three hundred ninety patients (31%) received previous targeted therapy: 264 with CDK4/6 inhibitor, 205 with mTOR inhibitor, and 79 with both. The most frequent chemotherapy agents were capecitabine (48.9%) and taxanes (paclitaxel, nab-paclitaxel, or docetaxel; 28.6%). After adjustment for all factors in a multivariate model, IBC and prior exposure to a CDK4/6 inhibitor were significantly associated with shorter TTF. Previous treatment with a CDK4/6 inhibitor had the strongest negative effect on chemotherapy TTF regardless of ET duration (adjusted hazard ratio [HR] 1.84; 95%CI 1.49-2.27; p &lt; 0.001). Capecitabine had significantly longer median TTF than taxanes regardless of whether patients had prior exposure to taxanes in (neo)adjuvant setting (6.1 vs 4.9 months; HR 0.64; 95%CI 0.55-0.75; p &lt; 0.001). Conversely, the median TTF for taxanes was shorter in patients who received prior (neo)adjuvant taxanes than in those who did not (4.5 vs 5.1 months). Conclusions: Previous exposure to CDK4/6 inhibitor had a negative predictive effect for the efficacy of chemotherapy. Capecitabine had the best efficacy against endocrine-resistant breast cancer.

Targeted degradation of activating estrogen receptor α ligand-binding domain mutations in human breast cancer.

Studies have identified several estrogen receptor α (ERα) ligand-binding domain (LBD) somatic mutations in endocrine therapy resistant, metastatic ER-positive breast cancers. The most common mutations, Tyr537Ser (Y537S) and Asp538Gly (D538G), are detected in ~ 30% of endocrine resistant metastatic breast cancer patients. These ESR1 mutations induce the agonist conformation of ERα, confer an estrogen-independent phenotype, and promote drug resistance to antiestrogens. ER-positive, estrogen-dependent MCF-7 cells were engineered to express either the Y537S or D538G mutants using CRISPR knock-in (cY537S and cD538G). These cells were used to screen several estrogen receptor degrader (ERD) compounds synthesized using the Proteolysis Targeting Chimeras (PROTAC) method to induce degradation of ERα via the ubiquitin-proteasome pathway. Wild-type MCF-7 and ERα LBD mutant cells were treated with ERD-148 (10pM-1µM) and assayed for cellular proliferation using the PrestoBlue cell viability assay. ERD-148 attenuated ER-dependent growth with IC50 values of 0.8, 10.5, and 6.1nM in MCF-7, cY537S, and cD538G cells, respectively. Western blot analysis showed that MCF-7 cells treated with 1nM ERD-148 for 24h exhibited reduced ERα protein expression as compared to the mutants. The ER-regulated gene, GREB1, demonstrated significant downregulation in parental and mutant cells after 24h of ERD-148 treatment at 10nM. Growth of the ER-negative, estrogen-independent MDA-MB-231 breast cancer cells was not inhibited by ERD-148 at the ~ IC90 observed in the ER-positive cells. ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ERα with comparable potency to Fulvestrant with marginal non-specific toxicity.

Abstract P5-11-06: Phase 1 study of TTC-352 in patients with metastatic breast cancer progressing on endocrine therapy

Abstract Background: TTC-352 is a selective human ER partial agonist (ShERPA) developed for treatment of ER+ breast cancer (BC). ShERPAs mimic the effects of estradiol (E2) in hormone-independent, endocrine-resistant BC cells, but since it has only partial agonist activity in ER+ cells, it could have an improved side effect profile. Thus, TTC-352 could be a promising option for endocrine-resistant BC. Methods: This is an open-label, accelerated dose escalation study with primary endpoint of maximum tolerated dose (MTD) that is evaluating up to five dose levels of TTC-352 in patients with ER+ endocrine-resistant metastatic BC (NCT03201913). The MTD of TTC-352 is determined using initial single-patient cohort escalations until grade 2 toxicity, then expansion to a modified-Fibonacci dose-escalation 3+3 design. The secondary objectives are: best response (BR), progression-free survival, overall survival, treatment tolerability, and pharmacokinetics (PK) of TTC-352. Patient population: patients with metastatic ER+ BC who received and progressed on at least two lines of endocrine therapy, with one that included a CDK4/CDK6 inhibitor, and with adequate hepatic, renal, and bone marrow function at screening. Results: This study completed enrollment of a total of 15 patients on June 5, 2019. Dose LevelTTC-352 Dose Level (mg)Number of SubjectsTwice-Daily (BID) DoseTotal Daily Dose115302230601360120141202401518036010 No dose limiting toxicity has been seen to date with any of the tested dose levels. There was one treatment-related grade 3 event of asymptomatic pulmonary embolism (30mg BID). The number and percentage (n, %) of patients with treatment-related grade 1 and grade 2 adverse events reported in two or more patients across all dose levels were diarrhea (9, 60), hot flush (4, 26), headache (3, 20), nausea (3, 20), abdominal pain (3, 20), rash (3, 20), blurred vision (2, 13) vomiting (2, 13), dizziness (2, 13), peripheral neuropathy (2, 13), breast pain (2, 13), uterine/vaginal hemorrhage (2, 13) and vaginal discharge (2, 13). BR in the 7 evaluable patients (patients with at least two 28-day cycles of treatment) showed that 3 patients had stable disease (SD) for 4, and 10 months, respectively; 1 patient continues study treatment with SD after 9 months; and 4 patients had progressive disease (PD) after completing two cycles of treatment. All 5 non-evaluable patients had PD prior to completing two cycles of treatment. PK: The intra- and inter-individual variability for AUC0-∞ hampered assessment of the relationship between dose and AUC0-∞., Half-life was 7.6-14.3 hrs. Trough TTC-352 plasma concentrations at steady-state (SS) remained well above the plasma concentrations associated with efficacy in animal models. PKC alpha expression in tumor tissue did not correlate with length of disease control with TTC-352 therapy, though the numbers are small and not all of the available specimens were metastatic. Conclusions: TTC 352 demonstrates manageable safety and early clinical evidence of antitumor activity in patients with BC progressing on endocrine therapy. Based upon SS TTC-352 plasma concentrations and tolerability, the 180mg BID dose is recommended for further testing. Citation Format: Ruth O'Regan, Randolph Hurley, Jasgit Sachdev, Jonathan Bleeker, Debra Tonetti, Gregory Thatcher, Robert Venuti, Arkadiusz Dudek. Phase 1 study of TTC-352 in patients with metastatic breast cancer progressing on endocrine therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-11-06.